Rossi’s Principles of Transfusion Medicine, Fourth Edition

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Rossi’s Principles of Transfusion Medicine

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4 Rossi’s Principles of Transfusion Medicine EDITED BY

Toby L. Simon Edward L. Snyder Bjarte G. Solheim Christopher P. Stowell Ronald G. Strauss Marian Petrides FOURTH EDITION

A John Wiley & Sons, Ltd., Publication

This edition © 2009 by AABB, published by Blackwell Publishing Ltd. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the publisher. Wiley-Blackwell Offices Registered office: John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial offices:

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2009

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Contents

Contributors, viii

Part II. Platelets, 149

Preface, xiii

10 Platelet Production, Kinetics, and Hemostasis, 149 Henry M. Rinder & Aaron Tomer

1 Transfusion in the New Millennium, 1 Ennio C. Rossi & Toby L. Simon

Section I. Blood Components and Derivatives, 15 Part I. Red Blood Cells, 17 2 Red Cell Production and Kinetics, 17 Mark J. Koury 3 Regulation of Oxygen Delivery by Red Cells and Red Cell Substitutes, 29 Christopher P. Stowell 4 Red Cell Metabolism and Preservation, 54 Bjarte G. Solheim & John R. Hess 5 Red Cell Immunology and Compatibility Testing, 69 W. John Judd

11 Platelet Immunology and Alloimmunization, 168 Brian R. Curtis & Janice G. McFarland 12 Preparation, Preservation, and Storage of Platelet Concentrates, 187 Ralph R. Vassallo, Jr. 13 Thrombocytopenia and Platelet Transfusion, 199 Peter W. Marks

Part III. White Blood Cells, 211 14 Neutrophil Production and Kinetics: Neutropenia and Neutrophilia, 211 Thomas H. Price 15 Neutrophil Collection and Transfusion, 219 Ronald G. Strauss 16 Leukocyte-Reduced Blood Components: Laboratory and Clinical Aspects, 228 Darrell J. Triulzi & Walter H. Dzik

6 Carbohydrate Blood Groups, 89 Eldad A. Hod, Patrice F. Spitalnik, & Steven L. Spitalnik

Part IV. Plasma, 247

7 Rh and LW Blood Group Antigens, 109 Connie M. Westhoff & Don L. Siegel

17 Composition and Mechanisms of Blood Coagulation and Fibrinolysis, 247 Peter Hellstern

8 Other Protein Blood Groups, 121 Petr Jarolim

18 Immunoglobulins, 260 Urs E. Nydegger

9 Anemia and Red Blood Cell Transfusion, 131 Jeffrey L. Carson & Paul Hébert

19 Preparation of Plasma Derivatives, 273 Toby L. Simon, Kirsten Seidel, & Albrecht Gröner

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Contents 20 Plasma Transfusion and Use of Albumin, 287 Simon J. Stanworth & Alan T. Tinmouth

33 Hematopoietic Progenitor Cells: Biology and Processing, 508 Thomas Leemhuis & Ronald A. Sacher

Part V. Cell Kinetics, 298

34 Hematopoietic Progenitor Cells: Autologous Transplantation, 521 Hooman H. Rashidi & Scott D. Rowley

21 Applications of Cellular Radiolabeling in Transfusion Medicine, 298 Larry J. Dumont, James P. AuBuchon, & Richard J. Davey

Section II. Clinical Practice, 319 Part I. Medical Patients, 321 22 Autoimmune Hemolytic Anemia and Paroxysmal Nocturnal Hemoglobinuria, 321 Thomas P. Duffy 23 Management of Immune Thrombocytopenia, 344 Donald M. Arnold, James W. Smith, & Theodore E. Warkentin 24 Bleeding from Acquired Coagulation Defects and Antithrombotic Therapy, 376 Thomas J. Raife, Jeffrey S. Rose, & Steven R. Lentz

Part II. Obstetric and Pediatric Patients, 391 25 Fetal and Neonatal Hematopoiesis, 391 Robert D. Christensen & Martha C. Sola-Visner 26 Obstetric Transfusion Practice, 406 Humphrey H.H. Kanhai, Jos J.M. van Roosmalen, & Anneke Brand 27 Hemolytic Disease of the Fetus and Newborn, 418 Bjarte G. Solheim & Morten Grönn 28 Congenital Disorders of Clotting Proteins and Hypercoagulable States in Pediatrics, 426 Linda J. Butros & Prasad Mathew 29 Management of Congenital Hemolytic Anemias, 448 Bruce I. Sharon

35 Hematopoietic Progenitor Cells: Allogeneic Transplantation, 542 Amin M. Alousi & Sergio A. Giralt 36 Umbilical Cord Blood: A Reliable Source of Stem and Progenitor Cells for Human Transplantation, 559 Suhag H. Parikh & Joanne Kurtzberg

Part IV. Surgery Patients, 566 37 Alternatives to Transfusion: Perioperative Blood Management, 566 Lynne Uhl 38 Blood Components to Achieve Hemostasis for Surgery and Invasive Procedures, 575 Walter H. Dzik 39 Transfusion Therapy in the Care of Trauma and Burn Patients, 589 Ellen C. Omi & Richard L. Gamelli 40 Transfusion Therapy in Solid-Organ Transplantation, 604 Glenn Ramsey

Section III. Apheresis, 615 41 Apheresis: Principles and Technology of Hemapheresis, 617 Ronald O. Gilcher & James W. Smith 42 Therapeutic Plasma Exchange, 629 Bruce C. McLeod 43 Specialized Therapeutic Hemapheresis and Phlebotomy, 652 Robert Weinstein

Section IV. Hazards of Transfusion, 681

30 Blood Component Transfusions for Infants, 470 Ronald G. Strauss

Part I. Hemovigilance, 683

Part III. Oncology Patients, 482

44 Overview of Hemovigilance, 683 Dorothy Stainsby, Jean-Claude Faber, & Jan Jørgensen

31 Transfusion Support for the Oncology Patient, 482 Christopher A. Tormey & Edward L. Snyder 32 Hematopoietic Growth Factors (Cytokines), 498 W. Conrad Liles & David C. Dale

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45 Immunomodulatory and Proinflammatory Effects of Allogeneic Blood Transfusion, 699 Eleftherios C. Vamvakas, José O. Bordin, & Morris A. Blajchman

Contents

Part II. Infectious Hazards of Transfusion, 718

Section V. Cell and Tissue Therapy, 885

46 Transfusion-Transmitted Hepatitis, 718 Harvey J. Alter & Juan I. Esteban-Mur

57 HLA Antigens and Alleles, 887 Thomas M. Williams

47 Retroviruses and Other Viruses, 746 John A. J. Barbara, & Brian C. Dow 48 Transfusion Transmission of Parasites, 760 Bryan R. Spencer 49 Bacterial Contamination of Blood Products, 773 Yara A. Park & Mark E. Brecher 50 Prion Diseases, 791 Marc L. Turner 51 Pathogen Inactivation, 801 Bjarte G. Solheim & Jerard Seghatchian

Part III. Noninfectious Hazards, 811 52 Hemolytic Transfusion Reactions, 811 Robertson D. Davenport 53 Febrile, Allergic, and Nonimmune Transfusion Reactions, 826 Gregory J. Pomper 54 Transfusion-Associated Graft-vs-Host Disease, 847 John E. Levine & James L. M. Ferrara 55 Transfusional Iron Overload, 858 Sujit Sheth 56 Transfusion-Related Acute Lung Injury, 870 Jonathan P. Wallis & Ulrich J. H. Sachs

58 Tissue Banking, 898 Jeanne V. Linden, William F. Zaloga, & A. Bradley Eisenbrey 59 Adoptive Immunotherapy, 920 Catherine M. Bollard & Helen E. Heslop 60 Gene Therapy in Transfusion Medicine, 936 Emanuela M. Bruscia & Diane S. Krause 61 Tissue Engineering and Regenerative Medicine, 950 Clay Quint & Laura Niklason

Section VI. Delivery of Transfusion and Transplantation Services, 973 62 Recruitment and Screening of Donors and the Collection, Processing, and Testing of Blood, 975 Kendall P. Crookston, Susan L. Wilkinson, & Toby L. Simon 63 Current Legal Issues in Transfusion Medicine, 993 Edward M. Mansfield, Thomas K. Berg, Kurt A. Leifheit, John Parker Sweeney & P. Robert Rigney 64 Current Good Manufacturing Practice, 1010 P. Ann Hoppe & Sheryl A. Kochman 65 Transplant Organizations and Networks in the Regulation of Cellular Therapy Programs, 1032 Dennis L. Confer 66 Hospital Transfusion Committee and Quality Assurance, 1041 Pamela Clark & Paul D. Mintz

Index, 1061

Companion PC - and, MAC - Compatible CD-ROMs CD-ROM 1: All chapters of the book with a full text search function.

5 Platelet Refractoriness Marian Petrides

CD-ROM 2: Interactive case studies for self-testing.

6 An Unexpected Panagglutinin Marian Petrides

CD-ROM 2: Cases

7 Postsurgical Bleeding Marian Petrides

1 Posttransfusion Hemolysis Marian Petrides & Nicole A. Pele 2 Child with Low Fibrinogen Marian Petrides & Donna Castellone 3 Head Injury, Sudden Dyspnea Marian Petrides 4 A Transfusion Fatality Marian Petrides

8 Jehovah’s Witness Surgery Christopher P. Stowell & Marian Petrides 9 Delayed Fever and Dark Urine Marian Petrides 10 Fever in Neutropenic Child Marian Petrides & Dava S. Cleveland-Noriega

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Contributors

Chapter Contributors Amin M. Alousi, MD Assistant Professor Stem Cell Transplantation The University of Texas M.D. Anderson Cancer Center Houston, Texas, USA Harvey J. Alter, MD Chief, Clinical Studies Associate Director for Research Department of Transfusion Medicine Warren Grant Magnuson Clinical Center National Institutes of Health Bethesda, Maryland, USA

Catherine M. Bollard, MD Associate Professor of Pediatrics, Immunology and Medicine Center for Cell and Gene Therapy Baylor College of Medicine, Texas Children’s Hospital and The Methodist Hospital Houston, Texas, USA José O. Bordin, MD Associate Professor Division of Hematology and Transfusion Medicine Universidade Federal de Sao Paulo Sao Paulo, Brazil

Assistant Professor Department of Medicine Michael G. DeGroote School of Medicine McMaster University Hamilton, Ontario, Canada

Anneke Brand, MD, PhD Professor of Transfusion Medicine Sanquin Division Southwest Department of Immunohematology and Blood Transfusion Leiden University Medical Center Leiden, The Netherlands

James P. AuBuchon, MD

Mark E. Brecher, MD

President and Chief Executive Officer Puget Sound Blood Center Seattle, Washington, USA

Director, McLendon Clinical Laboratories, University of North Carolina Hospitals; Professor and Vice Chair for Clinical Services Department of Pathology and Laboratory Medicine University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Donald M. Arnold, MD

John A.J. Barbara, MA, MSc, PhD, FIBiol, FRCPath Emeritus Microbiology Consultant to National Blood Service London, UK; Visiting Professor University of West of England Bristol, UK

Thomas K. Berg, JD Partner Hinshaw & Culbertson LLP Minneapolis, Minnesota, USA

Morris A. Blajchman, MD, FRCP(C) Professor Emeritus Departments of Pathology and Medicine McMaster University; Medical Director Canadian Blood Services Hamilton, Ontario, Canada

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Emanuela M. Bruscia, PhD Associate Research Scientist Department of Pediatrics, Yale University New Haven, Connecticut, USA Linda J. Butros, MD Assistent Professor of Pediatrics Pediatric Hematology/Oncology University of New Mexico Albuquerque, New Mexico, USA

Robert D. Christensen, MD Director of Research Department of Neonatology Intermountain Health Care Ogden, Utah, USA

Pamela Clark, MD, JD Associate Professor of Pathology Division of Clinical Pathology; Associate Director Blood Bank and Transfusion Medicine Services University of Virginia Health System Charlottesville, Virginia, USA

Dennis L. Confer, MD Clinical Professor of Medicine University of Minnesota; Chief Medical Officer National Marrow Donor Program Minneapolis, Minnesota, USA Kendall P. Crookston, MD, PhD Associate Professor University of New Mexico Department of Pathology; Associate Medical Director Tricore Reference Laboratories; Medical Director United Blood Services of New Mexico Albuquerque, New Mexico, USA Brian R. Curtis, MSTM, MT(ASCP)SBB Technical Director Platelet and Neutrophil Immunology Laboratory BloodCenter of Wisconsin Milwaukee, Wisconsin, USA

Jeffrey L. Carson, MD

David C. Dale, MD Professor of Medicine Department of Medicine University of Washington Seattle, Washington, USA

Richard C. Reynolds Professor of Medicine Chief, Division of General Internal Medicine Robert Wood Johnson Medical School University of Medicine and Dentistry of New Jersey New Brunswick, New Jersey, USA

Robertson D. Davenport, MD Associate Professor Department of Pathology The University of Michigan Medical School Ann Arbor, Michigan, USA

Contributors

Richard J. Davey, MD Professor of Pathology and Laboratory Medicine Weill Cornell Medical College; Director of Transfusion Medicine The Methodist Hospital Houston, Texas, USA

Director, Burn and Shock Trauma Institute Chief, Burn Center Loyola University Medical Center Strich School of Medicine Maywood, Illinois, USA

Brian C. Dow, BSc, PhD, CSci, FIBMS

Medical Director Emeritus Oklahoma Blood Institute Oklahoma City, Oklahoma, USA

Consultant Clinical Microbiologist; Head of National Microbiology Reference Unit Scottish National Blood Transfusion Service Glasgow, Scotland, UK

Thomas P. Duffy, MD Professor of Medicine Department of Internal Medicine and Hematology Yale University School of Medicine New Haven, Connecticut, USA

Ronald O. Gilcher, MD

Sergio A. Giralt, MD, MPH Professor, Stem Cell Transplantation and Lymphoma/Myeloma The University of Texas M.D. Anderson Cancer Center Houston, Texas, USA

Larry J. Dumont, MBA, PhD

Albrecht Gröner, PhD

Director, Cell Labeling Laboratory Assistant Professor Dartmouth Medical School Dartmouth-Hitchcock Medical Center Lebanon, New Hampshire, USA

Head of Virology CSL Behring GmbH Marburg, Germany

Walter H. Dzik, MD Co-Director, Blood Transfusion Service Massachusetts General Hospital; Associate Professor of Pathology Harvard Medical School Boston, Massachusetts, USA

A. Bradley Eisenbrey, MD, PhD Laboratory Director Gift of Life Michigan Ann Arbor, Michigan; Assistant Professor of Pathology Wayne State University School of Medicine Detroit, Michigan, USA Juan I. Esteban-Mur, MD Section Head Liver Unit, Department of Medicine Hospital Universitari Vall d’Hebron Universitat Autonoma de Barcelona Centro de Investigaciones Biomédicas en Red (Ciberehd) Instituto de Salud Carlos III Barcelona, Spain

Jean-Claude Faber, MD Director (Retired) Blood Transfusion Service Luxembourg Red Cross Luxembourg, Luxembourg

James L.M. Ferrara, MD Professor of Pediatrics and Internal Medicine Director, Combined Blood and Marrow Transplantation Program University of Michigan Ann Arbor, Michigan, USA

Richard L. Gamelli, MD, FACS The Robert J. Freeark Professor and Chair Department of Surgery

Morten Grönn, MD Head Physician Division of Paediatrics Rikshospitalet University Hospital Oslo, Norway

Paul Hébert, MD Professor of Medicine, Surgery, Anesthesiology, and Epidemiology, University of Ottawa; Critical Care Physician; The Ottawa Hospital; Senior Scientist, Ottawa Health Research Institute Ottawa, Ontario Canada

Peter Hellstern, MD Director and Professor Institute of Hemostaseology and Transfusion Medicine Academic City Hospital Ludwigshafen, Germany

Helen E. Heslop, MD Professor of Medicine and Pediatrics and Dan L. Duncan Chair Center for Cell and Gene Therapy Baylor College of Medicine, Texas Children’s Hospital and The Methodist Hospital Houston, Texas, USA John R. Hess, MD, MPH Professor Blood Bank University of Maryland Medical Center Baltimore, Maryland, USA Eldad A. Hod, MD Transfusion Medicine Fellow Department of Pathology and Cell Biology Columbia University Medical Center New York, New York, USA

P. Ann Hoppe, MT(ASCP)SBB President Hoppe Regulatory Consultants, LLC Decatur, Georgia, USA Petr Jarolim, MD, PhD Director, Clinical Chemistry Brigham and Women’s Hospital and the Dana Farber Cancer Institute; Associate Professor of Pathology Harvard Medical School Boston, Massachusetts, USA Jan Jørgensen, MD Director (Retired) Blood Transfusion Centre Aarhus University Hospital Aarhus, Denmark

W. John Judd, FIBMS, MIBiol Emeritus Professor of Immunohematology Department of Pathology University Hospitals University of Michigan Ann Arbor, Michigan, USA Humphrey H.H. Kanhai, MD, PhD Professor of Obstetrics and Fetal Medicine Department of Obstetrics Leiden University Medical Center Leiden, The Netherlands Sheryl A. Kochman, MT(ASCP) Chief, Devices Review Branch Division of Blood Applications Office of Blood Research and Review Center for Biologics Evaluation and Research Food and Drug Administration Rockville, Maryland, USA

Mark J. Koury, MD Professor of Medicine Division of Hematology/Oncology Vanderbilt University and VA Tennessee Valley Healthcare System Nashville, Tennessee, USA Diane S. Krause, MD, PhD Professor, Laboratory Medicine and Pathology Yale University School of Medicine New Haven, Connecticut, USA Joanne Kurtzberg, MD Susan Dees Distinguished Professor of Pediatrics Professor of Pathology Chief, Pediatric Blood and Marrow Transplant Program Director, Carolinas Cord Blood Bank Co-Director, Stem Cell Laboratory Duke University Medical Center Durham, North Carolina, USA

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Contributors

Thomas Leemhuis, PhD Director Cellular Therapies Division Hoxworth Blood Center University of Cincinnati Academic Health Center Cincinnati, Ohio, USA Kurt A. Leifheit, JD Associate Hinshaw & Culbertson LLP Chicago, Illinois, USA Steven R. Lentz, MD, PhD Hamilton Professor of Hematology; Director Division of Hematology, Oncology, and Blood and Marrow Transplantation; Professor of Medicine Department of Internal Medicine Carver College of Medicine University of Iowa Iowa City, Iowa, USA

John E. Levine, MD, MS Professor of Pediatrics and Internal Medicine Clinical Director, Pediatric Blood and Marrow Stem Cell Transplantation Program University of Michigan Ann Arbor, Michigan, USA W. Conrad Liles, MD, PhD Professor and Vice-Chair of Medicine Director, Division of Infectious Diseases University of Toronto Toronto, Ontario, Canada

University of New Mexico Albuquerque, New Mexico, USA

Janice G. McFarland, MD Director Platelet and Neutrophil Immunology Laboratory BloodCenter of Wisconsin Milwaukee, Wisconsin, USA

Bruce C. McLeod, MD Professor of Medicine and Pathology Rush Medical College; Director, Blood Center Rush University Medical Center Chicago, Illinois, USA

Paul D. Mintz, MD Professor of Pathology and Internal Medicine; Chief, Division of Clinical Pathology; Director, Clinical Laboratories and Transfusion Medicine Services; University of Virginia Health System Charlottesville, Virginia, USA Laura Niklason, MD, PhD Associate Professor Anesthesiology and Biomedical Engineering Yale University School of Medicine New Haven, Connecticut, USA

Urs E. Nydegger, MD Professor Emeritus University of Bern; Consultant Transfusion Therapy Consultancy Bern, Switzerland

Jeanne V. Linden, MD, MPH Director, Blood and Tissue Resources Wadsworth Center New York State Department of Health; Clinical Associate Professor Albany Medical College; Adjunct Associate Professor School of Public Health State University of New York at Albany Albany, New York, USA

Edward M. Mansfield, JD Equity Member Belin Lamson McCormick Zumbach Flynn PC Des Moines, Iowa, USA

Ellen C. Omi, MD Assistant Professor of Surgery Division of Critical Care, Trauma and Burn Loyola University Medical Center Stritch School of Medicine Maywood, Illinois, USA Suhag H. Parikh, MD Assistant Professor Pediatric Stem Cell Transplant Program Duke University Medical Center Durham, North Carolina, USA

Yara A. Park, MD Peter W. Marks, MD, PhD Associate Professor of Medicine Section of Hematology Yale University School of Medicine New Haven, Connecticut, USA

Assistant Professor Department of Pathology and Laboratory Medicine University of North Carolina at Chapel Hill Chapel Hill, North Carolina, USA

Prasad Mathew, MD Professor of Pediatrics Pediatric Hematology/Oncology

Gregory J. Pomper, MD Assistant Professor of Pathology Department of Pathology

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Wake Forest University School of Medicine Winston-Salem, North Carolina, USA

Thomas H. Price, MD Executive Vice-President and Medical Director Puget Sound Blood Center; Professor of Medicine University of Washington Seattle, Washington, USA Clay Quint, MD Fellow, Biomedical Engineering Yale University School of Medicine New Haven, Connecticut, USA Thomas J. Raife, MD Associate Professor of Pathology; Director Division of Transfusion Medicine and Blood Banking Department of Pathology Carver College of Medicine, University of Iowa Iowa City, Iowa, USA Glenn Ramsey, MD Professor of Pathology Northwestern University School of Medicine Chicago, Illinois, USA Hooman H. Rashidi, MD Chief Resident Department of Pathology and Laboratory Medicine Yale University School of Medicine New Haven, Connecticut, USA P. Robert Rigney, JD Chief Executive Officer American Association of Tissue Banks McLean, Virginia, USA

Henry M. Rinder, MD Associate Professor of Laboratory Medicine and Internal Medicine (Hematology) Yale University School of Medicine; Director, Hematology Laboratories Yale-New Haven Hospital New Haven, Connecticut, USA

Jos J.M. van Roosmalen, MD, PhD Professor of International Safemotherhood Department of Obstetrics Leiden University Medical Center Leiden, The Netherlands; Section of Health Care and Culture VU University Medical Center Amsterdam, The Netherlands Jeffrey S. Rose, MD Hematology/Oncology Fellow Department of Internal Medicine Carver College of Medicine University of Iowa Iowa City, Iowa, USA

Contributors

Ennio C. Rossi, MD

James W. Smith, MD, PhD

Christopher P. Stowell, MD, PhD

Professor Emeritus of Medicine Northwestern University School of Medicine Chicago, Illinois, USA

Medical Director Oklahoma Blood Institute Oklahoma City, Oklahoma, USA

Scott D. Rowley, MD

James W. Smith, MT, BSc Coordinator McMaster Platelet Immunology Laboratory Michael G. DeGroote School of Medicine McMaster University Hamilton, Ontario, Canada

Director, Blood Transfusion Service Massachusetts General Hospital; Assistant Professor of Pathology Harvard Medical School Boston, Massachusetts, USA

Chief, Adult Blood and Marrow Stem Cell Transplantation Program Hackensack University Medical Center Hackensack, New Jersey, USA

Ronald A. Sacher, MD, FRCPC Professor of Internal Medicine and Pathology Director, Hoxworth Blood Center University of Cincinnati Academic Health Center Cincinnati, Ohio, USA Ulrich J.H. Sachs, MD Head, The Platelet and Granulocyte Laboratory Institute for Clinical Immunology and Transfusion Medicine Justus Liebig University Giessen, Germany Jerard Seghatchian, MD Consultant Blood Components Technology and Haemostasis/Thrombosis Consultancy London, UK

Kirsten Seidel, MBChB, MD, DTransM, DTM⫹H, DPH Corporate Medical Director ZLB Plasma Services GmbH, a CSL Behring Company Marburg, Germany

Bruce I. Sharon, MD Associate Professor Department of Pediatrics University of Illinois College of Medicine and University of Illinois Hospital Chicago, Illinois, USA

Sujit Sheth, MD Associate Clinical Professor Department of Pediatrics Columbia University Medical Center New York, New York, USA Don L. Siegel, PhD, MD Vice-Chair and Professor Pathology and Laboratory Medicine; Chief, Division of Transfusion Medicine Department of Pathology and Laboratory Medicine University of Pennsylvania School of Medicine Philadelphia, Pennsylvania, USA Toby L. Simon, MD Corporate Medical Director ZLB Plasma, a CSL Behring Company Boca Raton, Florida, USA; Clinical Professor Department of Pathology University of New Mexico School of Medicine Albuquerque, New Mexico, USA

Edward L. Snyder, MD Professor of Laboratory Medicine Yale University School of Medicine; Director Blood Bank/Apheresis Service Yale-New Haven Hospital New Haven, Connecticut, USA

Martha C. Sola-Visner, MD Assistant Professor of Pediatrics Harvard Medical School; Physician in Medicine Childrens Hospital Boston, Massachusetts, USA

Bjarte G. Solheim, MD, PhD, MHA Professor Emeritus Institute of Immunology Rikshospitalet University Hospital and University of Oslo Oslo, Norway

Bryan R. Spencer, MPH Manager of Blood Research American Red Cross—New England Region Dedham, Massachusetts, USA

Patrice F. Spitalnik, MD Assistant Professor of Clinical Pathology Department of Pathology and Cell Biology Columbia University Medical Center New York, New York, USA

Steven L. Spitalnik, MD Professor of Pathology; Vice Chairman of Laboratory Medicine Department of Pathology and Cell Biology Columbia University Medical Center New York, New York, USA

Dorothy Stainsby, FRCP, FRCPath National Medical Coordinator (Retired) Serious Hazards of Transfusion Manchester Blood Centre Manchester, UK

Ronald G. Strauss, MD Professor Emeritus, Pathology and Pediatrics University of Iowa College of Medicine Iowa City, Iowa, USA John Parker Sweeney, JD Attorney at Law Womble Carlyle Sandridge & Rice PLLC Baltimore, Maryland, USA Alan T. Tinmouth, MD, MSc (Clin Epi), FRCP(C) Assistant Professor Faculty of Medicine University of Ottawa; Director Adult Regional Hemophilia/Bleeding Disorders Comprehensive Care Clinic Ottawa Hospital University of Ottawa Centre for Transfusion Research Clinical Epidemiology Ottawa Health Research Institute Ottawa, Ontario, Canada

Aaron Tomer, MD Professor of Medicine Faculty of Health Sciences Ben-Gurion University of the Negev; Director Blood Bank and Transfusion Medicine Soroka University Medical Center Beer-Sheva, Israel Christopher A. Tormey, MD Instructor Department of Laboratory Medicine Yale University School of Medicine Yale-New Haven Hospital New Haven, Connecticut, USA Darrell J. Triulzi, MD Director, Division of Transfusion Medicine Professor, Department of Pathology University of Pittsburgh Pittsburgh, Pennsylvania, USA

Simon J. Stanworth, MA, MRCP, DPhil, MRCPath Consultant Haematologist Department of Transfusion Medicine John Radcliffe Hospital Headington, Oxford, UK

Marc L. Turner, MB, ChB, MBA, PhD, FRCP, FRCPath Professor of Cellular Therapy University of Edinburgh; Clinical Director/Consultant Haematologist

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Contributors Edinburgh Blood Transfusion Centre Royal Infirmary of Edinburgh Edinburgh, Scotland, UK

Chief, Division of Transfusion Medicine UMass Memorial Medical Center Worcester, Massachusetts, USA

Contributors to Cases

Lynne Uhl, MD

Connie M. Westhoff, PhD, MT(ASCP)SBB

Assistant Professor of Pathology Harvard Medical School; Director Division of Laboratory and Transfusion Service Beth Israel Deaconess Medical Center Boston, Massachusetts, USA

Scientific Director Molecular Blood Group and Platelet Testing Laboratory American Red Cross, Penn-Jersey Region; Adjunct Assistant Professor, Division of Transfusion Medicine Department of Pathology and Laboratory Medicine University of Pennsylvania School of Medicine Philadelphia, Pennsylvania, USA

Instructor Department of Pathology Mount Sinai Medical Center New York, New York, USA

Donna D. Castellone, MS, MT(ASCP)SH

Eleftherios C. Vamvakas, MD, PhD Vice-Chair and Director of Clinical Pathology Department of Pathology and Laboratory Medicine Cedars-Sinai Medical Center Los Angeles, California, USA Ralph R. Vassallo, Jr., MD, FACP Heritage Division Chief Medical Officer American Red Cross Blood Services; Adjunct Associate Professor of Medicine University of Pennsylvania School of Medicine Philadelphia, Pennsylvania, USA

Jonathan P. Wallis, MBBS, FRCPath Consultant Haematologist Department of Haematology Freeman Hospital Newcastle upon Tyne, UK

Theodore E. Warkentin, MD, FRCP(C), FACP Professor Department of Pathology and Molecular Medicine Department of Medicine Michael G. DeGroote School of Medicine McMaster University Hamilton, Ontario, Canada Robert Weinstein, MD Professor of Medicine and Pathology University of Massachusetts Medical School;

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Susan L. Wilkinson, EdD Associate Professor Clinical Transfusion Medicine Associate Director Hoxworth Blood Center University of Cincinnati Academic Health Center Cincinnati, Ohio, USA

Thomas M. Williams, MD Professor and Chair Department of Pathology University of New Mexico School of Medicine; Director HLA and Molecular Diagnostics TriCore Reference Laboratories Albuquerque, New Mexico, USA

William F. Zaloga, DO Associate Director, Blood and Tissue Resources Wadsworth Center New York State Department of Health; Adjunct Assistant Professor Albany Medical College Albany, New York, USA

Dava S. Cleveland-Noriega, DO Transfusion Medicine/Blood Banking Fellow Department of Pathology University of Texas Southwestern Medical Center Dallas, Texas, USA Nicole A. Pele, DO Resident Physician Department of Pathology and Anatomical Sciences University of Missouri—Columbia Columbia, Missouri, USA Marian Petrides, MD Associate Professor of Clinical Pathology University of Missouri School of Medicine; Medical Director Transfusion Service and Coagulation Laboratory University of Missouri Health Care Columbia, Missouri, USA Christopher P. Stowell, MD, PhD Director, Blood Transfusion Service Massachusetts General Hospital; Assistant Professor of Pathology Harvard Medical School Boston, Massachusetts, USA

4

Preface

More than 6 years have elapsed since the third edition of Rossi’s Principles of Transfusion Medicine was published. During that time, the field has continued to be dynamic and many advances have been made. Molecular techniques are increasingly utilized; measurements of cell kinetics have been further refined; new treatments for bleeding disorders have been developed; progenitor cell transplantation protocols have matured; hemovigilance efforts have become more organized globally; pathogen inactivation and apheresis techniques have advanced; new treatments have emerged for iron overload; gene therapy and regenerative medicine are having an impact on the field; and regulation has become more complex in a global environment. As expected, some controversies remain unresolved. For example, the effect of red cell age on clinical outcome and whether transfusion itself affects patient recovery are at the center of recent articles that commanded attention from both professionals and the public. At the same time, pharmacologic agents intended to reduce the need for transfusion have also become subjects of controversy. Whether recombinant human erythropoietin has deleterious effects on patients, the potential for cell-free hemoglobin solutions to cause more harm than good, and the balance between efficacy and toxicity of recombinant Factor VIIa are being discussed at the national level of health-care decision-making. This edition provides updated comprehensive assessments of the subjects that remain important to the daily work of those who practice transfusion medicine today. The contents include information on controversies that are still percolating, as well as those new developments that have been widely adopted in practice. This volume also features those scientific areas that represent the frontiers of transfusion medicine such as cellular and gene therapy and regenerative medicine. Section I reviews the preparation and use of components and derivatives of blood used in clinical practice. Section II provides information and approaches to the major clinical issues related to transfusion in medical, obstetric, pediatric, oncology, and surgery patients, including transplant recipients. Section III focuses

attention on both donor and patient apheresis. Section IV introduces the major concerns related to hazards of transfusion and covers the infectious and noninfectious risks in separate chapters. Section V covers the broad issues related to cells and tissue therapy in our field. Section VI contains an overview of the logistic and regulatory aspects of the profession. The fourth edition is an example of both continuity and change. We have retained the general organizational plan of the prior edition. The comprehensive scope has been retained with emphasis on the scientific and clinical over the technical. Our objective is to create the best single reference for the professional who is managing transfusion issues both at the bedside and at the interface of the laboratory and the clinic. Yet at the same time, the volume is intended to provide a helpful resource for trainees. New chapters that have either combined material from several chapters in prior editions or ventured into new areas include cell kinetics, obstetric practice, cord blood, transfusion alternatives, regenerative medicine, hemovigilance, iron overload, and transplant organization and regulation. Once again, case studies have been included to enhance the educational aspects, but with this edition they are in an electronic format. In addition to the CD-ROM containing the case studies a second CD-ROM contains the 66 book chapters as searchable pdf files. Four of the editors from the last edition have returned along with many of the expert chapter authors. This edition also welcomes new chapter contributors with fresh approaches and two new editors—Bjarte G. Solheim, who has led the effort to include more international perspectives, and Marian Petrides, who developed the software for, and managed the transition to, the interactive case study format. Joining the team effort for this edition are publishing staff at AABB Press and Wiley-Blackwell. In a work of this magnitude, there are many to whom we owe words of appreciation. Clearly, thanks are due to the authors for their superb chapters, AABB Press staff for their expertise and assistance, and personnel at Wiley-Blackwell for their professional and collegial efforts on behalf of the book. Just as

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Preface important, the editors thank our families, colleagues, employers, teachers, and students for the support that sustained us throughout the development process. All of us have chosen to work in this field because of our commitment to improving and saving the lives of those in our communities who can benefit from transfusion therapies. Some have used the analogy of those who work backstage in a theatrical production; the actors take the bows but the play can go on only with the contributions of the unseen workers behind the curtains. When the surgeons, oncologists, pediatricians, obstetricians, intensive care specialists, other physicians, and nurses are successful in treating the patients, those of us in transfusion

xiv

medicine and related therapies can take satisfaction in the work we do. Our hope is that this book will help support those who can make a difference in transfusion medicine and serve as an example of the teamwork so urgently needed to move all of health care forward for the benefit of the global community.

Toby L. Simon, MD Edward L. Snyder, MD Bjarte G. Solheim, MD, PhD, MHA Christopher P. Stowell, MD, PhD Ronald G. Strauss, MD Marian Petrides, MD

1

Transfusion in the New Millennium Ennio C. Rossi1 & Toby L. Simon2 1 2

Professor Emeritus of Medicine, Northwestern University School of Medicine, Chicago, Illinois, USA Corporate Medical Director, ZLB Plasma, a CSL Behring Company, Boca Raton, Florida, and Clinical Professor, University of New Mexico School of Medicine, Albuquerque, New Mexico, USA

Prehistoric man left drawings of himself pierced by arrows.1 This means he was as aware of blood as he was of his own limbs. The flint implements he used as tools and weapons distinguished him from other creatures and contributed to the violence of his era. As he hunted food and fought enemies, he observed bleeding and the properties of blood. A cut, received or inflicted, yielded a vivid red color. If the cut was shallow, there was little blood. But if the cut was deep, a red torrent flowing from the stricken victim quickly led to death, with shed blood congealed and darkening in the sun. Fatal hemorrhage was commonplace. Nonetheless, the sight must have been fearful and possibly existential as life flowed red out of the body of an enemy or a wounded animal.2 It is no wonder, then, that at the dawn of recorded history, blood was already celebrated in religious rites and rituals as a life-giving force. The cultural expressions of primitive and ancient societies, though separated by time or space, can be strikingly similar. Whether these expressions emerged independently or were diffused about the world by unknown voyagers will probably always remain clouded in mystery.2 Nonetheless, there is a common thread in the ancient rituals that celebrate blood as a mystical vital principle. In Leviticus 17:11, “the life of the flesh is in the blood,” and the Chinese Neiching (circa 1000 BC) claims the blood contains the soul.2 Pre-Columbian North American Indians bled their bodies “of its greatest power” as self-punishment,3 Egyptians took blood baths as a recuperative measure, and Romans drank the blood of fallen gladiators in an effort to cure epilepsy.4 The Romans also practiced a ceremony called taurobolium—a blood bath for spiritual restoration. A citizen seeking spiritual rebirth descended into a pit or fossa sanguinis. Above him on a platform, a priest sacrificed a bull, and the animal’s blood cascaded down in a shower upon the beneficiary. Then, in a powerful visual image, the subject emerged up from the other end of the pit, covered with blood and reborn.1

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

The legend of Medea and Aeson taken from Ovid’s Metamorphoses and quoted in Bulfinch’s Mythology5 also ascribed rejuvenating powers to blood. Jason asked Medea to “take some years off his life and add them to those of his father Aeson.” Medea, however, pursued an alternative course. She prepared a cauldron with the blood of a sacrificed black sheep. To this, she added magic herbs, hoarfrost gathered by moonlight, the entrails of a wolf, and many other things “without a name.” The boiling cauldron was stirred with a withered olive branch, which became green and full of leaves and young olives when it was withdrawn. Seeing that all was ready, Medea cut the throat of the old man and let out all his blood, and poured into his mouth and into his wound the juices of her cauldron. As soon as he had imbibed them, his hair and beard laid by their whiteness and assumed the blackness of youth; his paleness and emaciation were gone; his veins were full of blood, his limbs of vigour and robustness. Aeson is amazed at himself and remembers that such as he now is, he was in his youthful days, 40 years before.

This legend seems to echo in the apocryphal story of Pope Innocent VIII, who is said to have received the blood of three young boys in 1492 while on his deathbed. As the story goes, a physician attempted to save the pope’s life by using blood drawn from three boys 10 years of age, all of whom died soon thereafter. Some 19th-century versions of this tale suggest the blood was transfused. However, earlier renditions more plausibly suggest that the blood was intended for a potion to be taken by mouth. In any event, there is no evidence the pope actually received any blood in any form.6,7 The folklore that flowed with blood was not accompanied by a great deal of accurate information. The ancient Greeks believed that blood formed in the heart and passed through the veins to the rest of the body, where it was consumed. Arteries were part of an independent system transporting air from the lungs. Although Erasistratos (circa 270 BC) had imagined the heart as a pump, his idea was ahead of its time. As long as veins and arteries were dead-end channels transporting blood and air, there was little need for a pump in the system. Although Galen (131-201 AD) finally proved that arteries contain blood, communication with

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Chapter 1: Transfusion in the New Millennium

the venous system was not suspected. Blood, formed in the liver, merely passed through the blood vessels and heart on its way to the periphery.1 These teachings remained in place for 1400 years until they were swept away in 1628 by Harvey’s discovery of the circulation. The realization that blood moved in a circulating stream opened the way to experiments on vascular infusion. In 1642 George von Wahrendorff injected wine8—and in 1656 Christopher Wren and Robert Boyle injected opium and other drugs9— intravenously into dogs. The latter studies, performed at Oxford, were the inspiration for Richard Lower’s experiments in animal transfusion.

The First Animal Transfusion Richard Lower (1631-1691) was a student at Oxford when Christopher Wren and Robert Boyle began their experiments on infusion. In due course, Lower joined their scientific group and studied the intravenous injection of opiates, emetics, and other substances into living animals.10 In time, the transfusion of blood itself became the objective. The announcement of the first successful transfusion, performed by Richard Lower at Oxford in February 1665, was published on November 19, 1666, in the Philosophical Transactions of the Royal Society Transactions (Transactions) in a short notation titled, “The Success of the Experiment of Transfusing the Blood of One Animal into Another.”11 The entire notation is as follows11: This experiment, hitherto look’d upon to be of an almost insurmountable difficulty, hath been of late very successfully perform’d not only at Oxford, by the directions of that expert anatomist Dr. Lower, but also in London, by order of the R. Society, at their publick meeting in Gresham Colledge: the Description of the particulars whereof, and the Method of Operation is referred to the next opportunity.

The December 17, 1666 issue of the Transactions contained the full description as promised.12 It was taken from a letter13 written by Lower to Robert Boyle on July 6, 1666, in which Lower described direct transfusion from the carotid artery of one dog to the jugular vein of another. After describing the insertion of quills into the blood vessels of the donor and recipient dogs, Lower wrote13: When you have done this you may lay the dogs on their side and fasten them densely together as best you may to insure the connection of the two quills. Quickly tighten the noose around the neck of the receiving animal as in venasection, or at all events compress the vein on the opposite side of the neck with your finger, then take out the stopper and open the upper jugular quill so that while the foreign blood is flowing into the lower quill, the animal’s own blood flows out from the upper into suitable receptacles—until at last the second animal, amid howls, faintings, and spasms, finally loses its life together with its vital fluid. When the tragedy is over, take both quills out of the jugular vein of the surviving animal, tie tightly with the former slipknots,

2

and divide the vein. After the vessel has been divided, sew up the skin, slacken the cords binding the dog, and let it jump down from the table. It shakes itself a little, as though aroused from sleep, and runs away lively and strong, more active and vigorous perhaps, with the blood of its fellow than its own.

These studies inevitably led to the transfusion of animal blood to humans. In England, this occurred on November 23, 1667, when Lower and Edmund King transfused sheep blood into a man named Arthur Coga.14 Described by Samuel Pepys as “a little frantic,” Coga was paid 20 shillings to accept this transfusion, with the expectation that it might have a beneficial “cooling” effect. One week later, Coga appeared before the society and claimed to be a new man, although Pepys concluded he was “cracked a little in the head.”13 However, this was not the first transfusion performed in a human. The credit for that accomplishment belongs to JeanBaptiste Denis (1635-1704), who had performed the first human transfusion several months earlier in Paris.

The First Animal-to-Human Transfusion The founding of the Royal Society in London in 1662 was followed in 1666 by the establishment of the Academie des Sciences in Paris under the patronage of King Louis XIV. The new Academie reviewed the English reports on transfusion with great interest. Denis probably read of Lower’s experiments in the Journal des Savants on January 31, 1667, and he began his own studies approximately 1 month later.15,16 The first human transfusion was then performed on June 15, 1667, when Denis administered the blood of a lamb to a 15-year-old boy (Fig 1-1). Although discovery of the circulation had suggested the idea of transfusion, indications for the procedure remained uninformed. Transfusion was still thought to alter behavior and possibly achieve rejuvenation. The blood of young dogs made old dogs seem frisky; the blood of lions was proposed as a cure for cowardice; and 5 months later, Arthur Coga would receive a transfusion of sheep blood because of its presumed “cooling” effect. Denis used animal blood for transfusion because he thought it was “less full of impurities”17: Sadness, Envy, Anger, Melancholy, Disquiet and generally all the Passions, are as so many causes which trouble the life of man, and corrupt the whole substance of the blood: Whereas the life of Brutes is much more regular, and less subject to all these miseries.

It is thus ironic that the symptoms of the first transfusion recipient may have been explained in part by profound anemia; the single transfusion of lamb blood may have produced temporary amelioration owing to increased oxygen transport. Denis described the case as follows17: On the 15 of this Moneth, we hapned upon a Youth aged between 15 and 16 years, who had for above two moneths bin tormented with a contumacious and violent fever, which obliged his Physitians to bleed him 20 times, in order to asswage the excessive heat.

Chapter 1: Transfusion in the New Millennium

Before this disease, he was not observed to be of a lumpish dull spirit, his memory was happy enough, and he seem’d chearful and nimble enough in body; but since the violence of this fever, his wit seem’d wholly sunk, his memory perfectly lost, and his body so heavy and drowsie that he was not fit for anything. I beheld him fall asleep as he sate at dinner, as he was eating his Breakfast, and in all occurrences where men seem most unlikely to sleep. If he went to bed at nine of the clock in the Evening, he needed to be wakened several times before he could be got to rise by nine the next morning, and he pass’d the rest of the day in an incredible stupidity. I attributed all these changes to the great evacuations of blood, the Physitians had been oblig’d to make for saving his life.

Three ounces of the boy’s blood were exchanged for 9 ounces of lamb arterial blood. Several hours later the boy arose, and “for the rest of the day, he spent it with much more liveliness than ordinary.” Thus the first human transfusion, which was heterologous, was accomplished without any evident unfavorable effect. This report stimulated a firestorm of controversy over priority of discovery.18,19 The letter by Denis was published in the Transactions on July 22, 1667, while the editor, Henry Oldenburg, was imprisoned in the Tower of London. Oldenburg, following some critical comments concerning the Anglo-Dutch War then in progress (1665-1667), had been arrested under a warrant issued June 20, 1667. After his release 2 months later, Oldenburg returned to his editorial post and found the letter published in his absence. He took offense at Denis’s opening statement, which claimed that the French had conceived of transfusion “about ten years agoe, in the illustrious Society of Virtuosi . . .” (Fig 1-1). This seemed to deny the English contributions to the field. Oldenburg cited these omissions in an issue of the Transactions published September 23, 1667, “for the Months of July, August, and September.” By numbering this issue 27 and beginning pagination with 489, Oldenburg attempted to suppress the letter by Denis.18 However, as is evident, this did not ultimately succeed. Nonetheless, subsequent events created even greater difficulties for Denis. Although the first two subjects who underwent transfusion by Denis were not adversely affected, the third and fourth recipients died. The death of the third subject was easily attributable to other causes. However, the fourth case initiated a sequence of events that put an end to transfusion for 150 years. Anthony du Mauroy was a 34-year-old man who suffered from intermittent bouts of maniacal behavior. On December 19, 1667, Denis and his assistant Paul Emmerez removed 10 ounces of the man’s blood and replaced it with 5 or 6 ounces of blood from the femoral artery of a calf. Failing to note any apparent improvement, they repeated the transfusion 2 days later. After the second transfusion, du Mauroy experienced a classic transfusion reaction20: His pulse rose presently, and soon after we observ’d a plentiful sweat over all his face. His pulse varied extremely at this instant, and he complain’d of great pains in his kidneys and that he was not well in his stomach.

Figure 1-1. The first human transfusion.17

Du Mauroy fell asleep at about 10 o’clock in the evening. He awoke the following morning and “made a great glass full of urine, of a colour as black, as if it had been mixed with the soot of chimneys.”20 Two months later, the patient again became maniacal, and his wife again sought transfusion therapy. Denis was reluctant but finally gave in to her urgings. However, the transfusion could not be accomplished, and du Mauroy died the next evening. The physicians of Paris strongly disapproved of the experiments in transfusion. Three of them approached du Mauroy’s widow and encouraged her to lodge a malpractice complaint against Denis. She instead went to Denis and attempted to extort money from him in return for her silence. Denis refused and filed a complaint before the Lieutenant in Criminal Causes. During the subsequent hearing, evidence was introduced to indicate that Madame du Mauroy had poisoned her husband with arsenic. In a judgment handed down at the Chatelet in Paris on April 17, 1668, Denis was exonerated, and the woman was held for trial. The court also stipulated “that for the future no Transfusion should

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Chapter 1: Transfusion in the New Millennium

be made upon any Human Body but by the approbation of the Physicians of the Parisian Faculty.”21 At this point, transfusion research went into decline, and within 10 years it was prohibited in both France and England.

The Beginnings of Modern Transfusion After the edict that ended transfusion in the 17th century, the technique lay dormant for 150 years. Stimulated by earlier experiments by Leacock, transfusion was “resuscitated” and placed on a rational basis by James Blundell (1790-1877), a London obstetrician who had received his medical degree from the University of Edinburgh.22 Soon after graduation, Blundell accepted a post in physiology and midwifery at Guy’s Hospital. It was there that he began the experiments on transfusion that led to its rebirth. The frequency of postpartum hemorrhage and death troubled Blundell. In 1818 he wrote23: A few months ago I was requested to visit a woman who was sinking under uterine hemorrhage . . . Her fate was decided, and notwithstanding every exertion of the medical attendants, she died in the course of two hours. Reflecting afterwards on this melancholy scene . . . , I could not forbear considering, that the patient might very probably have been saved by transfusion; and that . . . the vessels might have been replenished by means of the syringe with facility and prompitude.

This opening statement introduced Blundell’s epoch-making study titled “Experiments on the Transfusion of Blood by the Syringe.”23 (See Fig 1-2.) Blundell described in detail a series of animal experiments. He demonstrated that a syringe could be

Figure 1-2. The beginnings of modern transfusion.23

4

used effectively to perform transfusion, that the lethal effects of arterial exsanguination could be reversed by the transfusion of either venous or arterial blood, and that the injection of 5 drams (20 cc) of air into the veins of a small dog was not fatal but transfusion across species ultimately was lethal to the recipient.23 Thus Blundell was the first to state clearly that only human blood should be used for human transfusion. The latter conclusion was confirmed in France by Dumas and Prevost, who demonstrated that the infusion of heterologous blood into an exsanguinated animal produced only temporary improvement and was followed by death within 6 days.24 These scientific studies provided the basis for Blundell’s subsequent efforts in clinical transfusion. The first well-documented transfusion with human blood took place on September 26, 1818.25 The patient was an extremely emaciated man in his mid-thirties who had pyloric obstruction caused by carcinoma. He received 12 to 14 ounces of blood in the course of 30 or 40 minutes. Despite initial apparent improvement, the patient died 2 days later. Transfusion in the treatment of women with postpartum hemorrhage was more successful. In all, Blundell performed 10 transfusions, of which five were successful. Three of the unsuccessful transfusions were performed on moribund patients; the fourth was performed on a patient with puerperal sepsis; and the fifth was performed on the aforementioned patient with terminal carcinoma. Four of the successful transfusions were given for postpartum hemorrhage, and the fifth was administered to a boy who bled after amputation.22 Blundell also devised various instruments for the performance of transfusion. They included an “impellor,” which collected blood in a warmed cup and “impelled” the blood into the recipient via an attached syringe, and a “gravitator”26 (Fig 1-3), which received blood and delivered it by gravity through a long vertical cannula. The writings of Blundell provided evidence against the use of animal blood in humans and established rational indications for transfusion. However, the gravitator (Fig 1-3) graphically demonstrated the technical problems that remained to be solved. Blood from the donor, typically the patient’s husband, flowed into a funnel-like device and down a flexible cannula into the patient’s vein “with as little exposure as possible to air, cold and inanimate surface.”25 The amount of blood transfused was estimated from the amount spilled into the apparatus by the donor. In this clinical atmosphere, charged with apprehension and anxiety, the amount of blood issuing from a donor easily could be overstated. Clotting within the apparatus then ensured that only a portion of that blood actually reached the patient. Thus the amount of blood actually transfused may have been seriously overestimated. This may explain the apparent absence of transfusion reactions. Alternatively, reactions may have been unrecognized. Patients who underwent transfusion frequently were agonal. As Blundell26 stated, “It seems right, as the operation now stands, to confine transfusion to the first class of cases only, namely, those in which there seems to be no hope for the patient, unless blood can be thrown into the veins.” Under these circumstances, “symptoms” associated with an “unsuccessful” transfusion might be ascribed to the agonal state rather than the

Chapter 1: Transfusion in the New Millennium

transfusion itself. For a time, the problem of coagulation during transfusion was circumvented by the use of defibrinated blood. This undoubtedly increased the amount of blood actually transfused. However, there were numerous deaths. Interestingly, these deaths were attributed to intravascular coagulation when in actuality they were probably fatal hemolytic reactions caused by the infusion of incompatible blood.27 Transfusion at the end of the 19th century, therefore, was neither safe nor efficient. The following description, written in 1884, illustrates this point28: Students, with smiling faces, are rapidly leaving the theatre of one of our metropolitan hospitals. The most brilliant operator of the day has just performed immediate transfusion with the greatest success. By means of a very beautiful instrument, the most complex and ingenious that modern science has yet produced, a skilful surgeon has transfused half a pint, or perhaps a pint, of blood from a healthy individual to a fellow creature profoundly collapsed from the effects of severe hemorrhage. Some little difficulty was experienced prior to the operation, as one of the many stop-cocks of the

transfusion apparatus was found to work stiffly; but this error was quickly rectified by a mechanic in attendance. Towards the close of the operation the blood-donor, a powerful and heavy young man, swooned. Two porters carried him on a stretcher into an adjoining room.

In the latter half of the 19th century, there were many attempts to render transfusion a more predictable and less arduous procedure. In 1869, Braxton-Hicks,29 using blood anticoagulated with phosphate solutions, performed a number of transfusions on women with obstetric bleeding. Many of the patients were in extremis, and ultimately all died. Unfortunately, a detailed description of terminal symptoms was not provided.29 Some investigators attempted to rejuvenate animal-to-human transfusion, and Oscar Hasse persisted in this approach despite disastrous results. Studies by Emil Ponfick and by Leonard Landois finally put an end to this practice. Ponfick, in carefully controlled studies, confirmed the lethality of heterologous transfusion and identified the resulting hemoglobinuria along with its donor erythrocyte source. Landois documented the poor results of animal-to-human transfusion and demonstrated the lysis of sheep erythrocytes by human serum in vitro.8 Frustration with blood as a transfusion product led to even more bizarre innovations. From 1873 to 1880, cow, goat, and even human milk was transfused as a blood substitute.30 The rationale derived from an earlier suggestion that the fat particles of milk could be converted into blood cells. Milk transfusion was particularly popular in the United States,30 where the practice of animal-to-human transfusion was recorded as late as 1890.31 Fortunately, these astonishing practices were discontinued when saline solutions were introduced as “a life-saving measure” and “a substitute for the transfusion of blood.”32 A passage from an article written by Bull in 188432 is particularly instructive: The danger from loss of blood, even to two-thirds of its whole volume, lies in the disturbed relationship between the calibre of the vessels and the quantity of the blood contained therein, and not in the diminished number of red blood-corpuscles; and . . . This danger concerns the volume of the injected fluids also, it being a matter of indifference whether they be albuminous or containing blood corpuscles or not . . . .

Mercifully, volume replacement with saline solutions deflected attention from the unpredictable and still dangerous practice of blood transfusion. Accordingly, transfusions were abandoned until interest was rekindled by the scientific and technical advances of the early 20th century.

The 20th Century

Figure 1-3. Blundell’s gravitator.26

The 20th century was ushered in by a truly monumental discovery. In 1900, Karl Landsteiner (1868-1943) observed that the sera of some persons agglutinated the red blood cells of others. This study, published in 1901 in the Wiener Klinische Wochenschrift33 (Fig 1-4), showed for the first time the cellular differences in

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Chapter 1: Transfusion in the New Millennium

Figure 1-4. Landsteiner’s description of blood groups.33

individuals from the same species. In his article, Landsteiner wrote34: In a number of cases (Group A) the serum reacts on the corpuscles of another group (B), but not, however, on those of group A, while, again, the corpuscles of A will be influenced likewise by serum B. The serum of the third group (C) agglutinates the corpuscles of A and B, while the corpuscles of C will not be influenced by the sera of A and B. The corpuscles are naturally apparently insensitive to the agglutinins which exist in the same serum.

With the identification of blood groups A, B, and C (subsequently renamed group O) by Landsteiner and of group AB by Decastello and Sturli,35 the stage was set for the performance of safe transfusion. For this work, Landsteiner somewhat belatedly received the Nobel Prize in 1930. But even that high recognition does not adequately express the true magnitude of Landsteiner’s discovery. His work was like a burst of light in a darkened room. He gave us our first glimpse of immunohematology and transplantation biology and provided the tools for important discoveries in genetics, anthropology, and forensic medicine. Viewed from this perspective, the identification of human blood groups is one of only a few scientific discoveries of the 20th century that changed all of our lives.34 Yet the translation of Landsteiner’s discovery into transfusion practice took many years. At the turn of the century, the effective transfer of blood from one person to another remained a formidable task. Clotting, still uncontrolled, quickly occluded transfusion devices and frustrated most efforts. In 1901 the methods used in transfusion were too primitive to demonstrate the importance of Landsteiner’s discovery. Indeed, the study of in-vitro red cell agglutination may have seemed rather remote from the technical problems that demanded attention. An intermediate step was needed before the importance of Landsteiner’s breakthrough could be perceived and the appropriate changes could be incorporated into practice. This process was initiated by Alexis Carrel (1873-1944), another Nobel laureate, who developed a surgical procedure that allowed direct transfusion through an arteriovenous anastomosis. Carrel36 introduced the technique of end-to-end vascular anastomosis with triple-threaded suture material. This procedure brought the ends of vessels in close apposition and preserved luminal continuity, thus avoiding leakage or thrombosis. This technique paved the way for successful organ transplantation and brought Carrel the Nobel Prize in 1912. It was also adapted by Carrel37 and others38,39 to the performance of transfusion. Crile38 introduced the use of a metal tube to facilitate placement of sutures, and Bernheim39 used a two-piece cannula to unite the artery to the vein (Fig 1-5). Because all of these procedures usually culminated in the

6

Figure 1-5. Direct transfusion by means of arteriovenous anastomosis through the two-pieced cannula of Bernheim.39

Figure 1-6. Report of a fatal transfusion reaction.41

sacrifice of the two vessels, they were not performed frequently. Direct transfusion was also fraught with danger. In a passage written two decades later, the procedure was recalled in the following manner40: The direct artery to vein anastomosis was the best method available but was often very difficult or even unsuccessful. And, what was almost as bad, one never knew how much blood one had transfused at any moment or when to stop (unless the donor collapsed). (I remember one such collapse in which the donor almost died—and the surgeon needed to be revived.)

Despite these many difficulties, direct transfusion through an arteriovenous anastomosis for the first time efficiently transferred blood from one person to another. The process also disclosed fatal hemolytic reactions that were undeniably caused by transfusion41 (Fig 1-6). However, the relation of these fatal reactions to Landsteiner’s discovery was not recognized until Reuben Ottenberg (1882-1959) demonstrated the importance of compatibility testing. Ottenberg’s interest in transfusion began in 1906 while he was an intern at German (now Lenox Hill) Hospital in New York. There Ottenberg learned of Landsteiner’s discovery and in 1907 began pretransfusion compatibility testing.42 Ottenberg accepted an appointment at Mount Sinai Hospital the next year and continued

Chapter 1: Transfusion in the New Millennium

Figure 1-8. Apparatus for Unger’s two-syringe, four-way stopcock method of indirect transfusion.49

Figure 1-7. Report of the importance of testing before transfusion.43

his studies on transfusion. In 1913, Ottenberg published the report that conclusively demonstrated the importance of preliminary blood testing for the prevention of transfusion “accidents”43 (Fig 17). This was not Ottenberg’s only contribution. He observed the mendelian inheritance of blood groups,44 and he was the first to recognize the relative unimportance of donor antibodies and consequently the “universal” utility of type O blood donors.45 Further advances in immunohematology were to occur in succeeding decades. The M, N, and P systems were described in the period between 1927 and 1947.46 The Rh system was discovered in connection with an unusual transfusion reaction. In 1939, Levine and Stetson47 described an immediate reaction in a group O woman who had received her husband’s group O blood soon after delivery of a stillborn fetus with erythroblastosis. This sequence of events suggested that the infant had inherited a red cell agglutinogen from the father that was foreign to the mother. At about the same time, Landsteiner and Wiener48 harvested a rhesus monkey red cell antibody from immunized guinea pigs and rabbits. This antibody agglutinated 85% of human red cell samples (Rh-positive) and left 15% (Rh-negative) unaffected. When the experimentally induced antibody was tested in parallel with the serum from Levine’s patient, a similar positive and negative distribution was observed, and the Rh system had been discovered. Other red cell antigen systems were subsequently described, but when Rh Immune Globulin was introduced as a preventive measure for hemolytic disease of the newborn, it became one of the major public health advances of the century. Despite the introduction of compatibility testing by Ottenberg, transfusion could not be performed frequently as long as arteriovenous anastomosis remained the procedure of choice. Using this method, Ottenberg needed 5 years (Fig 1-7) to accumulate the 128 transfusions he reported in his study on pretransfusion testing.43 New techniques, such as Unger’s two-syringe method introduced in 191549 (Fig 1-8), eventually put an end to transfusion by means of arteriovenous anastomosis. However, transfusions did not become commonplace until anticoagulants were developed and direct methods of transfusion were rendered obsolete.

Anticoagulants, the Blood Bank, and Component Therapy The anticoagulant action of sodium citrate completely transformed the practice of transfusion. Early reports from Belgium50 and Argentina51 were followed by the work of Lewisohn52 that established the optimal citrate concentration for anticoagulation. The work of Weil53 then demonstrated the feasibility of refrigerated storage. Subsequently, Rous and Turner54 developed the anticoagulant solution that was used during World War I.55 Despite its very large volume, this solution remained the anticoagulant of choice until World War II, when Loutit and Mollison56 developed an acid-citrate-dextrose (ACD) solution. Used in a ratio of 70 mL ACD to 450 mL blood, ACD provided 3 to 4 weeks of preservation of a more concentrated red cell infusion. Thus, the two world wars were the stimuli for the development of citrate anticoagulants and the introduction of indirect transfusion.46 For the first time, the donation process could be separated, in time and place, from the actual transfusion. Blood drawn and set aside now awaited the emergence of systems of storage and distribution. Again, it was the provision of medical support during armed conflict that stimulated these developments. A blood transfusion service, organized by the Republican Army during the Spanish Civil War (1936-1939), collected 9000 L of blood in citrate-dextrose anticoagulant for the treatment of battle casualties.57 At about that same time, Fantus58 began operation of the first hospital blood bank at Cook County Hospital in Chicago. His interest had been stimulated by Yudin’s report59 on the use of cadaveric blood in Russia. Apart from certain scruples attached to the use of cadaveric blood, Fantus reasoned that a transfusion service based on such a limited source of supply would be impractical. Accordingly, he established the principle of a “blood bank” from which blood could be withdrawn, provided it had previously been deposited. As Fantus58 himself stated, “just as one cannot draw money from a bank unless one has deposited some, so the blood preservation department cannot supply blood unless as much comes in as goes out. The term ‘blood bank’ is not a mere metaphor.” The development of anticoagulants and the concept of blood banks provided an infrastructure upon which a more elaborate blood services organization could be built. World War II was the catalyst for these further developments.

7

Chapter 1: Transfusion in the New Millennium

At the beginning of World War II, blood procurement programs were greatly expanded.46 In Great Britain an efficient system had been developed through the organization of regional centers. When the war started, these centers, already in place, were able to increase their level of operation. In the United States the use of plasma in the management of shock had led to the development of plasma collection facilities.60 The efficient longterm storage of plasma had been further facilitated by the process of lyophilization developed by Flosdorf and Mudd and the introduction of ABO-independent “universal” plasma produced by pooling of several thousand units of plasma.61 In 1940, the United States organized a program for the collection of blood and the shipment of plasma to Europe. The American Red Cross, through its local chapters, participated in the project, which collected 13 million units by the end of the war.46 The national program of the American Red Cross ceased at the end of the war. However, many of the local chapters continued to help recruit donors for local blood banks, and in 1948, the first regional Red Cross blood center was begun in Rochester, New York. By 1949-1950 in the United States, the blood procurement system included 1500 hospital blood banks, 1100 of which performed all blood bank functions. There were 46 nonhospital blood banks and 31 Red Cross regional blood centers. By 1962, these numbers had grown to 4400 hospital blood banks, 123 nonhospital blood banks, and 55 American Red Cross regional blood centers, and the number of units collected had grown to between 5 and 6 million per year.62 During this time, blood was collected through steel needles and rubber tubing into rubber-stoppered bottles. After washing and resterilization, the materials were reused. On occasion, “vacuum bottles” were used to speed up the collection. However, the high incidence of pyrogenic reactions soon led to the development of disposable plastic blood collection equipment. In a classic article written in 1952, Walter and Murphy63 described a closed, gravity technique for whole blood preservation. They used a laminar flow phlebotomy needle, an interval donor tube, and a collapsible bag of polyvinyl resin designed so that the unit could be assembled and ready for use after sterilization with steam. The polyvinyl resin was chemically inert to biologic fluids and nonirritating to tissue. Soon thereafter, Gibson et al64 demonstrated that plastic systems were more flexible and allowed removal of plasma after sedimentation or centrifugation. In time, glass was replaced with plastic, and component therapy began to emerge. This development was enhanced by the US military’s need to reduce the weight and breakage of blood bottles during shipment in the Korean War. Component and derivative therapy began during World War II when Edwin J. Cohn and his collaborators developed the cold ethanol method of plasma fractionation.65 As a result of their work, albumin, globulin, and fibrinogen became available for clinical use. As plastic equipment replaced glass, component separation became a more widespread practice, and the introduction of automated cell separators provided even greater capabilities in this area.

8

Clotting factor concentrates for the treatment of patients with hemophilia and other hemorrhagic disorders also were developed during the postwar era. Although antihemophilic globulin had been described in 1937,66 unconcentrated plasma was the only therapeutic material until Pool discovered that Factor VIII could be harvested in the cryoprecipitable fraction of blood.67 This resulted in the development of cryoprecipitate, which was introduced in 1965 for the management of hemophilia. Pool showed that cryoprecipitate could be made in a closed-bag system and urged its harvest from as many donations as possible. The development of cryoprecipitate and other concentrates was the dawn of a golden age in the care of patients with hemophilia. Self-infusion programs, made possible by technologic advances in plasma fractionation, allowed early therapy and greatly reduced disability and unemployment. This golden age came abruptly to an end with the appearance of the AIDS virus.

Transfusion in the Age of Technology In contrast to the long ledger of lives lost in previous centuries because of the lack of blood, transfusion in the 20th century saved countless lives. In 1937, during those early halcyon days of transfusion, Ottenberg wrote40: Today transfusion has become so safe and so easy to do that it is seldom omitted in any case in which it may be of benefit. Indeed the chief problem it presents is the finding of the large sums of money needed for the professional donors who now provide most of the blood.

It is ironic that Ottenberg’s statement should refer to paid donors and foreshadow difficulties yet to come. However, experience to that point had not revealed the problem of viral disease transmission. More transfusions would have to be administered before that problem would be perceived. After the introduction of anticoagulants, blood transfusions were given in progressively increasing numbers. At Mount Sinai Hospital in New York, the number of blood transfusions administered between 1923 and 1953 increased 20-fold68,69 (Table 1-1). This increase was particularly notable after the establishment of blood banks. It was during this period that Beeson wrote his classic description of transfusion-transmitted hepatitis70 (Fig 1-9). He had been alerted to the problem by the outbreaks of jaundice that followed inoculation programs with human serum during World War II. Thus blood transfusion entered a new era. Blood components not only saved lives but also transmitted disease. The discovery of the Australian antigen71 and the subsequent definition of hepatitis A virus and B virus (HBV) still left residual non-A and non-B disease,72 a gap that has been largely filled by the discovery of the hepatitis C virus (HCV).73 However, it was the outbreak of AIDS that galvanized public attention to blood transfusion. The AIDS epidemic was first recognized in the United States, and the first case of AIDS associated with transfusion was observed in a 20-month-old infant.74 Subsequently the suspicion that AIDS

Chapter 1: Transfusion in the New Millennium

Table 1-1. Increase in the Number of Blood Transfusions at Mount Sinai Hospital, New York, 1923-1953 Year

No. of Transfusions

1923 1932 1935 1938 1941 1952 1953

143 477 794 (Blood bank started) 2097 2874 3179

Adapted from Lewisohn.68

Figure 1-9. The first description of posttransfusion hepatitis. Used with permission from Beeson (JAMA).70 Copyright 1943, American Medical Association.

could be transmitted by means of transfusion was confirmed.75 The human immunodeficiency virus (HIV) was identified,76 and an effective test to detect the HIV antibody was developed.76

Concern for Blood Safety Since 1943, transfusion therapy has been shadowed by the specter of disease transmission. In that year, Beeson described posttransfusion hepatitis and unveiled a problem that has grown with time. As transfusion increased, so did disease transmission. In 1962, the connection between paid donations and posttransfusion hepatitis was made.77 A decade later, the National Blood Policy mandated a voluntary donation system in the United States. And yet, blood usage continued to increase. Concern about posttransfusion hepatitis was not sufficient to decrease the number of transfusions. Although the use of whole blood declined as blood components became more popular, total blood use in the United States doubled between 1971 and 1980 (Table 1-2).78-84 This pattern changed as the emergence of AIDS exposed all segments of society to a revealing light. AIDS probably arose in Africa in the 1960s and spread quietly for years before it was detected. By 1980 an estimated 100,000 persons were infected, and by 1981, when the first cases were

Table 1-2. Transfusions in the United States (in Millions of Units)78-84 Year

Whole Blood and Red Blood Cells

Platelets

Plasma

Total

1971 1979 1980 1982 1984 1986 1987 1989 1992 1994 1997 1999 2001 2004 2006

6.32 9.47 9.99 11.47 11.98 12.16 11.61 12.06 11.31 11.11 11.52 12.39 13.90 14.18 14.65

0.41 2.22 3.19 4.18 5.53 6.30 6.38 7.26 8.33 7.87 9.04 9.05 10.20 9.88 10.39

0.18 1.29 1.54 1.95 2.26 2.18 2.06 2.16 2.26 2.62 3.32 3.32 3.93 4.09 4.01

6.91 12.98 14.72 17.60 19.77 20.64 20.05 21.48 21.90 21.60 23.88 24.76 28.03 28.15 29.05

reported, a worldwide pandemic lay just beneath the surface.85 The initial response of the public and officials seemed trifling and insufficient as the outbreak grew to proportions few had foreseen. Criticism was levied against the news media for initially ignoring the story, the government for delay in acknowledging the problem, gay civil rights groups for resistance to epidemiologic measures, research scientists for unseemly competition, blood services for delayed response in a time of crisis, and the US Food and Drug Administration (FDA) for inadequate regulatory activity. Historians with the perspective of time will determine whether there really were more villains than the virus itself.86 Improved donor screening and increased donation testing have greatly decreased the risk of disease transmission and rendered the blood supply safer than it has ever been.86 Nonetheless, the realization that transfusion can transmit an almost invariably fatal disease had a chilling effect on the public. Two major changes in blood services have occurred in the aftermath of the AIDS epidemic. The FDA, using pharmaceutical manufacturing criteria not “tailored to . . . blood banks,” has become more aggressive in regulatory actions against blood collection establishments.87 And, finally, blood use moderated for approximately 10 years. Through the 1980s and early 1990s, red cell and plasma transfusion peaked and began to stabilize (Table 1-2). Only platelet use and human progenitor cell transplantation, driven by the demands of cancer chemotherapy, continued to increase.79-81 Educational programs to encourage judicious use of blood have been initiated, and they have been favorably received by practicing physicians. Relentless public pressure for a “zero risk” blood supply resulted in dividends through continued scientific and technologic improvements. Enhanced sensitivity and better use of serologic testing, along with improved scrutiny of donors, resulted in major reductions in risk of transmitted disease by the mid-1990s.88 Discovery that pools of units subjected to nucleic acid testing almost closed

9

Chapter 1: Transfusion in the New Millennium

the window for HIV and HCV virus resulted in application of this testing for both whole blood and plasma donations beginning between 1998 and 2000.89 This, combined with virus reduction and inactivation of the final product, resulted in plasma derivatives that have not transmitted AIDS or hepatitis since 1994.90 For whole blood and platelet components, risks have become low. A solvent/ detergent-treated fresh frozen plasma component is used in Europe but not in the United States. With the reduction in the risk of viral transmission, the focus in the developed world has shifted to transfusion-related acute lung injury—possibly from recipient-directed leukocyte antibodies and lipid mediators in transfused plasma—and bacterial infection primarily occurring in room-temperature stored platelets. So that incremental gains can be made against these risks, the use of male plasma only and the culture of platelets before they are released are being discussed and considered in some areas and implemented in others. Geographic exclusions have been aimed at reducing the potential for variant CreutzfeldtJakob disease (vCJD) transmission by transfusion, although in the United States such occurrence seems highly unlikely. In many countries, universal leukocyte reduction has been a response to the vCJD risk. Ironically universal application of leukocyte reduction is probably ineffective for vCJD but has stimulated a controversy in the United States over its use for preventing other problems.91 Finally, focus on the understanding, management, and prevention of medical errors in general might lead to progress against remaining nemesis hemolytic transfusion reactions caused by mistransfusion. Bar code technology at the bedside, commonly applied to prevent errors in medication administration, has shown efficacy in reducing transfusion errors.92 Radio-frequency identification shows further promise in error-prone situations such as operating rooms.93 Transfusion safety officers and hemovigilance systems are other initiatives being considered or instituted. “Zero risk” has still not been achieved. Emerging global infections such as West Nile virus, Chagas’ disease, and Chikungunya virus remain future potential threats and have encouraged further test development. Nevertheless, increased public and physician confidence in the safety of the blood supply (Table 1-3) combined with both increased aggressiveness of therapies and aging of the population resulted in increased blood use by 1999. For 2001 through 2006, total red cell transfusions in the United States increased by 5.4% and total platelet and plasma transfusions rose by 2% each. A significant decrease was seen in autologous transfusions (47.4%).84

Current Megatrends In the developed world, no cost has been spared in meeting public demands for blood safety. Service fees charged to hospitals by independent blood centers in the United States (Fig 1-10) illustrate the effect each new safety development has had on the cost of Red Blood Cells (RBCs). As the new millennium began, the

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Table 1-3. Risk Estimates per Unit of Red Blood Cells Transfused in the United States, Ranked by Frequency Type of Risk

Estimated Occurrence

Urticaria Red cell allommunization Febrile reaction TRALI Hemolytic reaction Transfusion to the wrong recipient Anaphylaxis Hepatitis B virus HTLV-I/II Hepatitis C virus HIV Malaria Bacterial contamination GVHD

1 in 50-100 1 in 100 1 in 300 1 in 5000 1 in 6000 1 in 14,000-19,000 1 in 20,000-50,000 1 in 100,000-200,000 1 in 641,000 1 in 1-2 million 1 in 2-3 million 1 in 4 million 1 in 5 million Very rare, no estimates

TRALI ⫽ transfusion-related acute lung injury; HTLV-I/II ⫽ human Tcell lymphotropic virus, types I and II; HIV ⫽ human immunodeficiency virus; GVHD⫽graft–vs–host disease Adapted from Klein et al.94

mean payment for RBC units was $100, and a leukocyte-reduced unit was $126. By 2005 those had risen to $157 and $188 respectively, with significant annual increases.95 One group of researchers (committed to programs to reduce blood use) has published data suggesting that the societal cost of a unit of RBCs is $1400 per unit taking into account not only the blood center fee but also hospital-related costs, costs of treating adverse reactions, litigation, lost productivity of donors, and hemovigilance.96 Although the figure might be overinflated, such work does highlight the ever increasing cost of this form of therapy to the patient and society. In the underdeveloped world, the picture is quite different. The greatest blood need is for women hemorrhaging during childbirth, infants and children with anemia caused by malaria, and victims of trauma. In 80 of 172 countries responding to a World Health Organization (WHO) survey, fewer than 1% of the population donate blood. In sub-Saharan Africa, fewer than 3 million units of blood are collected annually for a population of more than 700 million. Of the 148 countries reporting data to WHO, 41 are unable to screen for minimum safety (HIV, HBV, HVC, and syphilis). WHO estimates that unsafe blood in these countries results in 16 million new infections with HBV, 5 million with HCV, and 160,000 with HIV each year (accounting for 5%-10% of the world’s HIV infections). Fortunately there is progress in some nations in achieving an all-volunteer supply and minimum screening. Thus, there are two drastically different pictures of blood safety and availability worldwide.97 Even in the developed world, availability remains a challenge. In the United States, the number in the population eligible to donate blood with all the new restrictions is 111 million, rather than the 177 million previously thought.98 Finding ways to motivate sufficient numbers of people to donate remains

America’s Blood Centers Safety Measures and Median Red Blood Cell Service Fees 1985-2007

Chagas‘ test

$225

Bacteria detection TRALI interventions (apheresis platelets) $200

Licensed HIV-1/HCV NAT

$175

Leukoreduction becomes widespread

$150

$125 HBc and ALT test $100

HCV 1.0

HIV-1 Ab test

Licensed WNV NAT WNV NAT (IND)

HIV-1/2 test

HTLV-I test $75 HIV-1/HCV NAT (IND) HCV 2.0 test $25 RBCs RBCs, LR

FDA cGMP guidelines

HIV-1 P24 antigen

HCV look-back

1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 $43

43

46

49

53

58

61

64

69

73

75

76

77

85

93

102

114

140

154

157

166

177

$111 115

73

116

117

117

121

125

134

153

171

178

188

195

205

Figure 1-10. Red Blood Cell service fees charged by community blood centers from 1985 to 2007, correlated with safety measures. Courtesy of America’s Blood Centers (modified). RBCs ⫽ Red Blood Cells; RBCs, LR ⫽ RBCs Leukocytes Reduced; HIV ⫽ human immunodeficiency virus (-1/2 ⫽ types 1 and 2); Ab ⫽ antibody; HBc ⫽ hepatitis B core (antigen); ALT ⫽ alanine aminotransferase; HTLV-I ⫽ human T-cell lymphotropic virus, type I; HCV ⫽ hepatitis C virus; FDA cGMP ⫽ Food and Drug Administration current good manufacturing practice; NAT ⫽ nucleic acid testing; IND ⫽ investigational new drug; WNV ⫽ West Nile virus.

11

Chapter 1: Transfusion in the New Millennium

$50

Chapter 1: Transfusion in the New Millennium

difficult. One suggestion has been to “personalize” the benefits. An example of this approach is having donors meet face-to-face with groups of recipients. Another response has been the use of cognitive interview evaluation and focus groups to define a more user-friendly questionnaire with computer-assisted techniques in many centers.99 More aggressive marketing to growing minority populations as well as continued use of various incentives seem to have increased donations in some locations. A clear need is for more group O red cells; populations of non-European ethnicity generally have an increased proportion of group O.100 Another approach to maintain adequate availability is to control usage by ensuring that blood is used appropriately. Some US blood centers have been successful in bringing their transfusion medicine expertise into the patient-care setting by providing transfusion services to hospitals. The model in Seattle, WA, has operated for decades.101,102 In the United Kingdom, liaison systems for blood centers to hospitals employing Web-based technology for supply chain management have been introduced.103,104 In Denmark, success has been reported using the Thromboelastograph (Haemoscope, Niles, IL) hemostatic system to manage coagulopathy in conjunction with treating physicians—something also done in many US hospitals.105 Other point-of-care tests to assess the state of the coagulation system and tissue oxygenation could also result in more accurately targeted component transfusion. Transfusion medicine specialists in hospitals—whether from pathology groups, blood center staff, or other areas such as anesthesiology or hematology—are critical to the successful use of blood transfusion in patient care. Although some advances in transfusion medicine at the end of the last millennium have been difficult to implement, such as use of hemoglobin solutions and some pathogen inactivation technologies, the field has continued to advance into new areas of stem cell biology, regenerative medicine, and cord blood banking. In addition, transfusion medicine specialists increasingly function in collaboration with surgeons, oncologists, and hematologists in treating the acutely ill patient with complex medical problems. With all the added sophistication, the optimal hemoglobin and platelet triggers and endpoints for transfusion remain unsettled. Clinicians are less likely to use oxygenation transport endpoints to determine the need for red cell transfusion but are beginning to look for other means to assess tissue oxygenation. If a patient’s hemoglobin is too high (even when below normal), complications such as thromboembolism can result. Too low an endpoint exposes some patients to the risk of tissue hypoxia. The clearest trend has been away from autologous transfusion, although some medical centers seek bloodless medicine and surgery combining pharmacotherapy (mainly erythropoietin and iron), blood recovery and reinfusion, and conservative triggers and endpoints.94 More conservative triggers and endpoints for platelet transfusion are becoming accepted, but approaches to alloimmunized patients and bacterial contamination are still in question.106 Also debatable is whether transfusion, through some poorly quantifiable mechanism such as immunomodulation, confers a poorer prognosis on acutely ill patients. From ancient times into the new millennium, blood has been a substance that fascinates mankind. Despite unresolved

12

controversies, blood transfusion remains of critical importance in the care of sick patients throughout the world.

Disclaimer The authors have disclosed no conflicts of interest.

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Chapter 1: Transfusion in the New Millennium

24. Transfusion and infusion. Lancet 1828;2:324-6. 25. Blundell J. Some account of a case of obstinate vomiting in which an attempt was made to prolong life by the injection of blood into the veins. Med Chir Trans 1819;10:296-311. 26. Blundell J. Observations on transfusions of blood. Lancet 1828;2:321-4. 27. Moss WL. A simple method for the indirect transfusion of blood. Am J Med Sci 1914;147:698-703. 28. Jennings CE. Transfusion: Its history, indications, and modes of application. New York: CH Goodwin, 1884:101. 29. Braxton-Hicks J. Cases of transfusion with some remarks on a new method of performing the operation. Guys Hosp Rep 1869;14:1-14. 30. Oberman HA. Early history of blood substitutes: Transfusion of milk. Transfusion 1969;9:74-7. 31. Schmidt PJ. Transfusion in America in the eighteenth and nineteenth centuries. N Engl J Med 1968;279:1319-20. 32. Bull WT. On the intra-venous injection of saline solutions as a substitute for transfusion of blood. Med Rec 1884;25:6-8. 33. Landsteiner K. Ueber Agglutinationserscheinungen normalen menschlichen Blutes. Wien Klin Wochenschr 1901;14:1132-4. 34. Dixon B. Of different bloods. Science 84 1984;5:65-7. 35. Decastello A, Sturli A. Ueber die Isoagglutinine im Serum gesunder und kranker Menschen. Munch Med Wochenschr 1902;49:1090-5. 36. Carrel A. The transplantation of organs: A preliminary communication. JAMA 1905;45:1645-6. 37. Walker LG Jr. Carrel’s direct transfusion of a five day old infant. Surg Gynecol Obstet 1973;137:494-6. 38. Crile GW. The technique of direct transfusion of blood. Ann Surg 1907;46:329-32. 39. Bernheim BM. Blood transfusion: Hemorrhage and the anaemias. Philadelphia: JB Lippincott, 1917:259. 40. Ottenberg R. Reminiscences of the history of blood transfusion. J Mt Sinai Hosp 1937;4:264-71. 41. Pepper W, Nisbet V. A case of fatal hemolysis following direct transfusion of blood by arteriovenous anastomosis. JAMA 1907;49:385-9. 42. Ottenberg R. Transfusion and arterial anastomosis. Ann Surg 1908;47:486-505. 43. Ottenberg R, Kaliski DJ. Accidents in transfusion: Their prevention by preliminary blood examination: Based on an experience of 128 transfusions. JAMA 1913;61:2138-40. 44. Epstein AA, Ottenberg R. A simple method of performing serum reactions. Proc N Y Pathol Soc 1908;8:117-23. 45. Ottenberg R. Studies in isoagglutination, I: Transfusion and the question of intravascular agglutination. J Exp Med 1911;13:425-38. 46. Diamond LK. The story of our blood groups. In: Wintrobe MM, ed. Blood, pure and eloquent. New York: McGraw-Hill, 1980:658-717. 47. Levine P, Stetson RE. An unusual case of intragroup agglutination. JAMA 1939;113:126-7. 48. Landsteiner K, Wiener AS. An agglutinable factor in human blood recognized by immune sera for rhesus blood. Proc Soc Exp Biol Med 1940;43:233. 49. Unger LJ. A new method of syringe transfusion. JAMA 1915;64:582-4. 50. Hustin A. Principe d’une nouvelle methode de transfusion. J Med Bruxelles 1914;12:436. 51. Agote L. Nuevo procediemento para la transfusion del sangre. An Inst Mod Clin Med Buenos Aires 1915;2:24-30. 52. Lewisohn R. A new and greatly simplified method of blood transfusion. Med Rec 1915;87:141-2.

53. Weil R. Sodium citrate in the transfusion of blood. JAMA 1915;64:425-6. 54. Rous P, Turner JR. The preservation of living red blood cells in vitro. J Exp Med 1916;23:219-48. 55. Robertson OH. Transfusion with preserved red blood cells. Br Med J 1918;1:691-5. 56. Loutit JF, Mollison PL. Advantages of a disodium-citrate-glucose mixture as a blood preservative. Br Med J 1943;2:744-5. 57. Duran-Jorda F. The Barcelona blood transfusion service. Lancet 1939;1:773-5. 58. Fantus B. The therapy of the Cook County Hospital. JAMA 1937;109:128-31. 59. Yudin SS. Transfusion of cadaver blood. JAMA 1936;106:997-9. 60. Strumia MM, McGraw JJ. The development of plasma preparations for transfusions. Ann Intern Med 1941;15:80-7. 61. Flosdorf EW, Mudd S. Procedure and apparatus for preservation in “lyophile” form of serum and other biological substances. J Immunol 1935;29:389-425. 62. Diamond LK. History of blood banking in the United States. JAMA 1965;193:40-5. 63. Walter CW, Murphy WP Jr. A closed gravity technique for the preservation of whole blood in ACD solution utilizing plastic equipment. Surg Gynecol Obstet 1952;94:687-92. 64. Gibson JG II, Sack T, Buckley ES Jr. The preservation of whole ACD blood, collected, stored and transfused in plastic equipment. Surg Gynecol Obstet 1952;95:113-19. 65. Cohn EJ. The separation of blood into fractions of therapeutic value. Ann Intern Med 1947;26:341-52. 66. Patek AJ, Taylor FHL. Hemophilia, II: Some properties of a substance obtained from normal human plasma effective in accelerating the coagulation of hemophilic blood. J Clin Invest 1937;16:113-24. 67. Pool JG, Shannon AE. Production of high-potency concentrates of antihemophilic globulin in a closed-bag system. N Engl J Med 1965;273:1443-7. 68. Lewisohn R. Blood transfusion: 50 years ago and today. Surg Gynecol Obstet 1955;101:362-8. 69. Rosenfeld RE. Early twentieth century origins of modern blood transfusion therapy. Mt Sinai J Med 1974;41:626-35. 70. Beeson PB. Jaundice occurring one to four months after transfusion of blood or plasma. JAMA 1943;121:1332-4. 71. Blumberg BS, Alter HJ, Visnich S. A “new” antigen in leukemia sera. JAMA 1965;191:541-6. 72. Feinstone SM, Kapikian AZ, Purcell RH, et al. Transfusion-associated hepatitis not due to viral hepatitis type A or B. N Engl J Med 1975;292:767-70. 73. Choo QL, Kuo G, Weiner AJ, et al. Isolation of a cDNA clone derived from a blood-borne non-A non-B viral hepatitis genome. Science 1989;244:359-62. 74. Ammann JA, Cowan MJ, Wara DW, et al. Acquired immunodeficiency in an infant: Possible transmission by means of blood products. Lancet 1983;1:956-8. 75. Curran JW, Lawrence DN, Jaffe H, et al. Acquired immunodeficiency syndrome (AIDS) associated with transfusions. N Engl J Med 1984;310:69-75. 76. Sarngadharan MG, Popovic M, Bruch L, et al. Antibodies reactive with a human T-lymphotropic retrovirus (HTLV-III) in the serum of patients with AIDS. Science 1984;224:506-8. 77. Allen JG, Sayman WA. Serum hepatitis from transfusions of blood. JAMA 1962;180:1079-85.

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Chapter 1: Transfusion in the New Millennium

78. Surgenor DM, Schnitzer SS. The nation’s blood resource: A summary report. National Institutes of Health (NIH) publication no. 85-2028. Bethesda, MD: US Department of Health and Human Services, Public Health Service, NIH, 1985. 79. Surgenor DM, Wallace EL, Hao SHS, et al. Collection and transfusion of blood in the United States, 1982-1988. N Engl J Med 1990;322:1646-51. 80. Wallace EL, Surgenor DM, Hao HS, et al. Collection and transfusion of blood and blood components in the United States, 1989. Transfusion 1993;33:139-44. 81. Wallace EL, Churchill DM, Surgenor GS, et al. Collection and transfusion of blood and blood components in the United States, 1994. Transfusion 1998;38:625-36. 82. Sullivan MT, McCullough J, Schreiber GB, Wallace EL. Blood collection and transfusion in the United Stated in 1997. Transfusion 2002;42:1253-60. 83. Sullivan MT, Wallace EL. Blood collection and transfusion in the United States in 1999. Transfusion 2005;45:141-8 84. US Department of Health and Human Services. The 2007 National Blood Collection and Utilization Survey Report. Washington, DC: DHHS, 2008. 85. Essex M. Origin of AIDS. In: DeVita VT, Hellman S, Rosenberg SA, eds. AIDS: Etiology, diagnosis, treatment, and prevention, 3rd ed. Philadelphia: JB Lippincott, 1992:3-11. 86. Starr D. Blood: An epic history of medicine and commerce. New York: Alfred A Knopf, 1998;147-357. 87. Solomon JM. The evolution of the current blood banking regulatory climate. Transfusion 1994;34:272-7. 88. Schreiber GB, Busch MP, Kleinman SH, et al. The risk of transfusion-transmitted viral infection. N Engl J Med 1996;334:1685-90. 89. Busch MP, Kleinman SH. Report of the interorganizational task force on nucleic acid amplification testing of blood donors: Nucleic acid amplification testing of blood donors for transfusion-transmitted infectious diseases. Transfusion 2000;40:143-59. 90. Tabor E. The epidemiology of virus transmission by plasma derivatives: Clinical studies verifying the lack of transmission of hepatitis B and C viruses and HIV type 1. Transfusion 1999;39:1160-8. 91. Goodnough LT. The case against universal WBC reduction (and for the practice of evidence-based medicine). Transfusion 2000;40:1522-7.

14

92. Murphy MF. Application of bar code technology at the bedside: The Oxford experience. Transfusion 2007:47(Aug Suppl): 120S-4S. 93. Dzik, S. Radio frequency identification for prevention of bedside errors. Transfusion 2007; 47(Aug Suppl):125S-9S. 94. Klein HG, Spahn DR, Carson JL. Red blood cell transfusion in clinical practice. Lancet 2007;370:415-26 95. MacPherson J, Mahoney CB, Katz L, et al. Contribution of blood to hospital revenue in the United States. Transfusion 2007;47(Aug Suppl):114S-16S. 96. Hoffman A, Gomhotz A, Theusinger OM, Spahn DR. Estimating the cost of blood: Past, present and future directions. Best Pract Res Clin Anaethesiol 2007;21:271-89. 97. Improving blood safety worldwide (editorial). The Lancet 2007;370:361. 98. Riley W, Schwei M, McCullough J. The United States potential blood donor pool: Estimating the prevalence of donor exclusion factors on the pool of potential donors. Transfusion 2007;47:1180-8. 99. Fridey JL, Townsend MJ, Kessler D, Gregory K. A question of clarity: Redesigning the American Association of Blood Banks blood donor history questionnaire—A chronology and methodology for donor screening. Transfus Med Rev 2007;21:181-204. 100. Simon TL. Where have all the donors gone? A personal reflection on the crisis in America’s volunteer blood program. Transfusion 2003;43:273-9. 101. Yazer M. The Pittsburgh centralized transfusion model: Less is more. Transfusion 2007;47(Aug Suppl):164S-9S. 102. Gottschall JL. Blood centers and hospitals: A model for clinical interactions and services. Transfusion 2007;47(Aug Suppl):172S-6S. 103. Allen T. Blood center and hospital relationships in England and North Wales: Their impact on the declining demand for red blood cells? Transfusion 2007;47(Aug Suppl):158S-63S. 104. Chapman J. Unlocking the essentials of effective blood inventory management. Transfusion 2007:47(Aug Suppl):190S-6S. 105. Johansson PI. The blood bank: From provider to partner in treatment of massively bleeding patients. Transfusion 2007:47(Aug Suppl):176S-81S. 106. Stroncek DF, Rebulla P. Platelet transfusions. Lancet 2007;370:427-38.

I

Blood Components and Derivatives

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PART I

2

Red Blood Cells

Red Cell Production and Kinetics Mark J. Koury Professor of Medicine, Division of Hematology/Oncology, Vanderbilt University; and Associate Chief of Staff for Education, VA Tennessee Valley Healthcare System, Nashville, Tennessee, USA

The main function of erythrocytes (red cells) is to transport oxygen from the lungs to the other tissues of the body. This delivery of oxygen is finely controlled by the number of circulating erythrocytes, which is a function of the rate of old erythrocyte removal from the circulation and the rate of new erythrocyte production. Circulating erythrocytes are maintained in an extremely narrow range because the normal marrow produces almost exactly the same number of new erythrocytes each day as is lost through senescence. This daily turnover of about 1% of circulating erythrocytes represents approximately 250 billion erythrocytes in a healthy adult. When much greater numbers of circulating erythrocytes are lost, such as with bleeding or hemolysis, the production of new erythrocytes increases rapidly to maintain the steady-state number of erythrocytes. This rapid expansion of erythrocyte production in response to bleeding or hemolysis is so well regulated that rebound polycythemia or overproduction of erythrocytes does not occur. This exquisitely controlled production of erythrocytes is mediated through a negative feedback mechanism that involves oxygen delivery to, and utilization by, the kidneys, the hormone erythropoietin (EPO) that is produced by specialized cells in the kidneys, and the erythroid progenitor cells in the marrow that respond to EPO. Normal red cell production also depends upon adequate supplies of specific nutrients, among which folate, vitamin B12, and iron are the most important in terms of clinically encountered anemias. Disorders of the hematopoietic system or other disease processes such as chronic inflammation also affect the erythropoietic process.

Erythropoiesis Erythropoiesis: A Component of Hematopoiesis Erythropoiesis, the process of erythrocyte production, is a component of the larger process of hematopoiesis in which a stem

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

cell proliferates and differentiates to form all the cell types in the blood and immune system.1 The endproducts of hematopoiesis include platelets, granulocytes, monocytes-macrophages, T lymphocytes, and B lymphocytes as well as erythrocytes. Thus, normally regulated hematopoiesis is required for effective hemostasis, inflammation, immune response, and tissue oxygenation. Among these various functions, tissue oxygenation is the first required during embryonic development and the most tightly regulated in postnatal life. Erythropoiesis has two sequential phases during development—the primitive phase, during weeks 3 to 8 of human gestation, when erythrocytes are produced in “blood islands” of the yolk sac, and the subsequent definitive phase in which erythrocytes are produced in the fetal liver and spleen and later in the marrow.2-4 In humans, the hemoglobin of the primitive erythrocytes contains embryonic ε- and ζ-globins, whereas the hemoglobin of the definitive erythrocytes contains adult α-globin and either fetal γ-globin from midgestation through the first few postnatal months or adult β-globin after the first few postnatal months. Embryologic studies demonstrate that the primitive and definitive phases of erythropoiesis arise from different stem cells in the early embryo.2,3,5,6 The primitive erythroid cells arise in the inner cell mass of the yolk sac blood islands, whereas the definitive erythroid cells arise from the aortogonadomesonephros region of the mesoderm. Current concepts of hematopoiesis are derived mainly from studies of mice and humans. These studies have included direct morphologic and immunologic analyses of cells in hematopoietic tissues, in-vitro culture of hematopoietic progenitor cells and hematopoietic stromal cells, transplantation studies with hematopoietic cells, and genetic studies of mice with natural mutations, transgene expressions, or targeted gene knockouts. Hematopoietic stem cells (HSCs) appear to be immediate progeny of an even more primitive stem cell, the hemangioblast, which can give rise to both hematopoietic cells and vascular cells during embryonic development.7 Studies with HSCs have led to a hematopoietic model (Fig 2-1) that applies to postnatal as well as prenatal hematopoiesis because turnover of both blood and immune cells requires new cell production throughout life. In this model, pluripotent HSCs, which have an incidence in

17

Section I: Part I

Hematopoietic stem cells

Lineage commitment stages

Mature blood cells

Maturation stages of committed progenitor cells

B-cell progenitors

B-Lymphocyte

T-cell progenitors

T-Lymphocyte

Lymphoid

Granulocyte progenitors

Monocytic-macrophage progenitors

Granulocyte

Monocyte

Myeloid Erythroid progenitors

Megakaryocytic progenitors

Erythrocytes

Platelets

Figure 2-1. Hematopoietic stem cell differentiation. Pluripotent hematopoietic stem cells (HSCs) can self-replicate (curved arrow) or give rise to progenitor cells committed to differentiation. Because of uncertainty about events leading to commitment and about self-replication of HSCs around the time of commitment, a hypothetical cell is shown at the border between the HSC and lineage commitment stages. Progeny of HSCs become committed to differentiation along specific cell lineages. After lineage commitment, progenitor cells mature and acquire the characteristics of terminally differentiated cells in blood (eg, erythroid cells produce hemoglobin and enucleate). During this maturation phase, lineage-specific hematopoietic growth factors act on progenitor cells. The stages of HSC differentiation are shown, but the proliferation that accompanies the differentiation is not. Proliferative potential decreases as cells progress through various stages toward mature blood cells. (Used with permission from Koury et al.8)

marrow of one per 104 to 105 nucleated cells, have two functions—self-replication and production of progenitor cells committed to differentiation into all lymphoid and myeloid cell types. Differentiation of HSCs requires a process called commitment in which the stem cell loses self-renewal capacity and is limited to either further differentiation or death. Commitment restricts the hematopoietic progenitor cells to differentiation along either the lymphoid or the myeloid pathway and subsequently to the specific, single-cell lineages (Fig 2-1). Differentiation involves cellular proliferation and additional commitment steps. Two possible mechanisms of hematopoietic cell commitment are a deterministic one and a stochastic one.1 In the deterministic mechanism, molecules from the cell’s environment react with specific receptors on or in the cell and thereby lead to changes in specific gene structure or expression that, in turn, determine the commitment status of that cell. These environmental molecules can be either soluble growth factors or fixed ligands on other cells or extracellular matrix components in the hematopoietic microenvironment. An example of deterministic commitment is Indian hedgehog upregulating the expression of bone morphogenic protein-4 (BMP-4) in the developing mesodermal cells.9 BMP-4, in turn, induces hematopoietic and endothelial differentiation, with the ventral portion of the developing dorsal aorta and the subjacent mesoderm inducing definitive hematopoiesis.9 In the stochastic mechanism, the fate of a hematopoietic cell is determined by the outcome of random intracellular events that

18

change the structure or expression of specific genes.10,11 Two examples of such changes in hematopoietic cells are immunoglobulin gene rearrangement in B-lymphocyte progenitors and binding of specific transcription factors such as TAL-1 or GATA-1 to the promoters of erythroid-specific genes.12,13 With the stochastic mechanism, environmental signals such as the concentration of a specific hematopoietic growth factor, or the interactions with marrow stromal cells, promote survival or proliferation but do not affect commitment. Thus, these environmental factors influence the size of hematopoietic cell populations that have had commitment decisions made by random intracellular events.

Stages of Erythropoiesis The stages of erythropoiesis from HSCs and extending through the mature erythrocytes are shown in Fig 2-2. Hematopoietic cell transplantation experiments with mice have demonstrated murine marrow cells that possess the properties of HSCs.14-16 Through analyses of murine HSCs and comparisons with human hematopoietic cell populations, a subset of hematopoietic cells that is highly enriched in human HSCs can be identified with cellular markers.1 This human HSC-enriched population displays the CD34 and CD90 (Thy 1) antigens and has low uptake of rhodamine-123 and weak expression of HLA-DR antigens. After erythroid lineage commitment, the first stage of recognized differentiation is the burst-forming units–erythroid (BFU-E)

Chapter 2: Red Cell Production and Kinetics

(A) Stage of erythroid differentiation

HSC

BFU-E

Pro EB

CFU-E

Baso EB

Poly EB

Ortho EB

RET

RBC

(B) Transcription factors for erythroid differentiation TAL-1 LMO-2 GATA-2 GATA-1 FOG RB EKLF

(C) Receptors for required hematopoietic growth factors c-KIT EPO-R IGF-1-R

(D) Proteins related to erthrocyte structure and function Kell Glycoprotein Rh Glycoprotein Glycophorin A ␣ - Spectrin ␤ - Spectrin Transferrin receptor ␦ - ALA - Synthetase globins Band 3/anion transporter Band 4.1 Figure 2-2. Cellular events in erythroid differentiation. (A) The relative sizes and the known or presumed morphologic appearances of erythroid cells at various stages of differentiation—pluripotent hematopoietic stem cells (HSCs); burst-forming unit–erythroid (BFU-E); colony-forming unit–erythroid (CFU-E); proerythroblasts (Pro EB); basophilic erythroblasts (Baso EB); polychromatophilic erythroblasts (Poly EB); orthochromatic erythroblasts (Ortho EB), reticulocytes (RET); erythrocytes (RBCs). (B) Erythroid transcription factors—basic helix-loop-helix factor (TAL-1); Lim-domain partner of TAL-1 (LMO-2); zinc finger factors that bind GATA sequences (GATA-1, GATA-2); GATA-1 partner, “friend of GATA” (FOG); retinoblastoma protein (RB); erythroid Krüppel-like factor (EKLF). (C) Receptors for hematopoietic growth factors—stem cell factor receptor (c-KIT); erythropoietin receptor (EPO-R); insulin-like growth factor-1 receptor (IGF-1-R). (D) Proteins related to erythrocyte structure and function. Periods of expression for erythroid-specific forms of proteins are shown. Transferrin receptors are present in all stages, but a period of large up-regulation is shown. Rh-associated glycoprotein (Rh50) is expressed before the proerythroblast stage, while the RhD and RhCE proteins are first expressed in the proerythroblast stage or later. For each transcription factor, growth factor receptor, and erythrocyte-related protein, the degree of expression can vary greatly during the period shown.

that proliferates and differentiates in semisolid tissue culture medium to form large colonies or groups of colonies called bursts of erythroblasts.17 Human BFU-Es need 2 to 3 weeks of culture to form bursts that contain as many as several thousand erythroblasts. The more immature BFU-Es need the longest culture period and form the largest bursts. The next well-defined stage of erythroid development is the colony-forming unit–erythroid (CFU-E).18 Human CFU-Es need 7 days in vitro to proliferate and differentiate into small colonies, usually composed of up to 64 erythroblasts. After the CFU-E stage is the proerythroblast

stage, the last erythroid progenitor with a nonspecific morphologic appearance in the differentiation scheme shown in Fig 2-2. The proerythroblast gives rise to the successive stages of the basophilic, polychromatophilic, and orthochromatic erythroblasts that are recognized by light microscopy of stained marrow samples. Orthochromatic erythroblasts enucleate, forming reticulocytes. Reticulocytes are very irregularly shaped cells containing residual organelles (the “reticulum”) that allow the cells to be distinguished from the more mature erythrocytes. The final stage of differentiation, the erythrocyte, is achieved after the reticulocytes

19

Section I: Part I

rid themselves of their residual organelles and remodel their irregular shapes to become uniform biconcave disks. Each progenitor cell loses proliferative potential as it differentiates along this continuum until it reaches the late erythroblast stages in which the cells do not divide. Thus, between the BFU-E and the CFU-E stages are a series of intermediate stages called mature burst-forming units, which need more than 1 week but less than 2 weeks in culture to develop into colonies with sizes intermediate between those of typical erythroid bursts and colonies.19 Similarly, the reticulocyte population has early, intermediate, and late stages of development based on their relative RNA content, which steadily declines with maturation.20

Requirements for Normal Erythroid Differentiation A series of intracellular and extracellular events are needed for successful completion of the erythroid differentiation scheme as shown in Fig 2-2. The intracellular events include the expression of 1) general hematopoietic and erythroid-specific transcription factors, 2) receptors for hematopoietic growth factors, and 3) proteins such as hemoglobin, membrane, and membrane cytoskeleton proteins found in the mature erythrocyte. Synthesis of hemoglobin begins as the erythroblasts are making the transition from the basophilic to the polychromatophilic stages. Among the erythroid membrane proteins, the Kell and the Rh glycoproteins are expressed before the CFU-E stage, while most of the other membrane proteins found on mature erythrocytes are expressed at the proerythroblast stage or later.21,22 At the proerythroblast stage, α- and β-spectrin are synthesized, but they are degraded in the cytoplasm rather than associating with the cytoskeleton.23 Glycophorin A is present in the plasma membrane of proerythroblasts,21,22 but the band 3/anion transporter protein is not made until the basophilic erythroblast stage, and band 4.1 does not begin to accumulate until the polychromatophilic stage.23 Once the band 3/anion transporter protein is present in the membrane, α- and β-spectrin become membrane associated in the characteristic 1:1 ratio.23 Extracellular requirements for erythroid differentitation include 1) the adequate supply of required hematopoietic growth factors, 2) a sufficient supply of nutrients required for progenitor cell proliferation and differentiation, and 3) the presence of stromal cell and matrix support. In Fig 2-2, the transcription factors, growth factor receptors, and hematopoietic growth factors necessary for normal erythropoiesis are shown for the period of differentiation when they are known to be needed. The principal growth factor that regulates erythropoiesis is EPO, which is discussed in detail in the following section. The nutrients that play important roles in erythropoiesis—folate, vitamin B12, and iron—are required at all stages of erythroid cell differentiation, but most importantly in the later stages. Deficiency of any of these three nutrients results in decreased erythrocyte production and anemia. The roles of these nutrients in erythropoiesis are described in later sections. HSCs and BFU-Es are found in the hematopoietic organs, but they also can circulate in the blood. Erythroid cells in the CFU-E through orthochromatic stages of

20

differentiation do not circulate and, in the hematopoietic tissue, they form specific structures with stromal macrophages termed erythroblastic islands.24 An erythroblastic island consists of a central macrophage surrounded by the adherent erythroid cells that are usually at about the same stage of differentiation within each individual island. Ablation of the stromal macrophages impairs the erythropoietic response to blood loss, indicating that erythroblastic islands have a functional role in red cell production.25 At least five surface membrane proteins can mediate erythroid-macrophage interactions in erythroblastic islands. 1. Vascular cell adhesion molecule 1 (VCAM-1) on marrow macrophages binds α4β1 integrin (also known as very late antigen-4 or VLA-4) on erythroblasts.26 2. αV component of integrins (eg, αV β1, αV β3, and αV β5) on marrow macrophages binds interstitial cell adhesion molecule 4 (ICAM-4, also known as LW glycoprotein) on erythroblasts.27 3. Erythroblast-macrophage protein (EMP), a transmembrane protein of both erythroblasts and macrophages, mediates interactions between these two cell types through a homophilic reaction.28 4. Sialoadhesin (CD169 or Siglec 1) on macrophages binds sialylated glycoproteins on erythroblasts.29 5. Hemoglobin-haptoglobin receptor (CD163) is a marrow macrophage surface glycoprotein that binds an unknown ligand on erythroblasts.30 Each of these adhesion protein pairs can be disrupted by antibodies and/or competing peptide ligands such that binding of erythroblasts to macrophages is inhibited. Because disruption of one of these binding protein pairs can inhibit attachment of erythroblasts to macrophages, the proteins may form functional binding complexes on the surfaces of macrophages and erythroblasts.

Erythropoietin Regulation of Erythropoietin Production by Tissue Hypoxia Under normal circumstances, the rate of erythrocyte production is controlled by the interaction of EPO with the EPO receptors on the erythroid progenitor cells. Erythropoietin is a 30.4-kD glycosylated protein, most of which is produced by the kidneys.31 The kidneys are a major component of the oxygenation-EPO negative feedback mechanism shown in Fig 2-3. The other components are the marrow, the blood erythrocytes, and EPO itself. The negative feedback characteristics account for the fine control of erythrocyte numbers that occur in each individual. The major determinant of oxygen delivery from the lungs to the peripheral tissues is the number of circulating erythrocytes. When erythrocyte numbers decrease, as occurs with bleeding or hemolysis, oxygen delivery decreases and the peripheral tissues become hypoxic. All tissues experience hypoxia, but the major ones that respond with EPO production are the kidneys and the liver.

Chapter 2: Red Cell Production and Kinetics

Renal cortex

Blood

Marrow

EPO

O2

Red cells Figure 2-3. The oxygenation-erythropoietin (EPO) negative feedback mechanism. The number of circulating red cells determines the amount of oxygen delivered from the lungs to other tissues. In the renal cortex, a specific subset of interstitial cells produce EPO when they perceive hypoxia. The EPO is immediately secreted into the blood and acts in the marrow to prevent the programmed death (apoptosis) of erythroid progenitor cells. Those erythroid progenitors that survive the EPO-dependent period of differentiation mature into reticulocytes (irregularly shaped anucleate cells in marrow and blood) and subsequently into erythrocytes. Increased numbers of erythrocytes resulting from increased plasma EPO levels deliver more oxygen to the kidneys and thereby lower the amount of EPO produced as renal hypoxia is relieved.

The same cells that sense hypoxia are the ones that produce EPO.32 The mechanism for sensing hypoxia in cells involves a family of specific transcription factors, hypoxia-inducible factors (HIFs).33,34 HIFs have two components, HIF-α and HIF-β, which form a complex that binds to a hypoxia-inducible transcription enhancer located in the region of various genes that include EPO, vascular endothelial growth factor (VEGF), and genes encoding several of the glycolytic enzymes.35 In the case of EPO, HIF-2α is the family member that binds to a 120-bp enhancer 3⬘ to the human EPO polyadenylation signal36,37 as shown in Fig 2-4(B). HIF-1β is constitutively produced, and its intracellular levels are not influenced by hypoxia. HIF-α components are constitutively produced, but under normoxic conditions, they are essentially undetectable because they are rapidly degraded by the ubiquitin-proteasome pathway,38 shown in Fig 2-4(A). However, when an EPO-producing cell experiences hypoxia, this rapid degradation ceases and intracellular HIF-2α levels promptly increase, as in Fig 2-4(B). Polyubiquitination of HIF-2α depends upon the von Hippel-Lindau protein (pVHL) interacting with those HIF-2α molecules that have hydroxylation of two specific proline residues.39-41 Hydroxylation of a specific asparagine residue42,43 in HIF-2α prevents its interaction with the p300 member of the HIF transcription complex, thereby preventing the formation of the active complex. These hydroxylations are directly linked to the oxygenation of the EPO-producing cell because the two non-heme iron-containing hydroxylases, which catalyze these proline and asparagine hydroxylation reactions respectively, use molecular oxygen as a substrate.35 Under

normoxic conditions, the hydroxylations and subsequent ubiquitination and interference with transcription complex formation proceed, but with hypoxia they do not. These posttranslational regulations of HIF-2α levels and activity are the key components in controlling EPO gene transcription. Components of this complex other than HIF-2α and HIF-1β include hepatocyte nuclear factor-4 (HNF-4) and the protein p300.44-46 Once hypoxia reaches the threshold that triggers EPO transcription, the resultant EPO messenger RNA is translated into the EPO glycoprotein, which is immediately secreted.31 When an individual cell is triggered to produce EPO, it does so in an all-or-nothing manner.32,47 Thus, EPO concentrations in the blood increase sharply within 2 hours after loss of blood, hemolysis, or a sudden decrease in atmospheric oxygen. The kidney cells that produce EPO are a subset of cortical interstitial cells adjacent to proximal tubules48,49 and appear to be fibroblasts50,51 (Fig 2-3). In mild anemia, small foci of the EPOproducing cells are present in the inner cortex. In moderate anemia, the EPO-producing cells are present in larger areas within the inner half of the cortex. In severe anemia, they are present throughout the renal cortex.47 These progressive increases in the areas of EPO production in the kidney correspond to increasing areas of renal cortical hypoxia that are a function of oxygen supply from the blood and local oxygen utilization by rapidly metabolizing cells, such as the adjacent proximal tubular epithelium.47,52 Thus, the rapid increase in EPO production after blood loss or hemolysis is not due to increased production by each EPO-producing cell but rather to the recruitment to active EPO production of increased numbers of cells with the potential to produce EPO.47 The number of cells actively producing EPO and the resultant plasma EPO levels increase exponentially with a linear decrease in hematocrit. This exponential increase characterizes most anemias except for those involving patients with renal disease or malignancies.53

Interaction of Erythropoietin and Erythroid Progenitor Cells EPO is carried through the blood to the marrow, where it binds to the specific transmembrane glycoprotein erythropoietin receptor (EPO-R).54 The mature, fully glycosylated EPO-R is displayed on the surface of erythroid progenitor cells at the CFU-E stage and persists through the late basophilic erythroblast stage with an average of approximately 1000 surface receptors per cell55,56 (Fig 2-2). The EPO-R is structurally homologous with receptors for numerous hematopoietic growth factors and cytokines including granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, thrombopoietin, the interleukins, growth hormone, and prolactin.57 The binding of EPO to EPO-Rs leads to three major events: 1) homodimerization and conformational alterations of EPORs, 2) initiation of intracellular signaling by the EPO-Rs, and 3) endocytosis of the EPO/EPO-R complexes, which are subsequently proteolyzed.45,55,58-61 Dimerization and structural changes of EPO-Rs after EPO binding induce both signaling and

21

Section I: Part I

OHOH P P

(A) Normoxia

pVHLⴙ Ub ligase

OHOH P P

N-OH

HIF-1␤

p300

Ubiquitinated HIF-2␣

N-OH

p300

HNF-4

Poly Ub

N-OH

OHOH P P

Proteasomal degradation

HIF-2␣ No HIF transcription complex formed

(B) Hypoxia

HIF-1␤

HIF-2␣

No ubiquitinated HIF-2␣

No proteasomal degradation

p300

HNF-4

p300

p300

p300

HIF transcription complexes

5’

3’ p3

00

⫹

Hypoxia-inducible enhancer

Promoter

EPO gene

EPO mRNA Figure 2-4. Induction of erythropoietin (EPO) gene transcription by hypoxia. (A) In cells capable of producing EPO, two components of hypoxia-inducible factor (HIF-2α and HIF-1β) are constitutively produced under normoxic conditions. However, the molecular oxygen present in the EPO-producing cells under normoxic conditions is used to hydroxylate two prolines and one asparagine in HIF-2α. The prolyl hydroxylations (P-OH) lead to recognition by von Hippel-Lindau protein (pVHL), which targets HIF-2α for polyubiquitination (Poly Ub) by ubiquitin ligase. The ubiqitinated HIF-2α is rapidly degraded in proteasomes. Asparaginyl hydroxylation (N-OH) inhibits the association of the HIF-2α with the p300 member of the HIF transcription complex, providing another means of blocking

the transcription of EPO. (B) When EPO-producing cells are hypoxic, HIF-2α is not hydroxylated and accumulates because it is not degraded by the ubiquitinproteasomal pathway. The HIF-2α forms heterodimers with HIF-1β. Because the asparagine hydroxylation of HIF-2α has not occurred when the EPO-producing cells are hypoxic, these heterodimers associate with two other components of the HIF transcription complex, hepatocyte nuclear factor-4 (HNF-4) and p300. The HIF transcription complex binds to the hypoxia-inducible enhancer 120 bp 3⬘ to the EPO polyadenylation signal and increases EPO promoter activity with a resultant increase in EPO transcription and accumulation of EPO messenger RNA accumulation.

endocytosis. The endocytosis and intracellular degradation of the EPO/EPO-R complexes appear to be the normal mechanisms for clearance of EPO from the blood.62,63 Like the other hematopoietic growth factor-cytokine receptors, EPO-Rs have no intrinsic enzyme activity,57 but they interact with several signal transduction pathways through Janus tyrosine kinase-2 (JAK2). JAK2 is physically associated with the cytoplasmic portion of EPO-Rs, where it chaperones EPO-Rs to the surface of the erythroid cell and is the initial enzyme activated by the conformational changes in the EPO-Rs produced by the binding of EPO.64,65 JAK2 is activated by phosphorylation. In turn, activated JAK2 phosphorylates tyrosines in EPORs and initiates signal transduction pathways that include signal

transduction and activator of transcription-5 (STAT5), RAS-rafMAP kinase, and phosphoinositol-3 kinase/Akt kinase (protein kinase B).45,55,58-61 In addition to activating these pathways that appear to lead to increased erythroid cell numbers, JAK2 also activates SHP-1 and SHP-2 phosphatases and suppressors of cytokine signaling-3 (SCOS-3) that control the phosphorylation state of the EPO-Rs and ultimately cease EPO-R signaling.66-68

22

Effects of Erythropoietin on Erythroid Progenitor Cells EPO has been proposed to be an inducer of proliferation and terminal differentiation, but the only effect of EPO on erythroid progenitor cells that has been well documented is the prevention of programmed death (apoptosis). Promotion of survival

Chapter 2: Red Cell Production and Kinetics

(A) Normal erythropoiesis

0.42

0.40

0.38

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(B) Elevated EPO Levels

0.58 (C) Decreased EPO

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(D) Ineffective erythropoiesis

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(E) Iron-deficient erythropoiesis HRI ⫺ 0.58

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Figure 2-5. Model of erythropoiesis based on suppression of programmed cell death (apoptosis) by erythropoietin (EPO) and heterogeneity in EPO dependence among erythroid cells. Between the (A) Normal erythropoiesis with an average survival rate of 40% in each of the EPO-dependent generations. Normal erythropoiesis produces about 250 billion new erythrocytes daily, despite the fact that a minority of all potential erythroid cells survive the EPO-dependent period. (B) Elevated EPO levels as found after acute blood loss or hemolysis increase average survival rates to 57% in each EPO-dependent generation. Daily erythrocyte production is increased three times the normal amount. (C) Decreased EPO levels as found in renal failure decrease average survival rate to 28% in each EPO-dependent generation. Daily erythrocyte production is one-third of normal.

(D) Ineffective erythropoiesis with high EPO levels increases rates of apoptosis caused by a pathologic process such as folate or vitamin B12 deficiency. High EPO levels in response to decreased erythrocyte production expand surviving cells in the early EPO-dependent generations, but the increased rates of apoptosis in the late EPO-dependent and post-EPO-dependent stages decrease daily erythrocyte production to one-third of normal. (E) Iron-deficient erythropoiesis with elevated EPO levels resulting in a similar increase to an average of 57% survival as seen in (B), but in the post-EPO-dependent period, when hemoglobin is synthesized, heme-regulated inhibitor (HRI) prevents apoptosis by inhibiting protein synthesis. The inhibited protein synthesis decreases the size of erythrocytes produced and reduces daily erythrocyte production to three-fourths of the normal numbers.

by EPO through the prevention of apoptosis has been found in several different systems, including CFU-Es from various hematopoietic tissues69-72 and in knockout mice that are either EPO null or EPO-R null.73,74 These studies all indicate that erythroid progenitor cells in CFU-E through basophilic erythroblast stages are dependent upon EPO for their survival. How EPO signaling prevents apoptosis is unknown, but one key modulator of apoptosis, the anti-apoptotic protein Bcl-XL, has been implicated in erythroid cell survival.75,76 Bcl-XL, however, does not mediate the anti-apoptotic effect of EPO on CFU-Es and proerythroblasts, but prevents the apoptosis of later stage erythroblasts.77 During the period of EPO dependence, individual erythroid cells in the same stage of differentiation from the same hematopoietic tissue display wide variation in degree of dependence on EPO for survival.78 Some of the erythroid cells need very low levels of EPO,

such as those in the plasma of patients with chronic renal failure. Other erythroid cells need very high levels of EPO, such as those in patients with acute blood loss or hemolysis. Thus, this broad spectrum of EPO requirements covers the more than 1000-fold range of EPO concentrations found in plasma under various physiologic and pathologic conditions. The mechanism responsible for this heterogeneity in EPO dependence is not known, but it does not appear to be caused by differences in either the numbers of EPO-Rs or the EPO-binding affinities of EPO-Rs in the dependent progenitors.78 A model that incorporates the wide range of plasma EPO levels and the wide heterogeneity in EPO dependence among the EPO-responsive cells of the hematopoietic tissues has been proposed to explain the regulation of erythrocyte production by EPO in various physiologic and pathologic conditions.79 In a version of this model in Fig 2-5, erythroid progenitor cells enter

23

Section I: Part I

an EPO-dependent period of differentiation (left of the dotted line in the figure) that extends from the CFU-E through the early erythroblast stages and encompassing three generations of cells. In Fig 2-5, the proportion of total cells that survive in a generation is shown immediately under the population. The surviving cells are represented by circles, each of which contains a large black dot representing an intact nucleus. The cells lost to apoptosis are shown by circles, each of which contains an X. The number of surviving cells in a generation results in twice that number for the total cells in the subsequent generation. Most cells reaching the CFU-E stage need more EPO than the low levels found in normal plasma to sustain them through the EPO-dependent period of differentiation. As a result, the approximately 250 billion erythrocytes produced daily by a normal, healthy adult are the progeny of a minority of all the erythroid progenitor cells that reach the CFU-E stage, shown in Fig 2-5(A). When blood loss, hemolysis, or decreased atmospheric oxygen is encountered, plasma EPO increases, allowing the survival of many of EPO-dependent progenitors that would die by apoptosis under normal conditions. This enhanced survival increases reticulocyte production within a few days after encountering blood loss or decreased atmospheric oxygen. The increased reticulocytosis leads to increasing erythrocyte numbers until oxygen delivery recovers to normal, and then plasma EPO levels decline toward normal. In pathologic states of decreased oxygen delivery, such as lung disease or cardiac diseases with right-toleft shunts, the persistently increased EPO levels allow greaterthan-normal survival of EPO-dependent cells such that the total number of erythrocytes is maintained in the polycythemic range. Conversely, when plasma EPO levels fall below normal because of decreased production in chronic renal disease, many erythroid progenitor cells that would survive the EPO-dependent period of differentiation under normal conditions die by apoptosis, resulting in anemia from decreased reticulocyte production, shown in Fig 2-5(C). Treatment of patients with renal failure with exogenously administered EPO rescues the erythroid progenitor cells that need normal plasma levels of EPO to survive.

Nutritional Requirements for Erythropoiesis Although erythropoiesis is finely regulated by the oxygenationEPO feedback mechanism, the erythropoietic process is frequently limited by an insufficient supply of three essential nutrients. These nutrients are folate, vitamin B12, and iron. Although all proliferating cell populations need folate and vitamin B12, the large number of erythrocytes that must be produced each day results in a large DNA synthesis requirement for erythropoiesis. Although iron also is needed by all proliferating cell populations, the erythroblasts need much more iron than any other cell type because they produce hemoglobin. When any of these three nutrients is not present in sufficient amounts for the erythroid cell population of the marrow, production of erythrocytes decreases, and anemia occurs. Through the hypoxia

24

feedback mechanism, these anemias increase EPO production,53 but increased EPO can only partially compensate for the decreased erythropoiesis caused by the specific nutrient deficiency. Administration of the deficient nutrient, however, results in resolution of anemia in each of the deficiency states. Folate deficiency results in decreased intracellular levels of all folate coenzymes in the erythroid progenitor cells. Deficiency of vitamin B12 results in trapping of folate in the methyltetrahydrofolate form, making it unavailable in the methylenetetrahydrofolate form required for thymidine synthesis or the formyltetrahydrofolate form required for purine synthesis.80 As a result, vitamin B12 deficiency, like folate deficiency, leads to decreased intracellular levels of the specific folate coenzymes needed for de novo synthesis of all of the deoxynucleotides used in DNA synthesis, except for deoxycytidine.81 The inability to synthesize DNA caused by an inadequate supply of thymidine and purines causes accumulation of erythroid progenitors in the S phase of the cell cycle, which is rapidly followed by the induction of apoptosis.82,83 The stages of erythroid differentiation that appear to be most susceptible to this apoptosis are at the end of the EPO-dependent stage and the beginning of the period of hemoglobin synthesis. EPO-induced expansion of the EPOdependent population at the CFU-E and proerythroblast stages leads to the presence of even greater numbers of these progenitor cells that subsequently undergo apoptosis just as they are beginning to produce hemoglobin.84 The resultant clinical disease is megaloblastic anemia. The prominent feature of megaloblastic anemia is ineffective erythropoiesis, shown in Fig 2-5(D). In ineffective erythropoiesis, progenitor cells in the EPO-dependent period expand in response to increased EPO levels. The number of reticulocytes formed, however, is less than normal because of the increased rates of pathologic apoptosis in the EPOdependent and post-EPO-dependent periods of differentiation. The increased death of erythroid cells in ineffective erythropoiesis is recognized clinically by increases in iron turnover, serum bilirubin, and serum lactate dehydrogenase. Erythroid progenitor cells are the greatest consumers of iron in the body. Iron uptake is mediated through transferrin receptors and tightly regulated by a complex intracellular system such that erythroid progenitor cells synthesize large amounts of hemoglobin without having an excess of iron, heme, or globins in the cells.85 Two-thirds of the body’s iron is in the hemoglobin of circulating erythrocytes, and iron deficiency almost always arises from blood loss. Two milliliters of blood contain 1 mg of iron, which is the amount normally absorbed each day from the diet. The iron absorption rate can be increased only slightly, so that chronic blood loss of just a few milliliters per day leads to iron deficiency. With iron deficiency anemia, serum EPO levels are elevated53 and the number of CFU-Es in the hematopoietic organs are increased while the numbers of erythroblasts in the marrow and the circulating reticulocytes are decreased.86 However, unlike megaloblastic anemia, ineffective erythropoiesis does not occur, and serum bilirubin and lactate dehydrogenase levels remain normal. The iron-deficient erythroid cells are

Chapter 2: Red Cell Production and Kinetics

spared from apoptosis by one of the regulators of protein synthesis, heme-regulated inhibitor (HRI).87 HRI is a protein kinase that phosphorylates eukaryotic translation initiation factor-2α (eIF-2α), thereby inhibiting translation of globin (and other proteins) in erythroblasts. When free heme is present in the erythroid cells, it binds HRI and inhibits its phosphorlylation of eIF-2α, thereby permitting active globin (and other) protein translation. Conversely, when erythroblasts are iron-deficient, they produce much less heme and HRI phosphorylation of eIF2α is uninhibited, leading to greatly decreased rates of protein synthesis. This decreased protein synthesis results in a much slower rate of erythrocyte production and microcytosis.87 The effect of iron deficiency and HRI on erythropoiesis is shown in Fig 2-5(E).

Influence of Pathologic States on Erythropoiesis The erythropoietic process can be influenced by diseases that are intrinsic to the erythropoietic cells or that secondarily affect the erythropoietic cells. Among the intrinsic diseases are several that decrease the number of erythropoietic cells. These include myelodysplasia, myeloid leukemia, aplastic anemia, pure red cell aplasia, and Fanconi anemia. Increased apoptosis of hematopoietic cells or failure to differentiate, in the case of acute leukemia, are considered the mechanisms of population reduction in these diseases. Conversely, polycythemia vera results in increased numbers of erythropoietic progenitor cells that have an increased sensitivity to EPO.88 This increased sensitivity to EPO has been found to be caused by mutations in the JAK2 kinase that is responsible for the signaling of the EPO-R.89 Indeed, the large majority of patients with polycythemia vera have a specific mutation in JAK2 kinase that keeps it in a constitutively active state. Because JAK2 also functions in the signaling of other hematopoietic growth factors, other hematopoietic lineages are involved in polycythemia vera as well as in other myeloproliferative disorders such as essential thrombocythemia and idiopathic myelofibrosis.89 In patients with polycythemia vera the EPO feedback mechanism shown in Fig 2-3 is functional as demonstrated by lower-than-normal EPO levels, but the erythroid progenitor cells in the EPO-dependent stages in polycythemia vera survive much better with the low EPO levels than do normal erythroid progenitors with normal EPO levels. Diseases that can secondarily decrease erythropoiesis include those that directly displace the hematopoietic cells in the marrow, such as metastatic neoplasms, lymphoid neoplasms, and myelofibrosis. However, the most common cause of secondary inhibition of erythropoiesis is anemia of chronic disease. This anemia occurs in chronic infections, neoplasms, and inflammatory diseases, and the prominent factor in its pathophysiology is excessive iron sequestration by macrophages. The major source of iron for erythropoiesis is macrophages, which store and process absorbed iron and recycled iron from the breakdown of

senescent erythrocytes.86 Macrophages export iron to plasma transferrin, which delivers it to the erythroblasts. The release of iron from the macrophages occurs through ferroportin, a membrane transport protein. Ferroportin also mediates the passage of iron from duodenal enterocytes into the plasma where it is carried by transferrin. The expression of ferroportin is regulated by hepcidin, a 25-amino acid hormone produced in the liver.90 Hepcidin production is induced by increased plasma iron and by cytokines produced in inflammation.90 Hepcidin binds to ferroportin on the plasma membranes of macrophages and duodenal enterocytes, thereby inducing the internalization and degradation of the ferroportin.90 Thus, increased hepcidin decreases ferroportin expression, resulting in the inhibition of iron export into the plasma by the macrophages and enterocytes. Therefore, as a result of hepcidin induction, less iron is available to erythroblasts and the erythropoietic process is suppressed. Although iron sequestration by macrophages plays a major role, other direct and indirect effects of inflammatory cytokines on the erythropoietic cells have been reported in the anemia of chronic disease. Another indirect effect is decreased EPO production by the kidneys (relative to the degree of anemia), while direct effects on erythroid cells include suppressed growth and increased apoptosis induced by inflammatory cytokines such as interferon-γ, tissue necrosis factor-α, and interleukin-1.91,92 Although the anemia of chronic disease may partially respond to high doses of exogenously administered EPO, resolution of the anemia requires effective treatment of the primary disease responsible for increased cytokine production.

Summary Erythropoiesis is a component of the larger process of hematopoiesis, in which a pluripotent HSC gives rise through proliferation and differentiation to all the mature cells of the blood and the immune system. Within the erythroid differentiation process, the rate of erythrocyte production is regulated by EPO. EPO acts by increasing the number of erythroid progenitors that can survive during a specific period when they are dependent on EPO for the prevention of apoptosis. Levels of EPO are controlled by oxygen delivery and utilization in the renal cortex, where hypoxia induces EPO production. This oxygen-EPO feedback mechanism results in finely controlled rates of erythrocyte production. This mechanism responds promptly to physiologic changes such as blood loss or changes in atmospheric oxygen. In pathologic conditions, it provides compensatory changes in the rates of erythrocyte production that partially correct the abnormal oxygen delivery to the peripheral tissues. Although the use of EPO in clinical medicine is routine for patients with the anemia of renal disease, further research is needed to understand the intracellular signaling by the EPO-R that leads to increased erythroid cell survival in disorders such as myelodysplasia and the anemia of chronic disease. Understanding of these EPO-related mechanisms as well as the roles of HRI in preventing apoptosis of

25

Section I: Part I

iron-deficient erythroblasts and hepcidin in inducing the anemia of chronic disease will facilitate treatment of these various anemias.

Disclaimer The author has disclosed no conflicts of interest.

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79. Koury MJ, Bondurant MC. Control of erythrocyte production: The roles of programmed cell death (apoptosis) and erythropoietin. Transfusion 1990;30:673-4. 80. Herbert V, Zalusky R. Interrelations of vitamin B12 and folic acid metabolism: Folic acid clearance studies. J Clin Invest 1962;41:1263-76. 81. Shane B, Stokstad ELR. Vitamin B12–folate interrelationships. Annu Rev Nutr 1985;5:115-41. 82. Koury MJ, Price JO, Hicks GG. Apoptosis in megaloblastic anemia occurs during DNA synthesis by a p53-independent, nucleosidereversible mechanism. Blood 2000;96:3249-55. 83. Koury MJ, Horne DW. Apoptosis mediates and thymidine prevents erythroblast destruction in folate deficiency anemia. Proc Natl Acad Sci U S A 1994;91:4067-71. 84. Koury MJ, Horne DW, Brown ZA, et al. Apoptosis of late stage erythroblasts in megaloblastic anemia: Association with DNA damage and macrocyte production. Blood 1997;89:4617-23. 85. Koury MJ, Ponka P. New insights into erythropoiesis: The roles of folate, vitamin B12, and iron. Annu Rev Nutr 2004;24:105-31.

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86. Kimura H, Finch CA, Adamson JW. Hematopoiesis in the rat: Quantitation of hematopoietic progenitors and the response to iron deficiency anemia. J Cell Physiol 1986;126:298-306. 87. Han AP, Yu C, Lu L, et al. Heme-regulated eIF2alpha kinase (HRI) is required for translational regulation and survival of erythroid precursors in iron deficiency. Embo J 2001;20:6909-18. 88. Eaves CJ, Eaves AC. Erythropoietin (Ep) dose-response curves for three classes of erythroid progenitors in normal human marrow and in patients with polycythemia vera. Blood 1978;52:1196-210. 89. James C, Ugo V, Casadevall N, et al. A JAK2 mutation in myeloproliferative disorders: pathogenesis and therapeutic and scientific prospects. Trends Mol Med 2005;11:546-54. 90. Ganz T. Molecular control of iron transport. J Am Soc Nephrol 2007;18:394-400. 91. Means RT Jr. Pathogenesis of the anemia of chronic disease: A cytokine-mediated anemia. Stem Cells 1995;13:32-7. 92. Weiss G. Iron and anemia of chronic disease. Kidney Int 1999; 55(Suppl 69):S12-S17.

3

Regulation of Oxygen Delivery by Red Cells and Red Cell Substitutes Christopher P. Stowell Director, Blood Transfusion Service, Massachusetts General Hospital, and Assistant Professor of Pathology, Harvard Medical School, Boston, Massachusetts, USA

The maintenance of tissue levels of O2 adequate to support aerobic respiration depends on four processes. The mechanics of breathing draw O2 into the alveolar space and expel CO2. These respiratory gases are exchanged between the alveoli and the red cells in the pulmonary capillaries followed by transport of the oxygen-rich red cells to the peripheral circulation. In the last process, O2 diffuses along a concentration gradient from the red cells, through the blood vessel wall, into the tissue, and eventually to the mitochondria, where its reduction along the respiratory chain drives the generation of energy in the form of adenosine triphosphate (ATP). The body stores of O2 are small relative to the rate of consumption and cannot be drawn down too far before weakening the concentration gradients that are key determinants of tissue oxygenation. Disruption of any one of these processes impairs cellular oxygenation; hence, they are tightly regulated by multiple, overlapping systems that operate on the systemic, regional, and tissue levels. This chapter focuses on the regulation of the last phase of this process, the transport of oxygenated red cells to the periphery and delivery of oxygen to tissues.

Regulation of Systemic Oxygen Delivery Some of the quantitative aspects of systemic oxygen dynamics are reviewed in Chapter 9, which points out that the amount of oxygen delivered is a function of the unit volume of blood flow (related to cardiac output) and the oxygen content of blood, which is determined by the concentration of hemoglobin and the degree to which it is saturated with O2. Ordinarily, the amount of oxygen delivered to the periphery exceeds what is required to sustain metabolic activity. Under resting conditions, only about 25% of all delivered O2 is consumed; hence, blood returning to the heart remains approximately 75% saturated. Even if delivery decreases because of reduced cardiac output or hemoglobin concentration,

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

or consumption increases because of exercise or fever, more oxygen can be extracted from the blood. However, at some threshold the tissues can no longer extract more oxygen and consumption becomes supply limited. To avoid reaching this point, systemic oxygen delivery is protected by regulation of the cardiac output, as well as the level and function of erythrocytic hemoglobin.1 In normovolemic anemia, decreased blood viscosity contributes to facilitated venous return with increased preload and decreased afterload.2 Anemia also results in increased synthesis of 2,3diphosphoglycerate (2,3-DPG) in the red cell, which increases the hemoglobin P50 and facilitates oxygen release. In addition, blood flow is redistributed to tissues with increased metabolic needs (eg, heart and muscle in exercise) and diverted from tissues whose activity can be reduced or whose baseline oxygen extraction ratio is low (eg, splanchnic beds). Overlapping regulatory systems sensitive to blood volume, pressure, and oxygen levels trigger hemodynamic and metabolic responses that serve to maintain blood flow and oxygen delivery to the periphery. (See Table 3-1.)

Central Nervous System Regulation of Hemodynamics The central nervous system (CNS) regulates hemodynamic responses to hypovolemia and hypotension by increasing cardiac output and vascular tone and shunting blood flow away from such tissues as the skin and gut. Cardiac output is increased because of increases in both heart rate and stroke volume in the awake patient3 but stroke volume alone in the anesthetized patient.4 This regulatory response is initiated in part by vascular mechanoreceptors sensitive to stretch. High-pressure baroreceptors are located in the carotid bodies and the aortic arch, and low-pressure receptors are found in the heart and lungs.5,6 The carotid sinus baroreceptors are located in the adventitia of the internal carotid artery, close to its juncture with the common carotid artery. Stretching of the arterial wall distorts sensory nerve endings and activates ion channels, causing membrane depolarization and the generation of an action potential proportionate to the degree of stretch. In the aortic arch, this signal transduction appears to be mediated by KCNQ K⫹ channels,7 a group of widely distributed, multimeric ion channels that play a major role in determining neuronal excitability as well as in the

29

Section I: Part I

Table 3-1. Systemic Control of Blood Flow Type of Control

Input

Effector Organ

Output

Nervous

High-pressure baroreceptors—aortic arch, carotid body Low-pressure baroreceptors—heart, lungs Oxygen sensors—carotid body, medulla

Medulla Medulla Medulla

Sympathetic/parasympathetic Sympathetic/parasympathetic Sympathetic/parasympathetic

Hormonal

Baroreceptors Baroreceptors—luminal [NaCl] Baroreceptors—aortic arch, carotid body Osmoreceptors—hypothalamus

Heart Juxtaglomerular cells of the kidney Posterior pituitary gland Posterior pituitary gland

BNP—vasodilation, natriuresis, diuresis Renin→→aldosterone—antidiuretic Vasopressin—vasoconstriction, antidiuretic Vasopressin—vasoconstriction, antidiuretic

BNP ⫽ B-type natriuretic peptide.

epileptic and arrhythmia syndromes that result from their malfunction.8 Afferent impulses from the carotid mechanoreceptors are transmitted via sensory fibers of the carotid sensory nerve that travel with the glossopharyngeal nerve and project to the nucleus of the solitary tract in the medulla.5 This nucleus also receives signals from the mechanoreceptors in the aortic arch and heart via nodose and vagal afferents, respectively. The baroreceptive neurons of the solitary tract nucleus in turn activate parasympathetic neurons of the dorsal vagal nucleus and the nucleus ambiguous. They also inhibit the sympathoexcitatory neurons of the rostral ventrolateral nucleus of the medulla.5,9 Thus, activation of the mechanoreceptors by increased stretch (increased blood volume or pressure) stimulates parasympathetic effects and inhibits sympathetic effects—resulting in decreased heart rate, vasodilation, and distribution of blood to skin and skeletal muscle. The neurons of the rostral ventrolateral nucleus are themselves sensitive to PO2, which shuts down a K⫹ channel and causes an increase in the spontaneous discharge rate.10,11 In addition, there is evidence that 1) nitric oxide (NO) acts as a neurotransmitter in the CNS, generally attenuating sympathetic tone by interacting with baroreceptor afferent inputs,12 and 2) NO from S-nitrosothiol (SNO) adducts in red cells regulates the ventilatory response to hypoxia in the nucleus of the solitary tract.13 The carotid bodies also contain oxygen sensors that detect systemic hypoxia and quickly stimulate breathing and raise blood pressure via activation of the autonomic nervous system.14 The O2 chemoreceptor tissue in the carotid body includes two cell types. Type I, or glomus, cells are of neural crest origin and produce various neurotransmitters. Type II, or sustentacular, cells play a supportive role and resemble glial cells. It is thought that the Type I cells act as the O2 sensors and trigger afferent nerve activation, which is transmitted to the CNS where it acts via autonomic control of respiratory rate and blood pressure. Although all cells respond to hypoxia, these responses usually evolve over a period of minutes to hours and require prolonged exposure to hypoxic conditions.15 The carotid body, however, responds within seconds to decreases in the arterial PO2 even as small as 20 mm Hg with excitation of the afferent nerves. This increased level of excitation is maintained if hypoxia persists and shows little adaptation.16 The identities of the molecular elements of the O2 sensor in Type I cells

30

are not clear, but candidates include several heme-containing proteins that use molecular O2, including cytochrome a3, nitric oxide synthase, heme oxygenase 2, and nicotinamide adenine dinucleotide phosphate oxidase.17-19 Type I cells also express several O2sensitive K⫹ channel proteins which are inhibited by hypoxia.20,21 There is evidence that multiple heme-containing proteins, may function as O2 sensors, each sensitive to a different dynamic range of PO2. These proteins interact with O2-sensitive K⫹ channel proteins (the “chemosome” hypothesis) to rapidly transduce the signal.22-24 Hypoxia leads to depolarization of Type I cells and release of neurotransmitters. The major neurotransmitter that mediates afferent nerve activation by hypoxia is ATP. Paradoxically, ATP also acts on the Type I cells in an autocrine fashion to inhibit the hypoxia-stimulated increase in intracellular Ca⫹⫹ concentration,25,26 and may release other inhibitory neurotransmitters as well.27,28 It has been suggested that the release of both excitatory and inhibitory neurotransmitters functions as a “push-pull” mechanism to prevent over-excitation and signal extinction of the afferent nerves. This mechanism may also explain the lack of adaptation in the presence of persistent hypoxia.29

Humoral Regulation of Blood Volume Both atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) are produced in myocytes; ANP largely in the atria and BNP in the ventricle.30 ANP is stored in perinuclear granules and released in response to increased atrial distension or pressure, as well as several neurotransmitters. BNP is not stored in granules but rather is synthesized and released episodically.31 Volume expansion or pressure overload of the ventricular wall prompts synthesis of precursor forms of BNP that are processed and released.32 Acting through receptor guanylate cyclases, these peptides have pleomorphic effects that generally contribute to peripheral vasodilation, natriuresis, and diuresis. BNP acts to inhibit the release of both antidiuretic hormone and aldosterone.33 Acting in concert with sympathetic nerve input, it also has direct effects on the kidney, stimulating glomerular filtration, natriuresis, and diuresis.34,35 In vascular smooth muscle cells, BNP, acting via the receptor guanylate cyclase and the Rho kinase system, stimulates dephosphorylation of myosin light chain, which promotes vasodilation.36

Chapter 3: Oxygen Delivery

Blood volume is also regulated directly by the kidney. The renin axis is stimulated by a baroreceptor mechanism as well as the macula densa system. The cells of the macula densa respond to decreases in luminal NaCl concentration by stimulating the secretion of renin from the juxtaglomerular cells of the kidney. The secretion of renin initiates a proteolytic cascade beginning with the cleavage of angiotensinogen from the liver to produce angiotensin I. Angiotensin I itself is cleaved by angiotensinconverting enzymes (found in the lung and other tissues) to angiotensin II, and then angiotensin III. Both angiotensins II and III stimulate the secretion of aldosterone and thereby reduce diuresis.37 The ability of the cells of the macula densa to sense NaCl levels depends on the function of the luminal Na⫹/K⫹/2Cl⫺ cotransporter, KKCC2.38,39 The affinity of this cotransporter for Cl⫺ is low; hence, uptake is very sensitive to small changes in Cl⫺ concentration. Prostaglandins have been implicated as mediators of macula-dependent renin secretion,40 partly on the basis of observations of impaired renin responses in the presence of COX-inhibitors and in COX-2 knockout mice.41-43 Within the kidney, local production of endothelin activates NO secretion, which not only affects vasomotor tone and blood flow but also promotes sodium excretion as well.44 In addition, increased intraluminal pressure is sensed by an as-yet-unidentified structure (possibly the juxtaglomerular cells themselves), which also acts through the release of prostacyclin (PGI2) to modulate renin release.45 Vascular volume is also regulated by the control of vasopressin over water metabolism. Arginine vasopressin (AVP) is synthesized as a prohormone in the magnocellular neurons in the supraoptic and paraventricular nuclei of the hypothalamus and transported to axon termini in the posterior pituitary gland.46 There, the prohormone is processed to AVP and released constitutively at low levels. The magnocellular neurons receive input from cardiovascular control centers in the medulla that receive impulses from the baroreceptors of the carotid bodies and the aortic arch. AVP secretion is stimulated when blood pressure decreases, markedly so in hypovolemic shock. The magnocellular neurons also are innervated by osmoreceptor centers in the anterior hypothalamus that trigger AVP release when plasma osmolality increases. AVP release is also stimulated by prostaglandins, angiotensin II, and norepinephrine through α1-receptors, but inhibited by NO, ANP, and norepinephrine binding to α2- and β-receptors.47,48 AVP binds to a family of receptors on a variety of tissues including vascular smooth muscle and the kidney, where it exerts pressor and antidiuretic effects, respectively.49 Binding of AVP to V1 receptors on vascular smooth muscle cells activates phospholipase C and stimulates the production of inositol triphosphate and diacyglycerol through the Gq/11 pathway.50 These second messengers in turn increase protein kinase C activity, leading to increased intracellular Ca⫹⫹ concentration and contraction of the actin-myosin complex. AVP also binds to V2 receptors on the distal convoluted tubule and collecting ducts of the kidney where, via a cyclic adenosine monophosphate (cAMP)-dependent

pathway, it leads to insertion of Aquaporin-2 water channel proteins into the membrane of the luminal surface of the collecting ducts, enhancing the reabsorption of water.51 Although not as actively regulated as the above systems, blood volume is also influenced by the balance between the hydrostatic and oncotic forces at the capillary level. The movement of water and proteins across the capillaries provides for a relatively passive, but significant, mechanism for regulating intravascular volume.

Erythropoiesis—Regulation of Oxygen Content Oxygen delivery can also be enhanced by increasing the amount of oxygen delivered. This can be accomplished quickly through red cell transfusion, or physiologically, although more slowly, through erythropoiesis. The production of erythropoietin by cortical interstitial cells of the kidney in response to hypoxia is covered in more detail in Chapter 2. Upregulation of the transcription of the erythropoietin gene occurs rapidly after the onset of hypoxia, and is mediated by hypoxia inducible factor1α (HIF), which forms a transcription factor complex with other proteins.52 Although the increase in erythropoietin production in response to hypoxia is prompt, the increase in the hematocrit is not seen for several days.Thus, this adaptation represents a longterm adjustment.

Regulation of Regional Oxygen Delivery Blood flow to different organs is tailored to their levels of metabolic activity at rest, and actively altered to meet episodic physiologic needs, such as exercise or digestion. Blood flow is controlled by a mix of intrinsic and extrinsic systems. The local, intrinsic control mechanisms that operate in all organs facilitate the appropriate matching of oxygen delivery to metabolic need, while central control mechanisms make it possible to prioritize the distribution of blood flow among different organs and to preserve flow to critical organs such as the brain and heart. (See Table 3-2.)

Coronary Circulation Blood flow through the coronary circulation is greatest in the left ventricle, about half of that level in the right ventricle, and only a quarter of the left ventricular rate in the atria. Myocardial O2 consumption can increase by about five-fold during peak work, but is matched by a comparable increase in blood flow.53 Even at rest, the myocardium extracts approximately 70% of the O2 delivered, which is much higher than any other resting tissue; thus, increased metabolic demands must be met by increased blood flow.54 In the resting state, only about half of the myocardial capillary beds are open to perfusion in the resting state55; however, in hyperemia, additional capillary beds are recruited to augment flow.56 Although the myocardial vasculature receives low-density innervation from the autonomic nervous system, blood flow is governed chiefly by intrinsic or metabolic regulatory mechanisms.57 During sympathetic stimulation of heart rate and cardiac

31

Section I: Part I

Table 3-2 Comparison of Oxygen Delivery to Selected Organs Organ or System

% of Cardiac Output

Oxygen Extraction

Control

Mechanism to Meet Demand

Distinctive Features

Coronary

5%

70%

Intrinsic

Increased flow

Flow varies in systole/diastole; O2 reserves depleted quickly

Cerebral

15%

35%

Intrinsic (dominant) and some extrinsic

Controls flow to other organs to protect itself

Constant pH is critical; responsive to CO2 levels

Pulmonary

10%

Skeletal/muscle

20%

Intrinsic and extrinsic 25-30% at rest; to 90% on demand

Intrinsic and extrinsic

Renal

20%

Intrinsic

Splanchnic

24%

CNS; organ-specific factors

Hypoxic pulmonary vasoconstriction; low pressure Arteriolar dilation; increased red cell flux through capillaries

O2 reserves depleted quickly with sustained vasocontraction High flow required

Increased flow; opening of capillary beds previously underperfused

Flow can double for several hours after a meal; intense vasoconstriction during exercise or hemorrhage

CNS ⫽ central nervous system

work, the weak sympathetic signal for vasoconstriction is overridden by intrinsic factors that increase blood flow to meet elevated O2 demands. Similarly, while vagal release of acetylcholine would be expected to stimulate vasodilation, the decreased metabolic demands of the myocardium under parasympathetic influence leads to vasoconstriction, as intrinsic systems for matching blood flow and cardiac activity supersede autonomic input. The nature of metabolic flow regulation of the coronary circulation is still unclear.58 Several models have been proposed to account for the autoregulatory behavior of the coronary circulation. It was recognized early that hypoxemia and hypercapnia both produce vasodilation, and in a synergistic manner.59 Prostaglandin-mediated vasodilation in response to low PO2 levels has been shown to play a role in epicardial arterioles, but it is probably not a major factor in the regulation of coronary blood flow in hypoxia.60,61 Hypercapnia (or the associated acidosis) has also been suggested to trigger autoregulatory responses from the arteriolar wall, or the subjacent myocardium via such mediators as adenosine, NO, or ATP-dependent K⫹ channel activity.62 However, while NO and ATP-dependent K⫹ channel activity may contribute to vasomotor tone in the resting coronary arteries, none of these three mediators are responsible for local metabolic control during exercise.57 It has also been hypothesized that release of ATP by red cells responding to hemoglobin desaturation63 or NO released from S-nitroso-hemoglobin64 might mediate a vasodilatory response in the coronary circulation. The myocardial circulation is also unique in that flow varies with the mechanical activity of the heart, with most flow occurring during diastole; during systole, blood flow through the coronary capillaries comes to a virtual standstill. Blood flow is also unevenly distributed throughout the myocardium.65 Compression forces are highest near the endomyocardium, and diminish progressively toward the epicardium.66 To compensate, the internally located arterioles are more dilated than those nearer the epicardium. One consequence of this local

32

adaptation is that the subendomyocardial arterioles have less capacity to compensate for increased myocardial activity or anemia by further vasodilating, making this tissue particularly vulnerable to ischemia. The very high rate of O2 utilization also makes the myocardium particularly sensitive to interruptions in blood flow. O2 reserves are quickly depleted and within minutes the myocardium must switch to anaerobic glycolysis—thus generating lactic acid and, as ATP stores are drawn down and not replenished, inorganic phosphate. This further impairs muscle contraction. One protective feature of the myocardial circulation in some mammals, including humans, is the existence of collateral circulation, and the ability to stimulate its growth in response to decreased blood flow.

Cerebral Circulation About 15% of the cardiac output perfuses the brain, which, at rest, extracts approximately 35% of the delivered O2. Because gray matter has a much higher rate of oxidative metabolism than white, it receives a disproportionate fraction of the blood flow. The maintenance of ion homeostasis and membrane potentials is energy intensive and consumes a large proportion of the ATP generated by the neurons, which depend on a constant supply of O2 and glucose. The O2 reserves of the brain are very limited so that even brief periods of ischemia can produce hypoxia and irreversible cell damage because glycolysis cannot meet the energy demands of the neuron. Cerebral blood flow is subject to both extrinsic and intrinsic regulation, although the latter is dominant, as is the case with the coronary circulation. Unlike other tissues, however, the brain also has the ability to control blood flow to other organs through autonomic output, and thus to protect its own blood supply. Extrinsic, autonomic innervation of resistance vessels in the brain has relatively weak effects on blood flow, and the blood-brain barrier excludes many humoral vasoactive molecules. Sympathetic input serves chiefly to shift the autoregulatory

Chapter 3: Oxygen Delivery

curve to higher pressures, thereby protecting the brain from sympathetic-driven increases in blood pressure.67 The parasympathetic system probably plays little part in autoregulation but has been implicated in ischemia and migraine.68 Cerebral resistance vessels and perivascular astrocytes both receive input directly from intrinsic CNS neurons (the neurovascular unit) located in the nucleus basalis, locus coeruleus, and raphe nucleus, which modulate vascular tone.67 The projections of these intrinsic neurons contain acetylcholine, nitric oxide synthase (NOS), norepinephrine or 5-hydroxytrypamine, depending on their source. Perivascular astrocytes respond to these transmitters, as well as glutamate, γ aminobutyric acid and ATP, and their endfeet are particularly rich in noradrenergic receptors, NOS, Ca⫹⫹-activated K⫹ channels, and purinergic receptors. These astrocytes release various vasoactive molecules such as ATP, adenosine, and NO, and exert both vasoconstrictive and vasodilatory influences via the release of 20-hydroxyeicosatetraenoic acid, or prostaglandin E2 and epoxyeicosatrienic acid, respectively.69 The level of NO appears to influence which of these vasoactive molecules is produced. Astrocytes also release K⫹ via a Ca⫹⫹-activated channel that causes hyperpolarization of smooth muscle cells and may be responsible for their relaxation, although its role in functional hyperemia is not completely understood.70,71 The autoregulatory mechanisms of the brain maintain adequate cerebral blood flow over a wide range of arterial blood pressures (between 60 and 140 mm Hg) and can adapt to even higher pressures in the face of chronic hypertension or acute sympathetic stimulation. These cerebral autoregulatory mechanisms also adapt to the considerable spatial and temporal heterogeneity of oxygen requirements72 as reflected in the wide variation in tissue PO2 and hemoglobin O2 saturation throughout the brain demonstrated by studies using blood oxygendependent functional magnetic resonance imaging.73 Increases in regional brain neural activity that increase metabolic demands trigger increased blood flow, probably by producing a temporary decrease in tissue PO2.74-76 Short-term responses to changes in cerebral PO2 appear to be mediated by O2-sensitive K⫹ channels that are reversibly inhibited by hypoxia.77,78 Gene transcription and protein synthesis may also be downregulated, the latter through inhibition of the function of eukaryotic initiation factors 1 and 2, as well as eukaryotic elongation factor 2,79-81 thereby limiting the metabolic demands of the neurons.82 Adaptation to persistent reduction in O2 supply requires changes in gene expression. Several O2-sensitive transcription factors have been shown to respond to hypoxia, leading to changes in gene expression among them HIF, nuclear factor kB, activator protein-1, and early growth response protein-1; however, the HIF system appears to play the central role in responses to changes in tissue PO2. Among the scores of HIF-regulated genes are those that affect the regulation of vascular tone, angiogenesis, erythropoiesis, cell metabolism, and pH regulation.83 The cerebral circulation is also distinctive from others in its responsiveness to levels of CO2. In fact, it appears to be even more sensitive to changes in arterial and tissue PCO2 than PO2.84

Even small increases in the PaCO2 stimulate vasodilation of cerebral arterioles.85 Hypercapnic vasodilation may be mediated through effects on tissue pH, which decreases as the PCO2 rises. Because neuronal function is markedly impaired by increased H⫹ concentration, maintenance of constant pH is a critical process. There is also evidence that hypercapnia triggers closing of ATP-sensitive K⫹ channels.86

Pulmonary Circulation In contrast to the circulation through the brain and heart, the pulmonary circulation is a low-pressure, high-flow-rate system. Pulmonary pressures and resistances are only about a fifth of those found in the systemic circulation, although the entire blood volume circulates through the lungs approximately every minute. Ordinarily, only about 500 mL of blood are in the pulmonary vasculature. However, the vasculature can accommodate much larger volumes, particularly in the pulmonary veins, without a marked increase in resistance, thereby protecting the lungs from edema in the event of reduced left ventricular function or volume overload.87 Unlike the coronary and cerebral circulations, blood flow through the lungs is affected by both extrinsic and intrinsic factors and is highly responsive to its environment. Sympathetic innervation increases pulmonary vascular tone, although parasympathetic stimulation has little effect.88 Some regulatory molecules also increase vascular tone, including angiotensin, serotonin, and prostaglandin F. Others, such as bradykinin and prostaglandin E1, reduce pulmonary resistance. Hypoxic pulmonary vasoconstriction is a paradoxical but distinctive feature of the pulmonary vasculature.89 Precapillary arterioles respond to the relative hypoxia of underventilated regions of the lung by constricting, thereby reducing flow and diverting it to better oxygenated alveoli. Pulmonary hypertension is prevented by the capacity of other vascular beds to absorb the diverted volume; however, widespread hypoxia may result in pulmonary hypertension.90 The mechanism for hypoxic pulmonary vasoconstriction is not completely understood. Hypoxia has been shown to stimulate a small increase in the intracellular concentration of Ca⫹⫹ in the smooth muscles of the pulmonary arteries. The effect of these small changes in Ca⫹⫹ on smooth muscle actin-myosin contraction may be enhanced by “sensitizers” such as the Rho kinase signal transduction system.89,91 It has also been observed that under conditions of hypoxia, NO consumption by red cells increases because of formation of hemoglobin Fe⫹⫹NO, which may potentiate other vasoconstrictive influences operative in this pulmonary reflex.92 The pulmonary vasoconstrictive response to hypoxia is enhanced by metabolic acidosis93 but muted by metabolic and respiratory alkalosis. This response is also decreased by various medications, including nitroprusside and some inhalational anesthetics, and in turn produces a decrease in the PaCO2.94 Circulation of blood through the lungs serves other purposes in addition to respiratory gas exchange with the alveoli and metabolic support of lung tissue. The lungs are also the site of the removal or inactivation of various vasoactive molecules

33

Section I: Part I

(eg, vasopressin, norepinephrine, acetylcholine), mediators of inflammation (eg, bradykinin, leukotrienes, prostaglandins E and F), and some medications (eg, propanolol, lidocaine, fentanyl) that in turn may have effects on vascular tone and blood flow. In addition, circulation of blood through the pulmonary bed serves to filter the venous return before its distribution through the arterial tree.

Skeletal Muscle Circulation At rest, the skeletal muscles of the body receive about 20% of the cardiac output. Skeletal muscle is heterogeneous, consisting of phasic fibers (glycolytic, rapid twitch) and tonic fibers (oxidative, postural). The majority of blood flow goes to the tonic fibers when the body is at rest, which is reflected in the higher density of the capillary beds around these muscle bundles. During exercise, however, the increased metabolic demands of the phasic fibers may be met with up to a 20-fold increase in blood flow.95,96 Because most of the capillary beds are perfused at rest, increased flow is achieved through arteriolar dilation and increased red cell flux through the capillaries.97-99 The hematocrit in the capillaries may rise from 10% to 15% at rest to 40% to 50% during hyperemia.99,100 Given the striking ability to increase flow to actively exercising muscle, central cardiovascular control is required to prevent systemic hypotension and inadequate flow to other tissues.101,102 Muscle can also extract more O2, increasing from a baseline oxygen extraction ratio of 25% to 30% to as much as 90%. As is the case for the myocardium, blood flow in skeletal muscle is episodically interrupted by contraction and compression of the arterial vasculature, although red cells are still present in the capillaries during contraction and oxygen extraction continues, albeit at a reduced rate.103 During periods of relaxation, the dilation of the arterioles and the opening of capillary beds also compensate for interruptions in flow. Indeed, there is evidence suggesting that contraction can activate a mechanosensitive response by the vascular endothelium or smooth muscle that stimulates vasodilation.104,105 When contraction is sustained, however, tissue O2 is quickly depleted, anaerobic glycolysis generates lactic acid, and ischemic pain is experienced. Unlike the myocardium, blood flow to skeletal muscle is controlled by both extrinsic and intrinsic factors.106 The arterioles in skeletal muscle are densely innervated with sympathetic nerves that maintain vascular tone and a tonic state of partial vasoconstriction. Arteriolar vasomotor tone is controlled centrally via reflex changes in autonomic output accompanying volition to exercise and in response to the arterial baroreflex. Additional sympathetic stimulation can produce greater vasoconstriction. In exercise, however, central autonomic sympathetic signals are overridden by intrinsic cues that trigger vasodilation. This occurs despite the fact that skeletal muscle also invokes the exercise pressor reflex, which augments blood pressure and heart rate through increased sympathetic outflow from the brainstem.107 Mechanical108 and/or chemical muscle receptors stimulated by muscle activity relay impulses to the dorsal horn of the spinal cord.109 These impulses project onto many of the same nuclei in

34

the medulla that mediate the baroreflex pathway, among them the solitary tract nucleus and the rostral venterolateral nucleus.110 Two other mechanisms have been implicated in the control of sympathetic output during exercise. Stimulation of the otoliths and vestibular system of the inner ear (by motion for example) enhances sympathetic activity of skeletal muscle and has been shown to produce vasoconstriction in the muscles of the forearm and calf.111,112 In addition, elevated or reduced muscle temperature, respectively, increase or decrease sympathetic outflow during forearm exercise.113,114

Renal Circulation The kidneys receive about 20% of the cardiac output, a volume that considerably exceeds the flow required for perfusion. In part, the high flow reflects the metabolic demands of the kidney. Tubular readsorption of sodium is an energy-intensive activity, consuming large amounts of O2. In addition, the kidneys require high flow to maintain hydrostatic pressure, which is the driving force for glomerular filtration. Renal blood flow is autoregulated to maintain a relatively constant rate of glomerular filtration over a wide range of arterial pressures by balancing the tone of the afferent and efferent arterioles serving the glomeruli. Two intrinsic systems interact to control blood flow through the kidneys.115 The smooth muscle of the renal afferent arterioles responds to systemic arterial pressure by constricting when pressures increase and dilating as pressures decrease, ensuring constant blood flow. This myogenic response functions rather slowly, and does not compensate for fluctuations in systemic pressures of short duration (seconds).116 The tubuloglomerular feedback mechanism is based on the macula densa responding to changes in blood flow with the release of vasoactive mediators, including adenosine, ATP, and NO, from the juxtaglomerular apparatus, in addition to renin as described above.117-119 These vasoactive mediators affect local flow.

Splanchnic Circulation The circulation to the organs of the splanchnic bed is much more subject to central control than the circulation in other areas of the body. This circulation is characterized by dual control by organ-specific factors as well as the CNS. The arterioles of the splanchnic organs are particularly well innervated by sympathetic nerves but are also highly sensitive to the vasoconstrictors vasopressin and angiotensin II. These signals are capable of triggering intense vasconstriction and reduction of flow under circumstances when blood must be diverted to other organs, as occurs in exercise or hemorrhage. In the absence of such demands, blood flow to the abdominal organs responds to local cues. Following meals, the metabolic needs of the digestive organs to support the secretion of acids and digestive enzymes or the adsorption and transport of nutrients, trigger increased blood flow to these tissues. This blood flow can double over a period of several hours following meals. The increased flow is largely achieved by opening capillary beds that were previously underperfused.120 Gastrointestinal hormones and neuropeptides

Chapter 3: Oxygen Delivery

released by cholinergic stimulation play a role in stimulating postprandial blood flow as do local mediators, including prostaglandins,121 adenosine,122 and NO.123 Long chain fatty acids stimulate hyperemia of the proximal small intestine,124 while bile acids evoke increased blood flow to the distal ileum.125 Glucose has the most marked hyperemic effect of any of the nutrient stimulators of blood flow.126 The liver receives about 75% of its blood supply through the portal vein, which provides relatively deoxygenated blood. The remainder, which comes from the hepatic artery, is comparatively oxygen-rich. Blood flow from these two sources is controlled reciprocally to prevent congestion of the hepatic sinusoids, which could cause excessive filtration of water and plasma proteins through their highly permeable barriers.127 A hyperdynamic splanchnic circulation with extensive portal-systemic shunting is typical of cirrhotic patients and plays a role in the development portal hypertension. The process is thought to be initiated by arterial vasodilation and has been related to NO overproduction.128

Regulation of Oxygen Delivery in the Microcirculation

tissue so there is no gradient to drive O2 into the tissues at that level.133 The arterioles also serve as a source of O2 to deoxygenated red cells flowing through capillaries135,136 and the capillaries themselves exchange O2 as well.136-138 The extensive diffusion of O2 among all of these elements of the microvascular unit is thought to be the basis for relatively homogeneous tissue oxygenation despite the considerable heterogeneity of blood flow through the capillaries.139 Under unstressed conditions, there is considerable temporal and spatial variability in blood flow through the capillary beds of the microcirculation. Depending on the tissue, only a quarter to a third of the capillaries are perfused at any time. When metabolic demands increase, however, a greater number of capillaries are perfused, increasing the effective area for O2 exchange. Blood flow is actively regulated at the level of the resistance vessels as well as by changes in the vascular tone of the microvascular unit. Vasodilation is associated with decreased O2 consumption by the arterial wall, which reflects decreased demand by smooth muscle, and thus a decreased O2 gradient across the arterial wall.130 More arterial O2 is therefore available to the adjacent tissue.130,140-143 A direct relation exists between intravascular PO2, arterial wall oxygen consumption, and microvascular tone.140,142,143

Regulation of Microcirculatory Blood Flow The Microcirculation The primary function of the microcirculation is to serve as a platform for the exchange of respiratory gases, nutrients, by-products of metabolic activity, and heat. The microvascular unit is composed of a reticulum of blood vessels with diameters less than 250 microns that lie between the arteries and the veins.1 These beds of small vessels are dense in tissues with high metabolic activity and relatively sparse in tissues whose energy demands are modest. The arterioles arborize into a network of vessels that progressively decrease in caliber to form the terminal arterioles, with diameters of around 10 microns. The capillaries, which consist of a single layer of epithelial cells, have diameters of 3 to 10 microns, and drain into the postcapillary venules, and then to the venules. Oxygen is delivered to the tissues by both convective and diffusive forces. Convection (blood flow) brings oxygenated blood to the microvascular unit. As oxygen is transferred from the incoming blood to the tissue, a longitudinal O2 concentration gradient is created parallel to the direction of flow as more O2 is extracted from the blood.129 A second, radial, O2 concentration gradient exists between the blood and the tissue surrounding the vessel, which is the driving force for O2 diffusion into the tissue.130-132 However, oxygen exchange in the microvascular unit involves more than the simple transfer of O2 from red cell to tissue. Most O2 is extracted from the blood at the level of the arterioles, particularly in skeletal muscle, with much of it being consumed by the arteriolar wall132 to support smooth muscle activity and endothelial cell metabolism, including synthesis of NO, which requires molecular O2.133,134 The O2 concentration in the capillaries is generally near equilibrium with the surrounding

Blood flow through the microcirculation is controlled largely by intrinsic mechanisms, including pressure flow autoregulation, active hyperemia, and reactive hyperemia.144 Pressure flow autoregulation refers to the ability of a tissue to maintain relatively constant blood flow over a wide range of systemic arterial blood pressures, vasodilating in response to elevated pressures and contracting when mean arterial pressure decreases. The effectiveness of this pressure-activated response is variable, but is generally robust in metabolically active tissues that must protect their oxygen supply. Active hyperemia is the ability of a tissue to meet metabolic demand by varying blood flow and the number of perfused capillaries, and is characteristic of tissue with episodic energy demands, such as skeletal muscle and the gastrointestinal tract.145 Reactive hyperemia is the transient increase in blood flow, above baseline levels, which occurs after release of blood flow occlusion. The period of reactive hyperemia is usually proportional to the period of reduced flow, presumably reflecting the intensity of the hypoxic driver. All of these autoregulatory responses have been attributed to myogenic or metabolic mechanisms, although elements of both probably contribute to all of these phenomena. The myogenic theory is based on the observation that vascular smooth muscle contracts when the tension or stretch of the arteriolar wall is increased and relaxes when tension drops. Smooth muscle cells respond to increased intraluminal pressure/ stretch by depolarizing. This depolarization leads to activation of L-type voltage gated Ca⫹⫹ channels, Ca⫹⫹-dependent activation of myosin light chain kinase, and actomyosin contraction.146 This intrinsic, myogenic response explains pressure flow autoregulation and reactive hyperemia, but cannot account for active hyperemia, or responses to increased metabolic demand.

35

Section I: Part I

The metabolic theory is based on the coupling of flow to the level of O2 consumption of the tissue, and the observation that many metabolic by-products, such as CO2, O2, K⫹, H⫹ and adenosine, have vasoactive effects. According to this paradigm, tissue PO2 levels are read by an oxygen sensor of unknown identity. This sensor modulates the release of mediators of vascular tone including endothelin, NO, prostaglandins, and others. The sensor might be in the tissue itself, although the very rapid adjustments to flow and its sensitivity to intravascular PO2 are not entirely consistent with this location. It has also been suggested that the vessel walls—particularly the arterioles, which are the sites of O2 consumption and transfer—and/or red cells may serve as the sensors.147 Endothelial cells play a key role in integrating and conducting local vasoregulatory stimuli.148 These cells act as signal transducers in their own right (eg, releasing NO in response to changes in shear stress)149 but also serve as conduits to communicate signals along blood vessels through intercellular connections formed with adjacent endothelial cells.150 Gap junction channels composed of connexins link endothelial cells to one another as well as to smooth muscle cells.151 Six connexin subunits, proteins with four transmembrane-spanning domains, combine to form a hemichannel. Alignment of hemichannels on adjacent cells produces a complete gap junction. Through these connections, endothelial cells transmit signals as far as 1 mm, reaching resistance arterioles and influencing vasoactivity,152,153 thereby accommodating increased flow through one capillary bed without diverting blood from another.148 This property is called the conducted vasomotor response. Endothelial cells exert their vasoregulatory effects through at least four mediators: endothelin, NO, prostaglandins, and endothelium-derived hyperpolarizing factor.

Endothelin Regulation of Microcirculatory Blood Flow Endothelin is a vasoconstrictor (usually) secreted by endothelial cells in response to a wide variety of signals.154 Endothelial cells synthesize three isoforms of endothelin, of which endothelin-1 (ET-1) is the most important in terms of effect on vascular tone.155 It is present in particularly large quantities in the pulmonary vasculature where its release is stimulated by hypoxia, but it is found throughout the vascular tree.156 Endothelin is synthesized as preproendothelin-1 and then processed to bigET-1 and ET-1 by ET-converting enzymes, a family of multiple metalloproteases.157 ET-1 is secreted at low levels constitutively, primarily toward the basolateral side of the endothelial cells, and so acts principally as a local mediator rather than as a circulating hormone. ET-1 secretion is stimulated by vasoconstrictors (angiotensin II, vasopressin, norepinephrine), thrombin, several inflammatory cytokines, and physicochemical factors (low shear stress, hypoxia). Its release is inhibited by vasodilators such as NO, prostaglandins E2 and I2, ANP, BNP, and bradykinin, as well as high levels of shear stress.158 ET-1 binds to one of two different G protein coupled receptor types found on the smooth muscle of blood vessels, the atrium,

36

and the ventricle. Binding of ET-1 to endothelin A receptors stimulates vasoconstriction via several signal transduction pathways.159 Activation of the phospholipase C and 1,4,5-inositol triphosphate pathways elevates intracellular Ca⫹⫹ concentration, which stimulates myosin light chain phosphorylation and contraction. ET-1 binding also inhibits myosin light chain phosphatase through activation of the Rho/Rho kinase pathway. Finally, ET-1 enhances phosphorylation of thin-filament associated protein through activation of c-Src, Janus tyrosine kinase, and protein tyrosine kinase, which stimulates extracellular signal-regulated kinases 1 and 2 (ERK1/2).160 Endothelin B receptors also mediate contraction of smooth muscle; however, they are found predominantly on endothelial cells.158 When ET-1 binds to B receptors on endothelial cells, it stimulates release of NO and prostacyclin and produces a vasodilatory response in the adjacent smooth muscle. Exogenous endothelin administered to rats has been shown to have an initial vasodilatory (hypotensive) effect followed by a prolonged vasoconstrictive (hypertensive) effect, reflecting the dual nature of the A and B receptors.161 ET-1 also stimulates production of vascular endothelial growth factor and potentiates the effects of platelet-derived growth factor and transforming growth factor-β.

Nitric Oxide Regulation of Microcirculatory Blood Flow NO, or endothelium-derived relaxing factor, is synthesized from L-arginine and molecular oxygen by the enzyme NOS in a number of tissues but most notably in the vascular endothelium. The three isoforms of NOS (endothelial, inducible, and neuronal) are all activated in response to several mediators, including acetylcholine, bradykinin, ATP, and adenosine diphosphate (ADP), via G protein coupled receptors on the endothelial cell membrane that increase the level of intracellular Ca⫹⫹. NOS is a dimeric enzyme with a catalytic heme group and a zinc-thiolate domain, which is primarily structural.162 It is localized principally in the caveolae of the plasmalemma and is inhibited by the major scaffold protein, caveolin-1, to which it is bound. When intracellular levels of Ca⫹⫹ increase, calcium binds to calmodulin, which in turn displaces NOS and permits it to become catalytically active.163 In the vessel wall, NO released by endothelial cells diffuses to smooth muscle where it binds to guanylate cyclase coupled receptors and stimulates cyclic guanosine monophosphate (cGMP) synthesis.164,165 The increase in cGMP level activates protein kinase G and produces muscle relaxation by opening K⫹ channels and/or decreasing the responsiveness of the contractile machinery to Ca⫹⫹,166 as well as modulating mitochondrial respiration.167 Most vascular endothelial cells constitutively secrete low levels of NO, with the notable exceptions of the cerebral and coronary vessels. In response to hypoxia, NO released by endothelial cells produces vasodilation, which results in redistribution of blood flow and the opening of more capillary beds.168 Another factor influencing the production of NO (and endothelin) is shear stress, a property of a moving fluid that is influenced directly by its viscosity. Endothelial cells respond to decreases in shear stress

Chapter 3: Oxygen Delivery

that occurs because of reduction of either flow velocity or blood viscosity (eg, anemia). In response to reduced shear stress, endothelial cells downregulate NO production and release, and increase endothelin release, resulting in vasoconstriction.169 In animal models of hemorrhagic shock or isovolemic hemodilution, restoration of mean arterial pressure and capillary perfusion was better achieved with asanguinous fluids with higher viscosity compared to those with lower viscosity.170,171 These effects were shown to be mediated by NO production based on direct measurement of NO by microelectrode and their abrogation by the presence of the NOS inhibitor N-nitro-L-arginine methyl ester, or in NOS-deficient mice.172 In the same animal systems, equivalent restoration of microvascular flow was achieved with red cells whether they contained oxyhemoglobin or methemoglobin. These results suggest that the maintenance of blood viscosity, rather than oxygen delivery, was primarily responsible for successful maintenance of tissue perfusion.173,174 NO has wide-ranging effects beyond the vascular wall. In addition to its influence on vascular smooth muscle, NO also modulates systemic hemodynamic responses to hypovolemia, anemia, and hypoxia.175 The vascular-dependent effects of NO on the heart include regulation of coronary tone, thromobogenicity, and angiogenesis. In addition, NO affects myocardial cell excitationcontraction coupling, mitochondrial respiration, and responses to autonomic input. Low levels of NO have a positive inotropic effect, while high levels have a negative inotropic effect. Negative chronotropic effects are mediated through the neuronal form of NOS in the cardiac ganglia, and the endothelial form in the myocardium.175 NO released into the vascular lumen inhibits platelet aggregation and adhesion as well as leukocyte adhesion to the endothelium by interfering with the function of the CD11/CD18.162,176

Prostaglandin Regulation of Microcirculatory Blood Flow Prostaglandins are another set of molecular signals that endothelial cells release to modulate the activity of vascular smooth muscle. Arachidonic acid is released from the plasma membrane through the action of phospholipase A2 and then metabolized to various eicosanoids by COX and other enzymes.177 The COX-1 isoform is expressed in endothelial cells of the vascular wall and is responsible for a key step in the pathways that lead to the synthesis of both vasodilators [such as prostacyclin (PGI2), prostaglandin E2 (PGE2), and prostaglandin D] and vasoconstrictors (such as prostaglandin F2 and thromboxane). The major products in endothelial cells, however, are the vasodilators PGI2 and PGE2. COX-2 is an inducible form of the enzyme that is also expressed by endothelial cells in inflammatory states and, in vitro, by prolonged periods (hours) of shear stress, stretch, hypoxia, or exposure to inflammatory cytokines, growth factors, or ET-1. Expression of COX-2 in response to these stimuli also leads to secretion of PGI2; however, it is not clear that there is a role for COX-2 in unstressed conditions.178 PGI2 diffuses from the endothelial cell to the smooth muscle. The surface membrane of the smooth muscle cell expresses

a family of eicosanoid receptors.179 The IP and EP2 receptors for PGI2 and PGE2, respectively, mediate vasodilation, whereas the TP receptors for thromboxane produce vasoconstriction. PGI2 binding to the G-protein-coupled IP receptor activates adenylate kinase and increases cAMP levels. cAMP stimulates protein kinase A activity, which opens K⫹ channels and causes membrane hyperpolarization; this, in turn, closes voltagesensitive Ca⫹⫹ channels. The decrease in intracellular Ca⫹⫹ concentration results in relaxation of the smooth muscle cell and vasodilation.166 PGI2 release is stimulated by bradykinin and hypoxia.178 Conversely, hyperoxia has been shown to inhibit synthesis and release of vasodilator prostaglandins from endothelial cells and permit vasoconstriction, an effect that is independent of NO.180

Endothelium-Derived Hyperpolarizing Factor in the Regulation of Microcirculatory Blood Flow Endothelium-derived hyperpolarizing factor (EDHF) is a term used to define an as-yet-unidentified molecule (or process) that originates with the endothelial cell and produces smooth muscle relaxation, independently of NO or prostaglandins.152 EDHF causes hyperpolarization of the smooth muscle membrane by activating K⫹ channels that, in turn, close voltage-sensitive Ca⫹⫹ channels and elicit relaxation, as described for NO and PGI2. The mechanism whereby the endothelial cell produces this hyperpolarization of the smooth muscle cell membrane has not been completely worked out. The one step of the process that is well understood is the response of endothelial cells to agonists such as ATP. ATP increases the concentration of intracellular Ca⫹⫹ via activation of phospholipase C and inositol triphosphate-gated Ca⫹⫹ channels.181 Several mechanisms have been proposed to explain how endothelial cells induce hyperpolarization of the smooth muscle cell membrane. One proposal is that the increase in endothelial cell intracellular Ca⫹⫹ leads to the synthesis and release of epoxyeicosatrienoic acids that open large-conductance, calcium-activated K⫹ channels in the smooth muscle membrane, causing hyperpolarization.182,183 K⫹ itself, released from activated endothelial cells, may activate the inwardly rectifying K⫹ channel (Kir) or Na/K ATPase in the smooth muscle cell, producing hyperpolarization.184,185 It has also been suggested that the gap junctions between endothelial cells and smooth muscle cells either transmit the EDHF or propagate the hyperpolarizing current from cell to cell.186,187 Finally, H2O2 (produced by superoxide dismutase from the superoxide ion generated by endothelial cell NO) has been shown to activate several K⫹ channels and elicit hyperpolarization.188 The physiologic role for EDHF is not completely understood.152 EDHF-like dilations have been demonstrated in a wide variety of arteries and arterioles in vitro and in vivo. It is particularly prominent in smaller vessels, and may even supersede NO in some tissue beds. These observations suggest that EDHF may be a significant factor in regulating flow through the microcirculation. EDHF may also play a role in conducted vasomotor response, a property of microcirculatory beds that is

37

Section I: Part I

Neurotransmitters

O2 CO2 NO

Vasoactive peptides

Nucleotides

Growth factors

Cytokines

Hormones

Mechanical stimuli

Endothelial cell endothelin NO prostaglandins EDHF

Other endothelial cells

Accessory cells Smooth muscle cells

Nerve termini

retained even when the production of prostaglandin and NO are inhibited.189,190

Coordination of Endothelial Control Mechanisms Endothelial cells play an integrative role in the regulation of blood flow and the microvascular level (Fig 3-1). They are the recipients of myriad inputs including neurotransmitters, growth factors, hormones, vasoactive peptides, respiratory gases, nucleotides, and cytokines, as well as mechanical stimuli such as shear stress, stretch, and gravity. They are in physical contact with other endothelial cells, smooth muscle cells, nerve endings and, in some tissues, accessory cells such as astrocytes in the CNS. The endothelial cells also send out signals of their own, including endothelin, NO, prostaglandins, EDHF, adenosine, K⫹, and others. The endothelial cell integrates all of this input and translates it into a coherent response that functions to adjust vascular tone. The relative contribution of the four main endothelial signals is not uniform through the vascular tree; NO serves as the major vasodilator in conduit arteries and EDHF is the major vasodilator in arterioles.191 In addition, NO tonically inhibits EDHF responses as well as prostaglandin output. These complex interactions provide for a stable but responsive platform for oxygen delivery in the dynamic environment of the microcirculation.

The Red Cell as a Regulator of Microcirculatory Blood Flow It has been hypothesized that the red cell, or the hemoglobin therein, is a regulator of hypoxic vasodilation.192 As hemoglobin deoxygenates, it undergoes a conformational change during the transition from the relaxed (R) to the tense (T) state; hence, it

38

Figure 3-1. Endothelial cells receive inputs from various local and systemic sources, which they coordinate into a cohesive response with outputs to the smooth muscle cells of the vascular wall, other endothelial cells, and in some cases, nerve termini and other accessory cells. They play a central role in the regulation of blood flow and oxygen delivery through the microcirculation. NO ⫽ nitric oxide; EDHF ⫽ endothelium-derived hyperpolarizing factor.

could function as an oxygen sensor in the physiologically relevant PO2 range. Several models have been proposed to explain how the conformational change accompanying hemoglobin deoxygenation could be translated into a signal for vasodilation. One model couples the R-T transition to release of ATP from the red cell, which then binds to purinergic receptors on endothelial cells, stimulating a vasodilatory response.193-196 The model is supported by observations of increased levels of ATP in venous blood after hypoxia, the secretion of ATP from hypoxic red cells in vitro, the effect of ATP on vasomotor tone, and the retrograde propagation of the vasodilatory signal from the capillaries to the precapillary arterioles.194 The conformation-induced release of ATP from red cells is mediated by the cystic fibrosis transmembrane conductance regulator.197 The second set of models proposes that the release of NO from the red cell in response to hemoglobin deoxygenation and the R-T transition is responsible for the vasodilatory effect. One mechanism of vasodilation is based on the release of NO from S-nitrosolated hemoglobin as it deoxygenates.198-200 Although free NO has a very short half-life, NO adducts to cysteine thiols in proteins, while readily reversible, are quite stable. The S-nitrosothiol adducts can exchange with the free NO pool or other SNO adducts (transnitrosation) and in that manner can be transported far from the site of origin.201,202 NO binds to the heme group of deoxygenated hemoglobin, but transfers to the β-93 cysteine upon reoxygenation202 and has been shown to be released in hypoxia.203 The anion exchange 1 macrocomplex may facilitate the exchange of gases, including NO, across the red cell membrane.204 A number of experimental observations of the enhancement of hypoxic vasodilation in the presence of red cells have also been reported, consistent with a role in regulating blood flow.165,194,205-206

Chapter 3: Oxygen Delivery

A second mechanism to explain NO release from red cells is based on the observation that hemoglobin has an intrinsic, allosterically regulated enzymatic activity that reduces nitrite 207-209 anion (NO⫺ Deoxyhemoglobin has more unli2 ) to NO. ganded heme groups than the oxygenated form and can bind more NO⫺ 2 ; however, the reduction rate constants are higher for oxyhemoglobin-mediated reduction. Maximal NO⫺ 2 reduction therefore occurs where there is a mix of oxygenated and deoxygenated heme groups in the same molecule, which occurs around the P50.209 The reaction consumes a proton, hence hypoxia (or hemoglobin deoxygenation to levels of 40% to 60%; ie, around the P50) and acidosis would be expected to favor the generation of NO by this route. Exogenous NO2⫺ has been shown to stimulate vasodilation in model systems,207-209 experimental animals, and humans.210-213 It is not clear if the NO⫺ 2 pool operates as an independent source of NO, or in fact acts as a source of Fe bound NO, which is then transferred to β-93 cysteine and eventually released from the red cell.198,214

The Microcirculation in Disease As pointed out in Chapter 9, there is a critical point at which the rate of oxygen delivery is no longer adequate to support metabolic needs (oxygen consumption). It has been shown in stressed animals (eg, inflammation, sepsis) that this point occurs at higher ratios of O2 delivered to O2 consumed, even when the latter is not elevated, suggesting that the ability to extract O2 from the blood is impaired in these pathologic states. Inadequate tissue oxygenation when gross oxygen delivery is adequate may reflect disordered microcirculation.215 Shunting of O2 from arterioles to venules has been described in sepsis and systemic inflammatory states, as has reduced tissue oxygenation when flow rates are high and red cell transit times through the arterioles and capillaries are too brief for effective O2 off-loading.216 Shunting of O2-rich blood around capillary beds also occurs when there is a decrease in the fraction of capillaries perfused, as has been demonstrated to occur in models of sepsis217-220 and shock,221 with increased O2 extraction by those capillaries that do remain open.222 It has been suggested that the increased heterogeneity (maldistribution) of blood flow and resulting tissue hypoxia that occurs in sepsis and systemic inflammatory states is an early step leading to organ failure.221,223,224227 As a result, several approaches to microvascular resuscitation have been advocated combining volume replacement, inotropes, vasodilating drugs, and restrained used of vasopressors.228 NO and prostacyclin in particular have been studied in human sepsis, and these vasodilators have been found to achieve some improvement in tissue perfusion and oxygen extraction.229,230 Capillary underperfusion in disease is thought to be the result of both vessel occlusion and impaired regulation of blood flow. One factor contributing to capillary occlusion is endothelial cell edema, which can be seen in sepsis.231 In addition, the activated and immature leukocytes characteristic of systemic inflammation are less malleable and may mechanically occlude capillaries. Or, the leukocytes may interact with upregulated adhesion molecules on endothelial cell membranes, impeding

their progress through the microcirculation.232 Red cells also exhibit reduced deformability in sepsis233-235 perhaps related to overproduction of NO.234 They also bind to endotoxin-exposed endothelial cells in vitro, suggesting that they, too, might occlude the microvasculature.236-238 Finally, disseminated intravascular coagulation could also occlude vessels with microthrombi.239 Arteriolar dysfunction also contributes to impaired blood distribution through the capillary beds in pathologic states. Arterioles exhibit abnormal responses to vasoregulatory signals in animal models of sepsis.240,241 In addition, it has been shown that communication between endothelial cells is disrupted by lipopolysaccharide in vitro, and in animal model systems.241-244 Impaired intercellular communication may be mediated by abnormal tyrosine phosphorylation of connexin 43, one of the gap junction proteins.245,246 Excess NO production seen in inflammatory states is associated with endothelial cell and smooth muscle cell injury and may be responsible for defective arteriolar responses to regulatory cues, which results in the abnormal distribution of blood flow seen in these pathologic conditions.247

Red Cell Transfusion and the Microcirculation The effects of red cell transfusion on the microcirculation are just beginning to be understood. Direct assessment of microvascular function in patients is difficult, although minimally invasive techniques are becoming more widely available for this application. The analytic tools that have been used in the clinical setting include the O2 microelectrode, near infrared spectroscopy (NIRS), orthogonal polarizing spectroscopy (OPS), and blood oxygen-dependent function magnetic resonance imaging (BOLDfMRI).248 The use of these techniques to assess the impact of red cell transfusion in humans has been limited.

PO2 Microelectrodes Direct measurements of tissue PO2 by microelectrode used in an observational study to assess brain tissue oxygenation in patients with subarachnoid hemorrhage or traumatic brain injury showed that the transfusion of one unit of Red Blood Cells (RBCs) increased tissue PO2 without changing the cerebral perfusion pressure.249 However, transfusion of one or two RBC units to patients after coronary artery bypass grafting did not raise the PO2 in the deltoid muscle as measured by microelectrode.250 Near Infrared Spectroscopy NIRS measures the degree of hemoglobin deoxygenation in the blood vessels in the sampled tissue and has been used widely as a monitoring tool in trauma,251,252 sepsis,253,254 brain injury and surgery,255,256 and cardiac surgery.257-259 NIRS has also been used to investigate the impact of blood loss on tissue oxygenation. Donation of 450 mL of whole blood was found to be associated with only a small decrease in cerebral blood oxygen saturation.260 During removal of 740 mL of whole blood from research subjects, there was a correlation between O2 saturation levels measured in the cerebrum and calf muscle with the volume of

39

Section I: Part I

blood loss.261 Similar results were obtained in acute normovolemic hemodilution.262 A few studies have investigated the effect of RBC transfusion on tissue oxygenation as assessed by NIRS. In a study in the neonatal intensive care unit (ICU), preterm neonates with symptomatic anemia were found to have higher fractional oxygen extraction, as determined by measurement of cerebral cortex tissue hemoglobin saturation by NIRS. When they received RBC transfusions, these infants showed a decrease in oxygen extraction to the levels seen in asymptomatic, anemic infants, and nonanemic infants.263 NIRS monitoring of low-birthweight infants showed improvement in cerebral cortex oxygenation following RBC transfusion.264 Both tissue oxygenation and cerebral blood flow improved when anemic, preterm infants received RBCs.265 Intraoperative transfusion of 84 RBC units to 29 adult patients were monitored by NIRS. RBC transfusion was associated with improved cerebral and peripheral (calf muscle) oxygen saturation readings that correlated with the increase in hemoglobin level.266 Decreases in cerebral blood oxygen saturation were noted in a high proportion of elderly patients undergoing abdominal surgery that correlated with the degree of blood loss; the decreases were correctable by RBC transfusion.267 Notably, these changes in oxygenation were present in the absence of any decrease in blood pressure or other clinical signs of anemia.

Orthogonal Polarizing Spectroscopy Orthogonal polarizing spectroscopy assesses blood flow through the microcirculation.268,269 It generates an image of the microcirculation based on the differential absorption and scattering of light, which distinguishes between patent blood vessels that contain red cells and vessels (including tissue) that do not. This technique can assess tissue perfusion in a manner that correlates with fractional capillary density. There are some limitations to OPS—chiefly, that the most suitable monitoring site in adults is the mucosa of the mouth (sublingual placement is common) and that it cannot be used on internal organs, except intraoperatively. It has been validated in animal systems and also against conventional capillary microscopy in human nail skinfold.270,271 Altered microcirculation was demonstrated by OPS in patients with severe sepsis, and the changes were more severe in nonsurvivors.227,272,273 Improvement in microcirculatory blood flow as assessed by OPS was also more strongly associated with good outcome than other changes in hemodynamic parameters or serum lactate levels.274 Transfusion of 10 to 15 mL/kg of RBCs to anemic, preterm infants was shown to increase functional capillary density in blood vessels of the upper arm as measured by OPS.275 The change was due to the perfusion of additional capillary beds because it was also shown that vessel diameter, red cell velocity, and flow rate did not change. Sublingual OPS was used to assess the effect of RBC transfusion in septic patients.276 RBC transfusion resulted in increased hematocrit, mean arterial pressure, and calculated oxygen delivery; however, microvascular perfusion was unchanged, and was found to show considerable inter-patient variability.

40

Blood Oxygen-Dependent Function Magnetic Resonance Imaging BOLDfMRI detects changes in the ratio of oxyhemoglobin to deoxyhemoglobin. It has been used extensively to determine tissue oxygenation and blood flow in many tissues including brain,277 kidney,278 heart,279 muscle,280 and tumors.281 It has not yet been used to assess hemorrhage or transfusion, but offers the advantage of being able to query any organ noninvasively.

Animal Models Direct assessment of the impact of RBC transfusion on the microcirculation has been studied more extensively in experimental animal systems. In a hemorrhagic shock model in rats, resuscitation with human red cells was associated with improved oxygenation of the mesenteric microcirculation as measured by O2-dependent quenching of palladium-porphyrin phosphorescence.282 Similar results were obtained when this system was adapted to an isovolemic exchange model.283 Experiments in the hamster skinfold system, where the microvascular circulation is visualized directly and can be coupled with O2 level determination using palladium-porphyrin phosphorescence, showed better maintenance of microvascular blood flow and tissue oxygenation after exchange transfusion with fresh RBC units, than with units that had been stored for 28 days.284 In a hemorrhagic shock model in the same hamster skinfold system, however, the same improvements in the microcirculation were obtained with fresh RBC units whether or not they contained functional hemoglobin or methemoglobin, suggesting that the rheologic effects of RBC transfusion were more important than the ability of the red cells to off-load O2.174,285

Effect of RBC Storage Changes on the Microcirculation During storage, RBC units undergo several changes. It has been suggested that these changes affect their ability to deliver O2. Although there has been considerable interest in defining the clinical impact of these storage changes, clinical studies and experiments in animal model systems have yielded conflicting results. Several observational studies in cardiac surgery, trauma, and ICU patients have shown an association between prolonged storage time and poor clinical outcomes284,286-290; however, several other studies, including a pilot randomized, controlled trial in ICU patients, have not found deleterious effects after the transfusion of longer storage RBC units.277,276,291-294 The effect of the storage changes on the microcirculation is also being evaluated. Among the characteristic changes that red cells undergo during storage is alteration of their shape from a biconcave disc to an echinocyte with protrusions from which lipid vesicles are shed. This shape change is associated with loss of deformability295,296 and increased osmotic fragility that results in hemoglobin loss to the plasma-anticoagulant-preservative phase.297 Given the ability of hemoglobin to scavenge NO, RBC units with free hemoglobin could trigger vasoconstriction and impede blood flow. In fact, there is evidence that hemoglobin scavenging of NO contributes to vaso-occlusive crisis in sickle cell disease.298 Reduced red cell deformability might also impede flow as a result of impaired

Chapter 3: Oxygen Delivery

transit through small vessels, although this effect has been shown to be mitigated by the shunting of rigid or misformed red cells to larger diameter vessels in the microcirculation.299 Stored red cells have been shown to adhere to endothelial cell layers in vitro and to small vessels in rat muscle, suggesting another mechanism whereby transfused red cells could occlude the microcirculation.300 Candidate adhesion molecules include the Lutheran and LW antigens,301 and the thombospondin receptor, CD36.300 Levels of 2,3-DPG decrease during red cell storage. The loss of this allosteric modifier is reflected in a decrease in the hemoglobin P50 from 29 mm Hg to approximately 20 mm Hg. After transfusion, the red cell replenishes its 2,3-DPG over a period of 24 hours.302 Increased O2 affinity has been suggested to impair oxygen offloading. No effect of experimentally altered P50 on global parameters of oxygen dynamics or the microcirculation was apparent in several studies in experimental animals.303-305 Conversely, in three studies in rats, tissue oxygenation (as assessed by palladium-porphyrin phosphorescence or microelectrode) and/or functional capillary density (as assessed by intravital microscopy) showed that tissue oxygenation and capillary perfusion were not as well maintained by transfusion of RBC units that had been stored for various periods and were presumably deficient in 2,3-DPG.283,306,307 Transfusion of red cells from mice with a low-affinity hemoglobin variant (hemoglobin Presbyterian) conferred a survival advantage in a mouse LPS-challenge sepsis model.308 The behavior of hemoglobin-based oxygen carriers (HBOCs) is different than that of intraerythrocytic hemoglobin. In two animal model systems, highaffinity hemoglobin preparations preserved functional capillary density and tissue oxygenation better than preparations formulated with lower-affinity hemoglobin.309,310 High oxygen affinity is thought to balance the enhanced propensity of the HBOCs, which are much smaller than red cells, to facilitate diffusion of O2 to the vessel wall and thereby avoid triggering autoregulatory vasoconstriction and impaired blood flow through the microcirculation.311 ATP levels in stored RBC units decrease to around 60% of initial levels after 5 weeks of storage, and are restored a few hours after transfusion, a process that involves the uptake of adenosine.312 ATP levels do correlate with posttransfusion red cell survival, but only at levels below 50%, which is usually reached only after the standard storage period of 42 days.313 However, restoration of ATP levels in stored RBC units improved tissue oxygenation in a model system,283 suggesting that ATP plays a role, whether as an energy source for a metabolic process or as a vasodilator, in oxygen delivery. It has recently been observed that red cell levels of SNOhemoglobin decline rapidly during storage, decreasing by 70% within a day of collection.314 These SNO-depleted red cells failed to induce vasodilation in isolated rabbit aorta rings or to promote blood flow through canine coronary arteries in vivo; however, these properties could be restored by re-nitrosolating the red cells by exposure to NO in solution. This change may explain, in part, the experimental observations of the failure of RBC transfusion to improve global and/or microcirculatory parameters of oxygenation.

Red Cell Substitutes Investigators have long sought a substitute for red cells that would successfully imitate its oxygen delivery capabilities. Interest in the development of materials that could be used to deliver O2 to tissues in place of red cells was initially stimulated in the 1980s by concerns about the adequacy of the blood supply and transfusion-transmitted infectious diseases. This interest has been sustained primarily because of the clinical potential for an effective red cell substitute and secondarily because of the insights into oxygen delivery and its regulation that have been gained through the study of these materials.315-317 The search for a red cell substitute has led to the investigation of three different classes of oxygen carriers: modified hemoglobin, perfluorocarbon emulsions, and liposome-encapsulated hemoglobin. The development of the latter remains in the preclinical stage,318 but several perfluorocarbon- and hemoglobin-based substitutes have advanced to the level of Phase III trials.

Hemoglobin-Based Oxygen Carriers Cell-free hemoglobin is an obvious candidate for a red cell substitute and has a number of desirable attributes, the first of which is its ability to carry high concentrations of O2 and CO2. The absence of the red cell membrane eliminates the complications created by the presence of blood group antigens. The purification procedures remove other cell components that could produce undesirable effects, and a variety of processes are used to eliminate or inactivate infectious agents. In its purified form, hemoglobin solutions are quite stable and can be stored for much longer periods than RBC units and under more permissive conditions (eg, room temperature). However, hemoglobin is a highly active molecule, and in its unmodified state, has the potential of producing a number of deleterious effects.319 These toxicities have been responsible for the failure of several candidate oxygen carriers in clinical trials and were recently addressed at a symposium sponsored by the National Heart, Lung, and Blood Institute. The symposium identified areas of particular concern requiring more research to sustain future development.320 In an effort to eliminate or mitigate some of these potential toxicities, all of the HBOCs being studied make use of hemoglobin preparations that have been chemically modified. One problem with free hemoglobin in plasma is that it dissociates into αβ dimers and is rapidly cleared by glomerular filtration. Not only does the hemoglobin leave the bloodstream too rapidly to be clinically useful, but also it is toxic to the renal tubular cells.321 Various modifications have been made to all HBOCs under study; these changes have had the effect of increasing the plasma half-life and eliminating nephrotoxicity. Three types of modifications have been used to achieve these ends: intramolecular stabilization of the hemoglobin tetramer; intermolecular cross-linking (polymerization of the tetramer); and surface conjugation of hemoglobin with other macromolecules such as polyethylene glycol (PEG).

41

Section I: Part I

A second concern with the hemoglobin molecule is its vasoactivity. The hypertensive effects of cell-free hemoglobin have been observed in animals322,323 and humans324 and are attributed, in part, to NO scavenging by the heme iron and the resulting vasoconstriction.325 In addition, the HBOCs are not S-nitrosylated, like hemoglobin in red cells, and therefore lack the vasodilatory activity of the intact red cell.326 However, in-vitro S-nitrosylation of a pegylated hemoglobin was recently shown to enhance its ability to promote vasodilation of the coronary arteries in dogs undergoing open-chest surgery.327 Other factors that play a role in the vasoactivity of HBOCs are the effects of their low viscosity328 on vascular tone.329,330 Delivery of O2 by the substitute may also exert local vasoconstrictive effects by triggering an O2-sensitive autoregulatory response.331,332 The degree of vasoactivity varies among the different HBOC compounds and is generally more prominent in preparations based on a hemoglobin species with small molecular radius (eg, stabilized hemoglobin tetramer) compared to a species with a large radius (eg, pegylated hemoglobin).333 Hemoglobin in solution auto-oxidizes to methemoglobin, which does not carry O2, thence to irreversibly altered metabolites including free radicals,334 which could have deleterious effects on tissues exposed to them. Methemoglobin formation has been measured in all of the HBOCs studied to date and found to be ⬍15% in all, with little tendency to accumulate during storage.335 The possibility that HBOCs might be immunogenic is low but is being assessed in the various clinical trials.336 Human hemoglobin is a poor immunogen, but chemically modified human hemoglobin, or bovine hemoglobin, which has 90% sequence homology with the human form, could conceivably be more highly immunogenic. Surface modification with macromolecules such as PEG and polyoxyethylene may shield hemoglobin from the immune system and reduce the frequency or magnitude of the response.337 The HBOCs under development have been based on hemoglobin from three sources: human, from outdated donor RBC units; bovine; and recombinant technology. Seven HBOCs, four of them based on human hemoglobin, have been studied in clinical trials, as have two HBOCs based on bovine hemoglobin and one using a recombinant human hemoglobin. (See Table 3-3.) Only three remain under active development, although a newer product based on lyophilized human hemoglobin, OxyVita (OxyVita, New Windsor, NY), is in preclinical testing.338 PolyHeme (Northfield Laboratories, Evanston, IL) is a preparation consisting of a glutaraldehyde polymerized human hemoglobin that has been pyridoxylated and extensively purified to remove residual hemoglobin tetramers.339 (See Table 3-4.) It is being developed as an alternative to RBC use in surgery and trauma.340,341 Trauma patients who received PolyHeme required fewer transfusions of banked blood.340 Northfield recently completed a pivotal Phase III clinical trial of the use of PolyHeme as an early intervention in trauma.342 The study was controversial for its waiver of individual informed consent, which has occasionally been used in clinical studies in trauma patients, as well as for the

42

study designs, which called for continued use of the study material even after patients arrived at the hospital and could have received banked blood. Although there was a trend for excess 1-day and 30-day mortality in the treatment arm, the differences were not statistically significant. Myocardial infarction was reported more frequently in patients receiving PolyHeme (p⬍0.05). The Biopure Corporation (Cambridge, MA) uses glutaraldehyde to polymerize bovine hemoglobin followed by extensive purification and virus inactivation steps to produce a substitute with less than 3% hemoglobin tetramers (Hemopure). The Biopure Corporation is the first to have obtained licensure for a blood substitute product. In 1997, a veterinary formulation of their HBOC, Oxyglobin, was licensed by the Food and Drug Administration (FDA) for use in animals and in 2001, Biopure announced that Hemopure had been approved for clinical use in South Africa. This product has been studied principally for perioperative use as a “bridge,” delaying the need for banked RBCs until the patient has been stabilized and blood loss curtailed.343-345 Biopure has reported in abstract form on the safety and efficacy of Hemopure in Phase II and III trials in orthopedic surgery, where it was shown to reduce the use of allogeneic RBCs346; however, the firm has announced that it is in the process of designing several Phase II trials in nonsurgical applications.347 Hemospan (Sangart, San Diego, CA) is a pegylated human hemoglobin designed specifically with a large molecular radius and high oxygen affinity to mitigate the diffusive transfer of O2, which is thought to trigger the vascular autoregulatory response responsible for the vasoactivity characteristic of many of the other HBOCs.310,348,349 This product has undergone Phase I and II clinical testing in Europe where it was found to have minimal hypertensive effects.350,351 A Phase II clinical trial in patients undergoing radical retropubic prostatectomy is being mounted in the United States and a Phase III trial in orthopedic surgery is open in Europe.352

Perfluorocarbon-Based Oxygen Carriers The perfluorocarbons (PFCs) are a class of compounds consisting of linear or cyclic carbon backbones that are highly substituted with fluorine.353 Most of the PFCs are liquid at room temperature and are highly soluble for respiratory gases including O2, N2, and CO2. The amount of gas that can be dissolved into a PFC is directly proportional to its ambient partial pressure. Theoretically, at a high enough PO2, a PFC could contain the same amount of O2 as blood. PFCs are not miscible in water, however, and must be prepared as emulsions for most biologic applications. The need to prepare the PFCs as emulsions decreases the O2-carrying capacity; therefore, patients must receive supplemental oxygen when a PFC emulsion is used. PFC emulsions are cleared by the reticuloendothelial system (RES), as are the HBOCs, but eventually are exhaled via the lungs. The half-lives in the RES of some of the first PFCs studied were quite prolonged, but the newer PFCs have half-lives in the RES on the order of a few days. The impact on the RES of the ingestion of large amounts of PFCs or the emulsifying

Table 3-3. Modified Hemoglobin-Based Red Cell Substitutes Reaching Clinical Trials* Product Class

Modification

Product (Manufacturer)

Hemoglobin Source

Trial Level

Application

Intramolecular crosslinked hemoglobin

Di-aspirin

HemAssist (Baxter)

Human

Phase II

Phase I

Septic shock, hemodialysis, hemorrhagic shock, cardiopulmonary bypass Acute blood loss—surgery, trauma Stroke Erythropoiesis—ESRD, refractory anemia

Phase II (discontinued)

ANH, acute blood loss, surgery

Phase III (discontinued) Recombinant di-α hemoglobin

Polymerized hemoglobin

Glutaraldehyde polymerization ⫹ pyridoxylation Glutaraldehyde polymerization

Glutaraldehyde polymerization o-raffinose

Surface conjugation

Optro (Somatogen Baxter)

Recombinant

PolyHeme (Northfield)

Human

Phase III

Trauma, surgery

Hemopure † (Biopure)

Bovine

Preclinical Phase I Phase II

Oxyglobin (Biopure)

Bovine

Phase III (on hold) Approved

Erythropoiesis Radiosensitizer, glioblastoma Sickle cell crisis, oncology, surgery—orthopedic, urological, vascular, cardiac, trauma Surgery—cardiac, orthopedic Veterinary—anemia, acute blood loss

Hemolink (Hemosol)

Human

Phase II

PEG

Hemospan (Sangart)

Human

Polyoxyethylene PEG

PHP (Ajinomoto/Apex) PEG Hemoglobin (Enzon)

Human Bovine

Phase III (discontinued)

Cardiopulmonary bypass—ANH, orthopedic surgery, acute blood loss, dialysis Cardiac surgery

Phase II Phase III Phase III Phase Ib (discontinued)

Prostatectomy, orthopedic surgery Orthopedic surgery NO-induced shock Radiosensitizer solid tumors

43

Chapter 3: Oxygen Delivery

*Information current as of May 2008. † Approved in South Africa. ESRD ⫽ end-stage renal disease; ANH ⫽ acute normovolemic hemodilution; PEG ⫽ polyethylene glycol; PHP ⫽ pyridoxalated hemoglobin polyoxyethylene; NO ⫽ nitric oxide.

Section I: Part I

Table 3-4. Characteristics of HBOCs in Phase III Clinical Trials Characteristic

Hemoglobin-Based Oxygen Carriers

Hemoglobin source Modification Molecular weight (kD) Molecular radius (nm) % tetramer (64 kD) P50 (mm Hg) % Methemoglobin Hemoglobin (g/dL) Colloid oncotic pressure (mm Hg) Change in mean arterial pressure

PolyHeme

Hemopure

Hemospan

Human Glutaraldehyde pyridoxal-5-phosphate 150 2.7 ⬍1% 28-30 ⬍3% 10 20-25 ↔

Bovine Glutaraldehyde

Human PEGylated

240 4.9 ⬍3% 38 ⬍3% 13 17 ↑ 15%

96 14.1 100% 6⫾2 ⬍5% 4.2 55 ↔

Table 3-5. Perfluorocarbon Red Cell Substitutes Reaching Clinical Trials* Product (Manufacturer)

Perfluorocarbon

Trial Level

Application

Fluosol-DA (Green Cross/Alpha)

Perfluorodecalin Perfluoropropylamine

Phase II (discontinued) Approved (withdrawn)

Acute blood loss PTCA

Oxygent (Alliance)

Perflubron

Phase II Phase III (discontinued)

CABG—ANH Ortho surgery—acute blood loss Cardiac surgery, surgery—ANH

Imagent GI (Alliance) Imavist (Alliance)

Perflubron Perflubron

Approved (withdrawn) Phase III (discontinued)

GI imaging Cardiac ultrasound

Liquivent (Alliance)

Perflubron (neat)

Phase Ib/II Phase II/III (discontinued)

Liquid ventilation—IRDS Liquid ventilation—pedi and adult ARDS

Oxyfluor (HemaGen/PFC)

Perfluorodichlorooctane

Phase II (discontinued)

Surgery, neuro-protectant/bypass

Oxycyte (Synthetic Blood Int.)

Perfluoro-t-butylcyclohexane

Phase IIb

Acute brain injury

*Information current as of May 2008. PCTA ⫽ percutaneous transluminal coronary angioplasty; CABG ⫽ coronary artery bypass graft; ANH ⫽ acute normovolemic hemodilution; GI ⫽ gastrointestinal; IRDS ⫽ infant respiratory distress syndrome; ARDS ⫽ acute respiratory distress syndrome.

agents remains to be determined. The clearance rate of PFC emulsions from the circulation increases as the emulsion particle sizes decrease. Thus, reproducibly and predictably preparing emulsions with the appropriate distribution of particle sizes is one of the technical challenges that the developers of these materials have faced. The plasma half-lives of the PFC emulsions that have been studied for clinical use are approximately 12 to 24 hours, which compare favorably with the half-lives of the HBOCs. Two PFC emulsions underwent clinical trials in the 1990s but development of both has been discontinued. (See Table 3-5.) Two new products are in preclinical or clinical testing. One, PherO2 (Sanguine, Pasadena, CA) is a reformulation of perfluorodecalin, one of the constituent PFCs in Fluosol, a product licensed in the 1980s for use in percutaneous transluminal coronary angioplasty to perfuse the vascular bed distal to the balloon, but which saw off-label use as a transfusion equivalent.354 Synthetic Blood

44

International (Costa Mesa, CA) announced that its PFC product, Oxycyte, was more effective at increasing brain oxygen levels in patients with traumatic brain injury than either 50% or 100% O2. The firm has announced plans for a Phase IIb study in 200 patients with traumatic brain injury.355

Red Cell Substitutes: Summary Although the various preparations that have been studied share the ability to transport oxygen, they are quite distinct from one another and certainly very different from red cells. If the compounds are to be used to best advantage, transfusion medicine specialists will need to be familiar with the capabilities and liabilities of the substitutes that find their way into the clinical arena. The development of these substitutes has shed considerable light on the mechanisms of oxygen delivery and its regulation, and forced reconsideration of some long-held assumptions. In order to adequately fulfill the role of consultant, the transfusion

Chapter 3: Oxygen Delivery

medicine specialist will need an even greater understanding of the physiology of red cell transfusion and oxygen delivery.

Conclusion A constant supply of oxygen to the tissues is essential to sustain the basic energy-producing machinery of the cell. Homeothermic organisms have evolved multiple, interlinked regulatory systems to ensure that oxygen delivery to the periphery meets the specific metabolic requirements of each tissue and is coordinated with the function of other systems.

Disclaimer The author has disclosed no conflicts of interest.

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226. Ayala A, Tu Y, Flye M, Chaudry I. Depressed splenic function after hemorrhage results from gastrointestinal tract stimulation of hepatic-mediator release. Correction with portacaval shunt. Arch Surg 1996;131:1209-14. 227. De Backer D, Creteur J, Preiser J, et al. Microvascular blood flow is altered in patients with sepsis. Am J Respir Crit Care Med 2002;166:98-104. 228. Bateman R, Walley K. Microvascular resuscitation as a therapeutic goal in severe sepsis. Crit Care 2005;9:S27-32. 229. Buwalda M, Ince C. Opening the microcirculation: Can vasodilators be useful in sepsis? Intensive Care Med 2002;28: 1208-17. 230. Dejam A, Hunter C, Tremonti C, et al. Nitrite infusion in humans and nonhuman primates: Endocrine effects, pharmacokinetics, and tolerance formation. Circulation 2007;116:1821-31. 231. Morisaki H, Bloos F, Keys J, et al. Compared with crystalloid, colloid therapy slows progression of extrapulmonary tissue injury in septic sheep. J Appl Physiol 1994;77:1507-18. 232. Yodice P, Astiz M, Kurian B, et al. Neutrophil rheologic changes in septic shock. Am J Respir Crit Care Med 1997;155:38-42. 233. Baskurt O, Gelmont D, Meiselman H. Red blood cell deformability in sepsis. Am J Respir Crit Care Med 1998;157:421-7. 234. Bateman R, Jagger J, Sharpe M, et al. Erythrocyte deformability is a nitric oxide-mediated factor in decreased capillary density sepsis. Am J Physiol Heart Circ Physiol 2001;280:H2848-56. 235. Condon M, Kim J, Deitch E, et al. Appearance of an erythrocyte population with decreased deformability and hemoglobin content following sepsis. Am J Physiol Heart Circ Physiol 2003;284: H2177-84. 236. Eichelbrönner O, Sielenkëmper A, Cepinskas G, et al. Endotoxin promotes adhesion of human erythrocytes to human vascular endothelial cells under conditions of flow. Crit Care Med 2000;28:1865-70. 237. Goddard C, Allard M, Hogg J, et al. Prolonged leukocyte transit time in coronary microcirculation of endotoxemic pigs. Am J Physiol Heart Circ Physiol 1995;269:H1389-97. 238. Goddard C, Poon B, Klut M, et al. Leukocyte activation does not mediate myocardial leukocyte retention during endotexemia in rabbits. Am J Physiol Heart Cirl Physiol 1998;275: H1548-57. 239. Levi M, de Jonge E, van der Poll T. Sepsis and disseminated intravascular coagulation. J Thromb Thrombolysis 2003; 16:43-7. 240. Hollenberg S, Tangora J, Piotrowski M, et al. Impaired microvascular vasoconstrictive responses to vasopressin in septic rats. Crit Care Med 1997;25:869-73. 241. Tyml K, Yu J, McCormack D. Capillary and interiolar responses to local vasodilators are impaired in a rat motel of sepsis. J Appl Physiol 1998;84:837-44. 242. Tyml K, Wang X, Lidington D, Ouellette Y. Lipopolysaccharide reduces intercellular coupling in vitro and arteriolar conducted response in vivo. Am J Physiol Heart Circ Physiol 2001;281: H1397-1406. 243. Lidington D, Ouellette Y, Tyml K. Endotoxin increases intercellular resistance in microvascular endothelial cells by a tyrosine kinase pathway. J Cell Physiol 2000:185:117-25. 244. Lidington D, Ouellette Y, Li F, Tyml K. Conducted vasoconstriction is reduced in a mouse model of sepsis. J Vasc Res 2003;40: 149-58.

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245. Lidington D, Tyml K, Wilson J. Communication of agonist-induced electrical responses along “capillaries” in vitro can be modulated by lipopolysaccharide, but not nitric oxide. J Vasc Res 2002;39: R50-6. 246. Lidington D, Tyml K, Ouellette Y. Lipopolysaccharide-induced reductions in cellular coupling correlate with tyrosine phosphorylation of connexin 43. J Cell Physiol 2002;193:373-9. 247. Wu F, Wilson J, Tyml K. Ascorbate inhibits iNOS expression and preserves vasoconstrictor responsiveness in skeletal muscle of septic mice. Am J Physiol Regul Integr Comp Physiol 2003;285: R50-6. 248. Vikram D, Zweier J, Kuppusamy P. Methods for noninvasive imaging of tissue hypoxia. Antioxid Redox Signal 2007;9:1745-56. 249. Smith M, Stiefel M, Magge S, et al. Packed red blood cell transfusion increases local cerebral oxygenation. Crit Care Med 2005;33: 1104-8. 250. Suttner S, Piper S, Kumle B, et al. The influence of allogeneic red blood cell transfusion compared with 100% oxygen ventilation on systemic oxygen transport and skeletal muscle oxygen tension after cardiac surgery. Anesth Analg 2004;99:2-11. 251. Nicks BA, Chang M, Bozeman WP. Near-infrared spectroscopy during resuscitation of trauma patients predicts future development of multiple organ dysfunction (abstract). Ann Emerg Med 2007;50(Suppl):S64. 252. Cohn SM, Nathans AB, Moore FA, et al. Tissue oxygen saturation predicts the development of organ dysfunction during traumatic shock resuscitation. J Trauma 2007;62:44-55. 253. Doreschug KC, Delsing AS, Schmidt GA, Haynes WG. Impairments in microvascular reactivity are related to organ failure in human sepsis. Am J Physiol Heart Circ Physiol 2007;293:1065-71. 254. Crawford J, Otero O, Goyal N, et al. Near infrared spectroscopy to assess systemic perfusion in the critically ill (abstract). Crit Care Med 2007;35(Suppl):A254. 255. Brawanski A, Faltermeier R, Rothoerl RD, Woertgen C. Comparison of near-infrared spectroscopy and tissue p(O2) time series in patients after severe head injury and aneurysmal subarachnoid hemorrhage. J Cereb Blood Flow Metab 2002;22: 605-11. 256. Mille T, Tachimiri ME, Klersy C, et al. Near-infrared spectroscopy during carotid enarterectomy: Which threshold value is critical? Eur J Vasc Endovasc Surg 2004;27:646-50. 257. Hoffman GM. Near-infrared spectroscopy should be used for all cardiopulmonary bypass. J Cardiovasc Vasc Anesth 2006;20: 606-12. 258. Hoffman GM, Ghanayem KS. Noninvasive monitoring of cardiac output: Benefits of NIRS technology. Congenit Cardiol Today 2007;5:1, 3-7. 259. Colasacco CG, Worthen M, Peterson BB, et al. NIRS monitoring to predict post-operative renal insufficiency following repair of congenital heart disease (abstract). Crit Care Med 2007;35(Suppl): A87. 260. Menke J, Stocker H, Sibrowski W. Cerebral oxygenation and hemodynamics during blood donation studies by near-infrared spectroscopy. Transfusion 2004;44:414-21. 261. Torella F, Cowley R, Thorniley M, McCollum C. Monitoring blood loss with near infrared spectroscopy. Comp Biochem Physiol 2002;132:199-20. 262. Torella F, Haynes SL, McCollum CN. Cerebral and peripheral nearinfrared spectroscopy: An alternative transfusion trigger? Vox Sang 2002;83:254-7.

263. Wardle SP, Weindling AM. Peripheral fractional oxygen extraction and other measures to guide blood transfusions in preterm infants. Semin Perinatol 2001;25:60-4. 264. Cerussi A, Van Woerkom R, Waffarn F, Tromberg B. Noninvasive monitoring of red blood cell transfusion in very low birthweight infants using diffuse optical spectroscopy. J Biomed Optics 2005;10:51401-9. 265. Liem KD, Hopman JC, Oeseburg B, et al. The effect of blood transfusion and haemodilution on cerebral oxygenation and haemodynamics in newborn infants investigated by near infrared spectroscopy. Eur J Pediatr 1997;156:305-10. 266. Torella F, Haynes SL, McCollum CN. Cerebral and peripheral oxygen saturation during red cell transfusion. J Surg Res 2003;110:217-21. 267. Green D. A retrospective study of changes in cerebral oxygenation using a cerebral oximeter in older patients undergoing prolonged major abdominal surgery. Eur J Anaesthesiol 2007;24:230-4. 268. Groner W, Winkelman J, Harris A, et al. Orthogonal polarization spectral imaging: A new method for study of the microcirculation. Nat Med 1999;5:1209-12. 269. Cerny V, Turek Z, Parizkova T. Orthogonal polarization spectral imaging. Physiol Res 2007;56:141-7. 270. Harris A, Sinitsina A, Messmer K. Validation of OPS imaging for microvascular measurements during isovolemic hemodilution and low hematocrits. Am J Physiol 2002;282:H1502-9. 271. Mathura K, Vollebregt K, Boer K, et al. Comparison of OPS imaging and conventional capillary microscopy to study the human microcirculation. J Appl Physiol 2001;91:74-8. 272. Boerma E, Kuiper M, Kingma W, et al. Disparity between skin perfusion and sublingual microcirculatory alterations in severe sepsis and septic shock: A prospective observational study. Intensive Care Med 2008; 34: 1294-8. 273. Trzeciak S, Dellinger R, Parrillo J, et al. Early microcirculatory perfusion derangements in patients with severe sepsis and septic shock: Relationship to hemodynamics, oxygen transport, and survival. Ann Emerg Med 2007;49:88-98. 274. Sakr Y, Dubois M, De Backer D, et al. Persistant microvasculatory alternations are associated with organ failure and death in patients with septic shock. Crit Care Med 2004;32:1825-31. 275. Genzel-Boroviczeny O, Christ F, Glas V. Blood transfusion increases functional capillary density in the skin of anemic preterm infants. Pediatr Res 2004;56:751-5. 276. Sakr Y, Chierego M, Piagnerelli M, et al. Microvascular response to red blood cell transfusion in patients with severe sepsis. Crit Care Med 2007;35:1639-44. 277. Ndubuizu O, LaManna J. Brain tissue oxygen concentration measurements. Antioxid Redox Signal 2007;9:1207-19. 278. Textor S, Glockner J, Lerman L, et al. The use of magnetic resonance to evaluate tissue oxygenation in renal artery stenosis. J Am Soc Nephrol 2008;19:780-8. 279. Egred M, Waiter G, Redpath T, et al. Blood oxygen level-dependent (BOLD) MRI: A novel technique for the assessment of myocardial ischemia as identified by nuclear imaging SPECT. Eur J Intern Med 2007;18:581-6. 280. Carlier P, Bertoldi D, Baligand C, et al. Muscle blood flow and oxygenation measured by NMR imaging and spectroscopy. NMR Biomed 2006;19:954-67. 281. Wardlaw G, Wong R, Noseworthy M. Identification of intratumour low frequency microvascular components via BOLD signal fractal dimension mapping. Phys Med 2008; 24: 87-91.

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Section I: Part I

282. van Bommel J, de Korte D, Lind A, et al. The effect of the transfusion of stored RBCs on intestinal microvascular oxygenation in the rat. Transfusion 2001;41:1515-23. 283. Raat N, Verhoeven A, Mik E, et al. The effect of storage time of human red cells on intestinal microcirculatory oxygenation in a rat isovolemic exchange model. Crit Care Med 2005;33:39-45. 284. Tsai AG, Cabrales P, Intaglietta M. Microvascular perfusion upon exchange transfusion with stored red blood cells in normovolemic anemic conditions. Transfusion 2004;44:1626-34. 285. Cabrales P, Intaglietta M, Tsai A. Transfusion restores blood viscosity and reinstates microvascular conditions from hemorrhagic shock independent of oxygen carrying capacity. Resuscitation 2007;75:124-34. 286. Zallen G, Offner P, Moore E, et al. Age of transfused blood is an independent risk factor for postinjury multiple organ failure. Am J Surg 1999;178:570-2. 287. Kristiansson M, Soop M, Saraste L, Sundqvist KG. Cytokines in stored red blood cell concentrates: Promoters of systemic inflammation and simulators of acute transfusion reactions? Acta Anaesthesiol Scand 1996;40:496-501. 288. Shanwell A, Kristiansson M, Remberger M, Ringden O. Generation of cytokines in red cell concentrates during storage is prevented by prestorage white cell reduction. Transfusion 1997;37: 678-84. 289. Vamvakas EC, Carven JH. Allogeneic blood transfusion and postoperative duration of mechanical ventilation: Effects of red cell supernatant, platelet supernatant, plasma components and total transfused fluid. Vox Sang 2002;8:141-9. 290. Vamvakas EC, Carven JH. Length of storage of transfused red cells and postoperative morbidity in patients undergoing coronary artery bypass graft surgery. Transfusion 2000; 40:101-9. 291. Walsh TS, McArdle F, McLellan SA, et al. Does the storage time of transfused red blood cells influence regional or global indexes of tissue oxygenation in anemic critically ill patients? Crit Care Med 2004;32:364-71. 292. Hebert PC, Chin-Yee I, Fergusson D, et al. A pilot trial evaluating the clinical effects of prolonged storage of red cells. Anesth Analg 2005;100:1433-8. 293. Van de Watering L, Lorinser J, Versteegh M, et al. Effects of storage time of red cell transfusions on the prognosis of coronary artery bypass patients. Transfusion 2006;46:1712-8. 294. Weiskopf R, Feiner J, Hopf H, et al. Fresh blood and aged stored blood are generally effective in immediately reversing anemiainduced brain oxygenation deficits in humans. Anesthesiology 2006;104:911-20. 295. Berezina T, Zaets S, Morgan C, et al. Influence of storage on red blood cell rheological properties. J Surg Res 2002;102:6-12. 296. Izzo P, Manicone A, Spagnuolo A, et al. Erythrocytes stored in CPD SAG-mannitol: Evaluation of their deformability. Clin Hemorheol Microcirc 1999;21:335-9. 297. Beutler E, Kuhl W, West C. The osmotic fragility of erythrocytes after prolonged liquid storage and after reinfusion. Blood 1982;59:1141-7. 298. Reiter C, Wang X, Tanus-Santos J, et al. Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease. Nat Med 2002;8:1383-9. 299. Parthasarathi K, Lipowsky H. Capillary recruitment in response to tissue hypoxia and its dependence on red blood cell deformability. Am J Physiol 1999;277:H2145-57.

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300. Ho J, Sibbald W, Chin-Yee I. Effects of storage on efficacy of red cell transfusion: When is it not safe? Crit Care Med 2003;31: S687-97. 301. Eyler C, Telen M. The Lutheran glycoprotein: A multifunctional adhesion receptor. Transfusion 2006;46:668-77. 302. Heaton A, Keegan T, Home S. In vivo regeneration of red cell 2, 3diphosphoglycerate following transfusion of DPG-depleted AS-1, AS-3 and CPDA-1 red cells. Br J Haematol 1989;71:131-6. 303. D’Almeida M, Gray D, Martin C, et al. Effect of prophylactic transfusion of stored RBCs on oxygen reserve in response to acute isovolemic hemorrhage in a rodent model. Transfusion 2001;41: 950-6. 304. Eichelbrönner O, D’Almeida M, Sielenkämper A, et al. Increasing P(50) does not improve DO(2CRIT) or systemic VO(2) in severe anemia. Am J Physiol Heart Circ Physiol 2002;283:H92-101. 305. Cabrales P, Tsai A, Intaglietta M. Modulation of perfusion and oxygenation by red blood cell oxygen affinity during acute anemia. Am J Resp Cell Mol Biol 2008;83:354-61. 306. Arslan E, Sierko E, Waters J, Siemionow M. Microcirculatory hemodynamics after acute blood loss followed by fresh and banked blood transfusion. Am J Surg 2005;190:456-62. 307. Gonzalez A, Yazici I, Kusza K, Siemionow M. Effects of fresh versus banked blood transfusion on microcirculatory hemodynamics and tissue oxygenation in the rat cremaster model. Surgery 2007;141:630-9. 308. Huang F, Nojiri H, Shimizu T, Shirasawa T. Beneficial effect of transfusion with low-affinity red blood cells in endotoxemia. Transfusion 2005;45:1785-90. 309. Nemoto M, Mito T, Brinigar W, et al. Salvage of focal cerebral ischemic damage by transfusion of high O2-affinity recombinant hemoglobin polymers in mouse. J Appl Physiol 2006;100: 1688-91. 310. Tsai A, Vandergriff K, Intaglietta M, Winslow R. Targeted O2 delivery by low-P50 hemoglobin: A new basis for O2 therapeutics. Am J Physiol Heart Circ Physiol 2003;285:H1411-9. 311. Winslow R. Red cell substitutes. Semin Hematol 2007;44:51-9. 312. Marikovsky Y. The cytoskeleton in ATP-depleted erythrocytes: The effect of shape transformation. Mech Ageing Dev 1996;86: 137-44. 313. Tinmouth A, Chin-Yee I. The clinical consequences of the red cell storage lesion. Transfus Med Rev 2001;15:91-107. 314. Reynolds J, Ahern G, Angelo M, et al. S-nitrosohemolgobin deficiency: A mechanism for loss of physiological activity in banked blood. Proc Natl Acad Sci U S A 2007;104:17058-62. 315. Jahr J, Walker V, Manoocheri K. Blood substitutes as pharmacotherapies in clinical practice. Curr Opin Anesthesiol 2007;20: 325-30. 316. Ness P, Cushing M. Oxygen therapeutics: Pursuit of an alternative to the donor red blood cell. Arch Pathol Lab Med 2007;131: 734-41. 317. Winslow R. Current status of oxygen carriers (“blood substitutes”): 2006. Vox Sang 2006;91:102-10. 318. Sakai H, Sou K, Horinuchi H, et al. Haemoglobin-vesicles as artificial oxygen carriers: Present situation and future vision. J Intern Med 2007;2263:4-15. 319. Buehler P, Alayash A. All hemoglobin-based oxygen carriers are not created equally. Biochim Biophys Acta 2008 (in press). 320. Estep T, Bucci E, Farmer M, et al. Basic science focus on blood substitutes: A summary of the NHLBI Division of Blood Diseases and

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Resources Working Group Workshop, March 1, 2006. Transfusion 2008; 48: 776-82. 321. Lieberthal W. Renal effects of hemoglobin-based blood substitutes. In: Rudolph AS, Rabinivici R, Feuerstein GZ, eds. Red blood cell substitutes: Basic principles and clinical applications. New York: Marcel Dekker, 1998:189-217. 322. Hess JR, Macdonald VW, Brinkley WW. Systemic and pulmonary hypertension after resuscitation with cell-free hemoglobin. J Appl Physiol 1993;74:1769-78. 323. Keipert PE, Gonzales A, Gomez CL, et al. Acute changes in systemic blood pressure and urine output of conscious rats following exchange transfusion with diaspirin-crosslinked hemoglobin solution. Transfusion 1993;33:701-8. 324. Przybelski RJ, Dailey EK, Birnbaum ML. The pressor effect of hemoglobin—good or bad? In: Winslow RM, Vandegriff KD, Intaglietta M, eds. Advances in blood substitutes: Industrial opportunities and medical challenges. Boston: Birkhäuser, 1997:71- 85. 325. Rioux F, Drapeau G, Marceau F. Recombinant human hemoglobin (rHb1.1) selectively inhibits vasorelaxation elicited by nitric oxide donors in rabbit isolated aortic rings. J Cardiovasc Pharmacol 1995;25:587-94. 326. Pawloski JR, Hess DT, Stamler JS. Export by red blood cells of nitric acid bioactivity. Nature 2001;409:622-6. 327. Asanuma H, Nakai K, Sanada S, et al. S-nitrosylated and pegylated hemoglobin, a newly developed artificial oxygen carrier, exerts cardioprotection against ischemic hearts. J Molec Cell Cardiol 2007;42:924-30. 328. Karmaker N, Dhar P. Effect of steady shear stress on fluid filtration through the rabbit arterial wall in the presence of macromolecules. Clin Exp Pharmacol Physiol 1996;23:299-304. 329. Schultz SC, Grady B, Cole F, et al. A role for endothelin and nitric oxide in the pressor response to diaspirin cross-linked hemoglobin. J Lab Clin Med 1993;122:301-8. 330. Gulati A, Singh S, Rebello S, Sharma AC. Effect of diaspirin crosslinked and stroma-reduced hemoglobin on mean arterial pressure and endothelin-1 concentration in rats. Life Sci 1995;56:1433-42. 331. Gulati A, Sharma AC, Burhop KE. Effect of stroma-free hemoglobin and diaspirin cross-linked hemoglobin on the regional circulation and systemic hemodynamics. Life Sci 1994;55:827-37. 332. Ulatowski JA, Nishikawa T, Matheson-Urbaitis B, et al. Regional blood flow alterations after bovine fumaryl ββ-crosslinked hemoglobin transfusion and nitric oxide synthase inhibition. Crit Care Med 1996;24:558-65. 333. Winslow RM, Gonzales A, Gonzales M, et al. Vascular resistance and the efficacy of red cell substitutes in a rat hemorrhage model. J Appl Physiol 1998;85:993-1003. 334. Vandegriff KD. Stability and toxicity of hemoglobin solutions. In: Winslow RM, Vandegriff KD, Intaglietta M, eds. Blood substitutes: Physiological basis of efficacy. Boston: Birkhäuser, 1995:105-31. 335. Light WR, Jacobs EE, Rentko VT, et al. Use of HBOC-201 as an oxygen therapeutic in the preclinical and clinical settings. In: Rudolph AS, Rabinovici R, Feuerstein GZ, eds. Red blood cell substitutes. New York: Marcel Dekker, 1998:421-36. 336. Patel MJ, Webb EJ, Shelbourn TE, et al. Absence of immunogenicity of diaspirin cross-linked hemoglobin in humans. Blood 1998;1:710-6. 337. Chang TMS, Lister C, Nishiya T, Varma R. Immunological effects of hemoglobin, encapsulated hemoglobin, polyhemoglobin and conjugated hemoglobin using different immunization

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4

Red Cell Metabolism and Preservation Bjarte G. Solheim1 & John R. Hess2 1 2

Professor Emeritus, Institute of Immunology, Rikshospitalet University Hospital of Oslo, Oslo, Norway Professor, Blood Bank, University of Maryland Medical Center, Baltimore, Maryland, USA

A human red cell, mature and released from the marrow, lacks a nucleus and mitochondria. It has a life span of 120 days, after which it is removed from the circulation in the natural course of aging. It can neither use oxygen for the extraction of energy, nor synthesize proteins or nucleotides. Its primary functions— transporting oxygen from the lungs to the tissues and carrying carbon dioxide back to the lungs—do not require the expenditure of energy. However, maintaining hemoglobin in an optimal state for delivery of oxygen and keeping a normal cell morphology do require active metabolism, and are prerequisites for function and successful transfusion. During standard storage at 4ºC, significant and, in part, reversible changes in red cell morphology and metabolism occur. One striking example is the change in red cell shape (Fig 4-1), which can be reversed to a large extent by metabolic rejuvenation of the cells. In order to optimize storage with the development of optimal strategies, it is essential to understand basic principles of red cell metabolism. Another example of a reversible change is the binding of oxygen. As detailed in Chapter 3, hemoglobin flips between a taut (low oxygen affinity) state (T) and a relaxed (high oxygen affinity) state (R). Therefore, the process of oxygenation causes the hemoglobin molecule, like the lungs, to “breathe.” However, oxygenation and deoxygenation of hemoglobin are not isolated events, because parallel to the intrasubunit and tetrameric conformational movements related to the T and the R states are changes in the binding of hemoglobin to its other ligands [hydrogen ions, carbon dioxide, chloride ions, 2,3-diphosphoglycerate (2,3-DPG), band 3 in the cell membrane, and nitric oxide]. Together with the influence of temperature, these ligands are mutually interactive in determining the conformational state of hemoglobin, and thus its oxygen affinity. Results obtained in the past decade by several research groups indicate that many intermolecular interactions may be Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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missed when hemoglobin is considered by itself without taking into account the cellular milieu in which it performs its main biological function.2,3 Thus, interaction of hemoglobin and band 3 is influenced by conformational changes that accompany ligand binding to hemoglobin, which modulates the function of band 3, the glycolytic pathway, and the structural architecture of the red cell membrane-cytoskeletal system (Fig 4-2). Human red cells move rapidly through the tissue and lung capillaries (transit time of 0.3-1 second). Cells in the tissue surrounding the microcapillary endothelium signal their requirement for oxygen by release of carbon dioxide and protons. To satisfy this requirement, oxygen must be released from red cells in the vicinity of the cells that signal their need for it.3 Thus, red cell carbon dioxide uptake and oxygen release must be completed within the brief time during which the red cell traverses the capillary and is in contact with endothelial cells. Moreover, the close contact of hemoglobin, band 3, and carbonic anhydrase in the cell membrane promotes the production of bicarbonate with release of protons. The release of protons via the Bohr effect (see Chapter 3) in turn releases oxygen from hemoglobin where it is needed in the tissues. In the following section, red cell metabolism is discussed taking into account intermolecular interactions of importance for the primary function of hemoglobin, which is the transport of oxygen and carbon dioxide.

Metabolism Metabolism of Glucose Under physiologic circumstances, the energy that the red cell requires is derived through the breakdown of glucose to lactate or pyruvate. The sequence of reactions is generally known as the glycolytic or the Embden-Meyerhof pathway.4 This pathway is phylogenetically very old, and the sequence of reactions is the same in bacteria, yeast, and vertebrates. Except for the exaggerated production of 2,3-DPG (also known as 2,3-bisphosphoglycerate) in the red cell, the pathway is the same in all tissues.

Chapter 4: Red Cell Metabolism and Preservation

(A)

1.0

(B)

0.8

(C)

0.6

(D)

0.4

(E)

0.2

(F)

0.0

Figure 4-1. Scanning electron micrographs showing representative red cells in various stages of shape change typical of prolonged storage.1 The cells progress from discocytes (A), through several stages of echinocytes (B-D), to spheroechinocytes (E), and finally to spherocytes (F). Scores based on the visual appearance (shown in the lower right of the individual images) can be assigned to several hundred individual stored cells and averaged to produce a morphology score for the unit. Such scores decrease in a linear manner during storage but can be substantially reversed by rejuvenation.

Glycophorin C Figure 4-2. Schemata of red cell components. In the past, it was believed that oxygen transport by hemoglobin and metabolism were cytosolic functions, and that the membrane and cytoskeleton merely enclosed them. It is now recognized that the attachments of the membrane to the cytoskeleton from band 3, through ankyrin (An) and proteins 4.2 and 4.1 to spectrin, and from glycophorin C through protein 4.1 and actin (Ac) to spectrin are destabilized by 2,3-diphosphoglycerate (2,3-DPG). 2,3-DPG released from deoxyhemoglobin (d-Hb) binds to band 3 and partially detaches the membrane from the cytoskeleton, allowing lateral movement of membrane structures. This could have implications for red cell flexibility in peripheral capillaries. Other abbreviations: O-Hb
oxygen-hemoglobin.

Band 3

Membrane 4.1

4.2

Ac

An 4.1

Spectrin

Cytoskeleton

Glucose

4 O2 d-Hb

o-Hb 2,3-DPG CO2

The reactions of the glycolytic pathway are shown in Fig 4-3. In a sequence of reactions, the six-carbon sugar glucose is phosphorylated, isomerized to fructose phosphate, phosphorylated again, and cleaved into three-carbon sugars. The three-carbon sugars are again phosphorylated. Finally, the carbohydrate-bound phosphate that has been gained is transferred to adenosine

2,3-DPG

Cytosol

Lactate, ATP NADH, NADPH

diphosphate (ADP), producing the high-energy compound, adenosine triphosphate (ATP). The synthesized ATP is used by ATPases for the pumping of ions against concentration gradients, for the phosphorylation of membrane proteins and lipids, and very importantly for the phosphorylation of glucose in the glycolytic pathway.5

55

Section I: Part I

Glucose ATP ADP

NADP NADPH

HK

G-6-PD

Glucose-6-P

6-Phosphogluconate

GPI Fructose-6-P ATP ADP

NADP NADPH

6-PGD PFK

Pentose shunt

Fructose-1,6 Di P Ribulose-5-P

Aldolase

Dihydroxyacetone-P

TPI

Glyceraldehyde-3-P Pi NAD GAPD

NADH

1,3-Diphosphoglycerate ADP

DPGM

2,3-Diphosphoglycerate

PGK

ATP

DPGP

3-Phosphoglycerate MPGM

Pi

2-Phosphoglycerate ENOLASE

2-Phosphoenolpyruvate ADP PK ATP

Pyruvate NADH NAD

LDH

Lactate

The 2,3-DPG Shunt (Rapoport-Luebering Shunt) Production of 2,3-DPG is the function of a shunt that branches from the main glycolytic pathway after the formation of 1,3diphosphoglycerate (1,3-DPG) and returns to it with the formation of 3-phosphoglycerate (3-PGA) (Fig 4-3). The pathway consists of the formation of 2,3-DPG from 1,3-DPG, followed by the dephosphorylation of 2,3-DPG to 3-PGA (Fig 4-4). Both reactions are catalyzed by the same enzyme and are balanced at physiologic pH.6 At higher pH, the enzyme acts only as a mutase, moving phosphate in 1,3-DPG from position 1 to position 2 in the molecule; at low pH it acts only as a phosphatase, transforming 2,3-DPG to 3-PGA. As the 2,3-DPG shunt bypasses one of the two ATP-making steps in glycolysis, 2,3-DPG is made at the expense of ATP. In storage systems, a high pH can shut down ATP production, while a lower than physiologic pH leads to a burst of ATP production driven by the breakdown of 2,3-DPG. The production of large quantities of 2,3-DPG is a unique feature of glycolysis in the red cell. Red cells contain approximately equimolar amounts of hemoglobin and 2,3-DPG. Binding of 2,3-DPG to the β subunits of deoxyhemoglobin serves to stabilize the T (low oxygen affinity) state of hemoglobin and thus shifts the oxygen equilibrium curve to the right (favoring dissociation of oxygen). In the R (high oxygen affinity) state, approximately 80% of 2,3-DPG is “free,” while in the T state over 80% of 2,3-DPG is bound to hemoglobin.7 In the “free” state 2,3-DPG at physiologic concentrations modulates

56

2,3-DPG shunt

Figure 4-3. The glycolytic pathway with the pentose and 2,3-DPG shunts of red cell metabolism. ATP
adenosine triphosphate; ADP
adenosine diphosphate; HK
hexokinase; NADP
nicotinamide adenine dinucleotide phosphate, oxidized form; NADPH
nicotinamide adenine dinucleotide phosphate, reduced form; 6-PGD
6phosphogluconate dehydrogenase; P
phosphate; G-6-PD
glucose-6-phosphate dehydrogenase; PFK
phosphofructokinase; TPI
triosephosphate isomerase; NAD
nicotinamide adenine dinucleotide, oxidized form; NADH
nicotinamide adenine dinucleotide, reduced form; GAPD
glyceraldehyde-3-phosphate dehydrogenase; PGK
phosphoglycerate kinase; DPGM
diphosphoglycerate mutase; DPGP
diphosphoglycerate phosphatase; MPGM
monophosphoglycerate mutase; PK
pyruvate kinase; LDH
lactate dehydrogenase.

properties of the red cell membrane.2 It binds directly to band 3 and thereby interferes negatively in the interactions between protein 4.1, 4.2, ankyrin, and band 3.8 In addition, 2,3-DPG releases spectrin from the membrane skeleton, and interferes negatively in the interactions between spectrin-actin-protein 4.1 and the glycophorin C complex.2,9,10 This decreases the number of connecting links between the cell membrane and the cytoskeleton and increases lateral mobility of integral membrane proteins.11 The rise and fall of 2,3-DPG concentrations with each pass through the circulatory system, therefore, results in repetitive destabilization and restabilization of the membranecytoskeleton architecture (Fig 4-2). “Free” 2,3-DPG increases cell flexibility by weakening the links between the membrane and the cytoskeleton, and facilitates gas exchange by allowing the red cell to slip into narrow capillaries and splenic sinusoids. However, further experiments are needed to clarify the physiologic implications of the interactions of 2,3-DPG, cell membrane proteins, and the cytoskeleton.

The Pentose Shunt (Hexose Monophosphate Shunt) Under normal, steady-state conditions, most glucose is metabolized in red cells by way of the glycolytic pathway, but there is another important metabolic pathway called the pentose shunt or hexose monophosphate shunt (Fig 4-3). Some of the glucose-6-phosphate (G-6-P) formed when glucose is phosphorylated in the hexokinase reaction may enter this pathway. Glucose-6-phosphate dehydrogenase (G-6-PD)

Chapter 4: Red Cell Metabolism and Preservation

Alternative Substrates for Red Cell Metabolism

GAP NAD

Alkali 3 PGA 2 PGA

Pi

Acid 2,3 DPG Phosphate

NADH 1,3 DPG ADP 2,3 DPG ATP

Glucose is the natural substrate for human red cell energy metabolism, but red cells are also capable of metabolizing other sugars (ie, fructose, mannose, galactose, and the three-carbon sugar dihydroxyacetone). However, none of these other sugars have proven to be useful in the design of blood preservatives. The presence in red cells of the enzyme nucleoside phosphorylase makes it possible for red cells to use nucleosides such as inosine to support ATP synthesis: Inosine  Pi → Ribose-1-P  Hypoxanthine

Pi 3 PGA 2,3 DPG 2 PGA

Acid Pyrosulfite Sulfite Dithionite Phosphoglycolate Phosphate-chloride Alkali 3 PGA PEP ATP

PEP Figure 4-4. Regulation of the formation and breakdown of 2,3-DPG. The steady-state concentration of 2,3-DPG is governed by the rate of its formation from 1,3-diphosphoglycerate and by its breakdown to 3-phosphoglycerate. Both reactions are catalyzed by the same multifunctional enzyme modulated by a variety of substances. Formation of 2,3 DPG by the mutase (DPGM) is enhanced by substances to the right of the upward arrow, and reduced by those to the right of the downward arrow. Similarly, arrows indicate substances modulating the breakdown of 2,3 DPG by the phosphatase (DPGP). GAP
glyceraldehyde3-P; 3 PGA
3-phosphoglycerate; 2 PGA
2-phosphoglycerate; PEP
2-phosphoenolpyruvate.

catalyzes the oxidation of G-6-P to 6-phosphogluconolactone, reducing nicotinamide adenine dinucleotide phosphate (NADP) to NADPH. After hydrolysis of the lactone to 6phosphogluconic acid, another oxidative step reduces additional NADP to NADPH, and releases carbon dioxide from the six-carbon compound, forming the pentose sugar ribose-1phosphate. After a series of rearrangements, two normal intermediates of the main glycolytic pathway—fructose-6-phosphate and glyceraldehyde-3-phosphate—are formed and rejoin the main metabolic stream. The pentose shunt is important to the red cell primarily as a source of NADPH. It is this reduced nucleotide that maintains glutathione (GSH) in its reduced form. Reduced GSH is important for the elimination of peroxide, protection of protein sulfhydryl (SH) groups and detoxification processes. The pentose shunt also plays an important role for the red cell by providing ribose-5-phosphate needed for the production of phosphoribosyl pyrophosphate (PRPP), an essential substrate for the synthesis of adenine nucleotides required for ATP synthesis (see below).

In this reaction, ribose-1-phosphate is formed without the expenditure of ATP. Ribose-1-phosphate is then readily converted to fructose-6-phosphate by the pentose shunt, which feeds into the glycolytic pathway leading to the generation of ATP. In the rejuvenation of red cells (see later) inosine allows ATP-depleted red cells to prime their metabolic pump (the glycolytic pathway). It has not been possible to include inosine in blood preservatives because the product of its metabolism, hypoxanthine, is rapidly converted to uric acid in the body. Because many patients who receive blood transfusions have impaired liver function or may already have hyperuricemia because of hereditary or acquired factors, a blood component that increases plasma uric acid level cannot be considered safe.12

Regulation of Energy Metabolism Rate of Glucose Metabolism In general, metabolic regulation is dependent on protein synthesis, which, in turn, is regulated by increasing or decreasing the rate of DNA transcription or the translation of messenger RNA. Red cells do not have this option. Instead, the rate of glucose metabolism is regulated by feedback mechanisms. However, in spite of extensive studies, our understanding of the control of glucose metabolism by red cells is still incomplete. The N-terminal cytoplasmic domain of band 3 binds hemoglobin, cytoskeletal proteins, and glycolytic enzymes.13 Based on current evidence, de Rosa et al2 point out that interaction between hemoglobin in the T state and band 3 causes a release of glycolytic enzymes, which results in increased activity of the main glycolytic pathway. On the other hand, hemoglobin in the R (highly oxygenated) state is associated with increased activity of the pentose shunt because the enzymes of the main glycolytic pathway show reduced activity when bound to band 3.14 The rate of glucose metabolism by red cells is influenced by many factors other than enzyme activity. Negative feedback mechanisms seem to be involved in the glycolytic pathway and 2,3-DPG is involved in several. Both hexokinase and phosphofructokinase are inhibited by hydrogen ions. Thus, one of the principal reasons for the markedly slow rate of glycolysis during blood storage is the accumulation of lactic acid in whole blood or the acidity of first-generation additive solutions.

57

Section I: Part I

2,3-DPG Concentration The concentration of 2,3-DPG depends on the rate of its formation and degradation. Many effectors determine whether the mutase or the phosphatase activity of the diphosphoglycerate mustase/phosphatase predominates (Fig 4-4). The hydrogen ion concentration is one of the most important physiologic modulators. At low pH, phosphatase activity is stimulated and mutase activity is inhibited. Thus, high pH favors 2,3-DPG maintenance and production during storage at the expense of ATP formation, whereas low pH leads to the rapid loss of 2,3-DPG with an increase in ATP production. Modulation of red cell metabolism by pH has been the principal means used to retard the decline of 2,3-DPG levels that occurs during liquid storage of red cells (see later). After depletion during storage, 2,3-DPG levels in transfused red cells return to 50% of normal in 7 hours and to almost 95% at 72 hours.15

synthesize GSH from its precursor amino acids. Adenine is able to enter the erythrocyte and purine nucleotides are synthesized through the adenine phosphoribosyl transferase reaction:

Pentose Shunt Activity The rate of the pentose pathway is influenced by the availability of NADP and the level of NADPH. Under oxidative stress NADPH is oxidized to NADP and the activity of the pentose pathway increases, which is consistent with the observation of increased pentose shunt activity when hemoglobin is in the R state.

Membrane Metabolism

Adenosine Triphosphate In the red cells, ATP is synthesized by the glycolytic pathway, but its regulation is complex. ATP is used in a number of different metabolic pathways—particularly by the kinases, which phosphorylate sugars (ie, hexokinase, phosphofructokinase) and proteins, and by ATPase-driven ion pumps (ie, Na-K-ATPase, Mg2ATPase, and Ca2-ATPase). Moreover, red cell membrane shape and rigidity are controlled by the ATP-dependent cytoskeleton. ATP is in equilibrium with ADP and adenosine monophosphate (AMP). As the level of ADP increases, some is converted to AMP. AMP, in turn, is deaminated in the AMP-deaminase reaction. The total red cell pool of adenine decreases during storage, which leads to the depletion of ATP if adenine is not added to the anticoagulant and/or the additive solution. Guanine Nucleotides Red cells contain and turn over guanine nucleotides, which perform at least two functions in red cells. First, guanine nucleotides play a role in the signal transduction of membrane shear into secretion of local vasodilators cyclic AMP and ATP.16 Second, high concentrations of guanosine triphosphate (GTP) inhibit red cell transglutaminase, a primitive coagulation system that also interacts with the cytoplasmic domain of band 3 and with protein 4.1.17 The GTP concentration is reduced when red cells age so that transglutaminase, which has a Factor XIII-like activity, can facilitate the removal of senescent red cells by binding them to fibrin clots.

Synthetic Processes Red cells are able to assemble only a limited repertoire of important molecules from simpler precursors. They retain the capacity to synthesize nucleotides through a “salvage pathway” and to

58

Adenine  PRPP → AMP  PP This reaction is critical in blood storage. The beneficial effect of addition of adenine to stored blood depends on it. PRPP, one of the substrates for the formation of AMP in this reaction, is synthesized from pentose-phosphate formed in the pentose shunt. Guanine nucleotides form in an analogous reaction and are catalyzed by a different enzyme, hypoxanthine-guanine phosphoribosyl transferase. Red cells also actively synthesize a number of other small molecules, including reduced glutathione, NAD, and S-adenosyl-L-methione.5

The red cell membrane is composed of a phospholipid bilayer containing cholesterol molecules and membrane proteins.18 The phospholipids are disposed with a predominance of phosphatidylcholine and sphingomyelin in the outer leaflet and phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine (PS) in the inner leaflet. Loss of phospholipid asymmetry results in exposure of PS, which is an important apoptopic marker on the red cell surface. Exposure of PS on the outer cell surface promotes red cell removal from the circulation, while the surface glycoprotein CD47, which decreases during storage, inhibits phagocytosis.19 The phospholipid asymmetry is maintained by the ATP-dependent flippase (aminophospholipid translocase) activity. This activity counteracts phospholipid scrambling, which moves PS from the inner to the outer cell surface. Flippase activity decreases during storage, but can be corrected by rejuvenation of the red cells.20 Phospholipid scrambling is normally low during storage, but can be enhanced by photodynamic treatment for pathogen inactivation.21 Maintaining the iron of hemoglobin in a reduced state is a prerequisite for effective oxygen transport. The enzyme involved is methemoglobin reductase, which reduces Fe3 to Fe2 by oxidizing the reduced form of nicotinamide adenine dinucleotide (NADH) to NAD. Protection of the SH groups of hemoglobin and membrane proteins from oxidation is also a crucial function. This is accomplished by maintaining adequate amounts of reduced GSH by oxydation of NADPH to NADP. The oxidation of GSH to GSSG catalyzed by peroxidase reduces H2O2 to H2O and Fe3 to Fe2. Adequate levels of ATP, NADH, NADPH, and 2,3-DPG for these metabolic functions are secured by the glycolytic pathway with its pentose and 2,3-DPG shunts. In addition, the synthesis of NAD and NADP from nicotinic acid must also occur. Membrane proteins such as glycophorin and band 3 extend through the lipid membrane. Branches of carbohydrates anchored to membrane proteins protrude from the outer surface of the membrane. The inner surface of the membrane is lined by the cytoskeleton, which is anchored to the membrane through the N-terminal cytoplasmic domain of band 3. The tetrameric form of band 3 binds ankyrin and constitutes with proteins 4.1

Chapter 4: Red Cell Metabolism and Preservation

and 4.2 a major attachment site for spectrin. A second attachment site for spectrin involves actin and protein 4.1 in the glycophorin C complex. Both attachments (Fig 4-2) are weakened when the concentration of “free” (non-hemoglobin-bound) 2,3DPG is increased; this could be of importance for cell flexibility and gas delivery.2 Membrane shape and deformability are controlled by the ATP-driven cytoskeleton. Because some capillaries in the microcirculation have a diameter of only half that of a red cell, loss of flexibility and deformability is a serious storage lesion responsible for removal of rigid cells (Fig 4-1). The red cell membrane also contains a number of transport proteins such as the glucose transporter, Ca2-ATPase, the Na- K-ATPase, the GSSG (oxidized glutathione) transport ATPases, and amino acid transporters, in addition to various other transport channels. A prime metabolic activity of the red cell is maintaining osmotic stability through the activity of its membrane pumps, which are ATP driven. The Na-K-ATPase is highly sensitive to changes in temperature and scarcely functions at 4ºC. During storage in the cold, sodium diffuses into the cells and potassium leaks out until a new equilibrium is reached. The leakage of potassium is further increased by irradiation. Increased potassium content in plasma or in the additive solution of stored Red Blood Cell (RBC) units presents a potential hazard to neonates, although under most other circumstances it can be ignored.15 Citrate in plasma increases potassium toxicity, while reducing red cell supernatant volume results in less potassium leakage before equilibrium is reached. Studies performed over 30 years ago indicate that restoration of potassium after transfusion is slow, and can take more than 6 days.15 Surprisingly, there are no reports on restoration of potassium after transfusing RBC units with modern anticoagulant and additive solutions. Although the macromolecules of the membrane are produced by red cell precursors, some of the components of the membrane are metabolically quite active. For example, cholesterol in the membrane exchanges readily with cholesterol in the plasma, phosphatidylinositol undergoes active phosphorylation, and some proteins are phosphorylated by protein kinases and dephosphorylated by phosphatases. These reactions may influence the functional status of membrane components, but their importance is not yet fully understood.

Summary In the circulation, red cells metabolize glucose by the glycolytic pathway with its pentose and 2,3-DPG shunts. The energy gained provides ATP to maintain ion and glucose concentration gradients between the plasma and erythrocyte and to secure red cell deformability. It also secures NADH to keep hemoglobin in the reduced state and NADPH to protect SH-groups on hemoglobin and membrane proteins. Finally, the production of 2,3DPG is important for the optimal dissociation of oxygen from hemoglobin while the rise and fall of non-hemoglobin-bound 2,3-DPG with each pass through the circulatory system induces repetitive changes in the membrane-cytoskeleton architecture,

which could have implications for red cell flexibility and gas transport.

Red Cell Preservation in Transfusion Medicine General Considerations and Principles RBCs are the most commonly transfused blood components, and their use in a variety of physical circumstances and clinical conditions has shaped their development as products. As an example, the US military’s need to reduce the weight and breakage of blood bottles during shipment led to the development and adoption of plastic blood bags.22 This led to closed-system component production and, fortuitously, exposure of red cells to the plasticizer diethylhexyl phthalate (DEHP), which reduced hemolysis during storage.23 Arguments between blood bankers, who wanted to remove the plasma to make albumin and coagulation factors, and surgeons, who wanted to keep the plasma so the blood could flow quickly in trauma patients, led to the development of additive solutions.24 Again, there were unforeseen consequences; the removal of plasma led to better RBC storage and reduced transfusion reactions. Regulatory agencies have also played a role. The US Food and Drug Administration (FDA) recognizes that 4 million patients a year receive RBCs and that the product must be safe for all.25 Adding antibiotics to blood bags to prevent bacterial contamination, which kills five patients a year in the country, may seem like a beneficial advance. However, the agency must consider the accompanying risk of exposing millions of patients to allergenic and toxic drugs, with the probability of greater harm than good being achieved. As a result of many competing forces, the development and adoption of new RBC storage technology has been slow.26 Five major improvements occurred in the last 50 years: 1) the addition of phosphate, 2) the use of plastic bags, 3) the addition of adenine, 4) the development of additive solutions, and 5) the use of leukocyte reduction, which reduces hemolysis. The reasons for this slow progress have had to do with the perception of little need for change, limited investment in change, lack of understanding of the red cell storage lesion, poor developmental strategies, and conservative regulatory stances.27 For 60 years, it has been a societal goal that RBC transfusion should be available, safe, effective, and cheap.24 Making red cells readily available requires 1) the ability to take liquid units out of the refrigerator and administer them immediately to critically ill or injured patients and 2) the ability to find rare units in frozen national or international inventory. Storage systems contribute to blood safety by isolating individual units in closed systems and reducing product breakdown and bacterial growth through cold storage. A major goal of storage systems is to maintain effectiveness by preserving the lifespan and function of fresh red cells to the greatest extent possible. Keeping blood components inexpensive remains a goal, but the social costs of unsafe blood are high, and the costs of additional testing have added significantly to production costs. At the present time, the costs of RBC units are

59

Section I: Part I

rising (see Chapter 1), and controlling those costs has been one factor in limiting progress on storage systems.

A Short History of RBC Storage Systems Rous and Turner developed the first red cell storage solution in 1916, a simple mixture of citrate and glucose.28 It was used initially to store rabbit red cells for heterophil agglutination testing for syphilis, but when the cells appeared to be intact 4 weeks later, they were reinfused back into the donor rabbits, raising the hematocrit without increasing the reticulocyte count or bile in the urine.29 A year later, Robertson used the Rous-Turner solution to build the first successful blood bank in the Harvard Medical Unit attached to the British Expeditionary Force in France.30 The major problem with the Rous-Turner solution was that it could not be heat sterilized, because the sugar caramelized; thus, there was a risk of bacterial contamination from the open mixing of the ingredients and addition of the solution to the bottles. In 1943, Loutit and Mollison solved this problem by lowering the pH of the solution to 5.0 to make acid-citrate-dextrose (ACD) solution.31 ACD could be autoclaved in sterile vacuum bottles and was used as the standard blood collecting solution in the United States and Britain for many years. It was made first to be used at a 1:4 ratio with collected whole blood and later concentrated to be used at a 1:7 ratio, as is now standard, to reduce the dilution of the blood. During research on why red cells seemed to do better in the lower volume ratio of the anticoagulant-nutrient solution, it became clear that the cells passively lose phosphate. This loss could be prevented by adding phosphate to the solution.31 Citrate-phosphate-dextrose (CPD) in a 1:7 volume ratio, 63 mL for 450 mL of whole blood, preserved red cells slightly better than the older ACD.32 CPD became the standard anticoagulant in the United States, but ACD persisted in Europe. Both were used for 21-day storage with recovery of 70% to 80% of the red cells 24 hours after reinfusion to their original donor. At about the same time, Gabrio and colleagues recognized that nucleotides were important for red cell metabolism, but almost a decade passed before researchers determined that adenine was the critical intermediate.33-36 In 1968, Shields formulated a mixture of CPD and adenine, CPDA-1, and showed that it markedly improved whole blood storage.37 However, during the subsequent 11 years that the FDA debated the safety of adenine, plastic bags revolutionized blood banking, and this excellent whole blood storage solution turned out to work less well with the red cells left behind when plasma was removed.38 Beutler and West went on to show that the higher the hematocrit of red cells in concentrates, the lower the red cell ATP concentrations and the in-vivo recovery.39 The obvious answer was to add back more volume and nutrients in the form of an “additive solution,” but the initial attempt to do this with a solution of bicarbonate, adenine, glucose, and phosphate (BAGP), did not work.40 CPDA- 1 was finally licensed as a 5-week storage solution in 1979, but only after Hogman had developed a simple additive solution of saline,

60

adenine, and glucose (SAG) that also worked for 5-week storage but without the adverse effects associated with the high pH.41 SAG went on to immediate use in Sweden. The use of SAG was associated with 1% hemolysis by the end of 5 weeks of storage, so mannitol was subsequently added (SAGM) as a “membrane stabilizer.”42 This reduced the hemolysis by more than 50%. Drawing whole blood into CPD, making component products, and storing the concentrate with an additional 100 mL of SAG-M (CPD/SAG-M) is the standard RBC storage system in Europe. Minimal variants of this basic solution, additive solution-1 (AS-1) and additive solution-5 (AS-5), are widely used in the United States. Also used is a more substantial variant using citrate and phosphate in the place of mannitol (AS-3 or SAG-CP). All of the variants appear to be equivalent and provide about 80% recovery with about 0.3% hemolysis after 6 weeks of storage.26 The most important limits of the first generation of additive solutions are the loss of membrane with resulting loss of deformability and viability that occur with prolonged storage. The progressive loss of 2,3-DPG may also be important in some situations, although the clinical impact is not well understood. More advanced additive solutions have been demonstrated in principle in small human trials, but only one is licensed and only in a few countries.26 They appear to hold the potential for 7 to 8 weeks of storage, but are considered to be attractive more for their ability to improve recovery, reduce membrane loss, and improve 2,3-DPG and/or ATP concentrations for all stored RBCs.

Collection and Separation Procedures The volume of whole blood removed for storage, processing, and transfusion was historically 450 mL (a pint) in Western countries. For some newer collection systems, this amount has been increased to 500 mL (a half-liter) to increase the collection with each donation and to offset the losses associated with leukocyte reduction by filtration. Products derived from either collection volumes, whether leukocyte-reduced or not, are considered one unit. The inter-donor differences in hematocrit and platelet count are sufficiently great that the yields of red cells, platelets, and plasma in components prepared from donor units of either collection volume show considerable overlap. Some have argued for a more standard definition of a unit, perhaps based on grams of hemoglobin, but it would make blood collection more difficult and wasteful.43 As noted above, the volume of anticoagulant-nutrient solution is normally 1/7th the volume of the collected blood, 63 mL for a 450-mL collection and 70 mL for a 500-mL collection. This volume ratio has been a standard for more than 50 years and was used with the anticoagulant-nutrient solutions ACD-A, CPD, and CPDA-1. There has long been a question of whether the first few drops of blood to enter the collection system are injured by their sudden immersion in the acid anticoagulant, but the red cells seem to tolerate this process with minimal hemolysis or loss of viability.44,45 At the end of collection, venous blood with a pH of about 7.35 has been mixed with anticoagulant-nutrient solution with a pH of 5.0 to 5.6 with a resulting pH about 7.05 in the

Chapter 4: Red Cell Metabolism and Preservation

mixture. The high buffer capacity of the hemoglobin molecule limits the effects of the acidic primary collection solution. Whole blood in anticoagulant-nutrient solution was licensed for storage for 3 or 5 weeks, but has now been largely replaced by separately stored blood components. Making components from whole blood not only serves more patients but also improves the storage of the individual blood elements. Red cells are best stored cold, platelets at room temperature, and plasma, frozen. Removing the white cells also improves RBC storage by removing a cell population with high energy requirements and potential for damaging red cells by released enzymes.46 Schemes for separating whole blood into components are based on centrifugation. The standard “platelet-rich plasma” method used in the United States involves performing a lowspeed “soft” spin to sediment the red cells against the pole of the bag opposite the connections to the satellite bags and then squeezing off the supernatant platelet-rich plasma from the top of the bag into the first satellite bag with pressure applied to the sides of the base of the bag. Thus, typically, 500 mL of whole blood with a hematocrit of 42%, consisting of 210 mL of red cells and 290 mL of plasma, is collected into 70 mL of anticoagulantnutrient solution, increasing the volume in the bag to 570 mL and reducing the hematocrit to 36%. Removing most of the supernatant plasma will increase the hematocrit in the bag to 80% to 90%, consisting of the original 210 mL of red cells and the remaining 22 to 45 mL of plasma/anticoagulant-nutrient solution mixture. Of the original 290 mL of plasma and 70 mL of anticoagulant-nutrient solution, about 90% is removed in this initial separation process. In Europe an initial hard spin is generally preferred in order to harvest buffy coat for the production of platelets and increase the volume of the separated plasma. As noted above, the red cells do better if most of the plasma volume and anticoagulant-nutrient solution are replaced. Recently, separation of whole blood into plasma and RBC by using a hollow-fiber filtration system has been described.47 Red cell parameters were similar to those obtained when routine centrifugation methods were used, and the filter did not cause hemolysis. Levels of plasma Factor VIII and Factor XI were slightly reduced with this prototype; however, there was no evidence of activation of the coagulation or complement systems and filters that do not interact with coagulation factors can be made.

Table 4-1. Compositions and Properties of Some of the Common Acid Citrate Preservative Solutions

Citric acid (in mM/L) Sodium citrate (in mM/L) Dextrose (in mM/L) Monosodium phosphate (in mM/L) Adenine (in mM/L) pH Volume ratio used (anticoagulant: blood in mL)

ACD-A

ACD-B

CPD

CPDA-1

CP2D

35 97 136 —

21 58 81 —

14 14 116 117 141 142 15.8 16

14 117 284 16

— 5.0 1:7

— — 1:4

— 2 5.6 5.6 1:7 1:7

— 5.6 1:7

ACD
acid-citrate-dextrose (two formulations, A and B); CPD
citratephosphate-dextrose; CPDA-1
citrate-phosphate-dextrose-adenine; CP2D
CPD with double dextrose.

The amounts of the nutrients in the anticoagulantnutrient solutions are critical depending on the intended storage times. There is enough glucose in whole blood to keep the red cells healthy for 4 days. Whole blood, stored in citrate alone for up to 4 days, was considered the safest form of storage before autoclavable solutions containing sugar were developed. The high glucose content of CP2D is necessary because it is used with an additive solution that does not contain sufficient glucose, AS-3. Phosphate exists at 1 to 1.3 mM/L in plasma, but needs to be present at higher concentrations in the nutrient solution to passively diffuse against the high concentrations in the red cells when 2,3-DPG breaks down. Adenine is needed for storage only beyond 3 weeks and is an ingredient of all of the additive solutions. In emergencies in remote locations, it may be necessary to collect fresh whole blood before new supplies of fully tested components are available. Under these circumstances, drawing whole blood into the primary collection bag containing the anticoagulant-nutrient solution and holding the whole blood at room temperature for up to 24 hours is associated with good shortterm preservation of function of the blood components.50 The national blood services of Finland and Israel collect all of their whole blood on one day and process it on the next, holding it at 20ºC overnight in the anticoagulant-nutrient solution before separating it into components.51,52

Anticoagulant-Nutrient Solutions Anticoagulant-nutrient solutions were developed from ACD to CPD to CPDA-1. This path is the result of the discovery of the critical nutrients for red cells during prolonged storage: dextrose, phosphate, and adenine. The recognition that RBC concentrates occasionally ran out of glucose led to development of CP2D, with twice the amount of glucose in CPD, and a never-licensed CPDA- 2 with a third more glucose than CPDA-1.48,49 The molar contents of the five licensed anticoagulant-nutrient solutions are shown in Table 4-1. Note that all of the anticoagulant-nutrient solutions are acidic, reducing the pH of the stored blood below 7.2 and leading to the rapid depletion of 2,3-DPG as it enters the glycolytic pathway.

Additive Solutions The first widely used additive solution, SAG, represented an attempt to replace the volume and sugar lost with plasma removal and add the adenine necessary for storage beyond 3 weeks.41 It was made with normal saline with 4.5% w/v glucose and 2 mM/L of adenine. Stored RBCs had good viability for 5 weeks but 1% hemolysis. In screening a large number of compounds for additives that would reduce the hemolysis, mannitol was identified as a compound that both reduced hemolysis and had an excellent safety record with intravenous infusion. The addition of 30 mM mannitol (SAG-M) reduced

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hemolysis and increased the osmolarity of the solution further.42 The solutions are made acidic to a pH of about 5.6 with hydrochloric acid to allow the glucose to be heat sterilized, but the solutions have essentially no buffer capacity so they do not make the RBC units much more acidic than they were when separated from plasma. SAG-M and its close relatives, AS-1 and AS-5, are the most widely used additive solutions. Other widely used first-generation additive solution systems include CP2D/AS-3 in the United States and Canada, and CPD/MAP (mannitol, adenine, phosphate) in Japan.53 Table 4-2 provides a comparison of the ingredients and concentrations of the more common of these first-generation additive solutions. Second-generation additive solutions started with attempts to rebalance the final suspending solution and a search for additional nutrients for the RBC units. With BAGP, Beutler attempted to preserve both ATP and 2,3-DPG by raising the pH.40 However, raising the pH substantially above 7.2 led to high concentrations

Table 4-2. Compositions (in mM/L) of the Common Licensed First-Generation RBC Additive Solutions

NaCl Phosphate Adenine Glucose Mannitol Citric acid Na3 citrate

SAG

SAG-M

AS-1

AS-3

AS-5

150 — 1.25 45 — — —

150 — 1.25 45 30 — —

154 — 2 111 41.2 — —

70 23 2 55 — 2 30

154 — 2 45 29 — —

SAG
saline-adenine-glucose; SAG-M
saline-adenine-glucose-mannitol; AS-1
Adsol (Fenwal, Lake, Zurich IL), a SAG-M variant formula; AS-3
Nutricel (Pall Medical, East Hills, NY); AS-5
Optisol (Terumo, Somerset, NJ), a SAG-M variant formula.

of 2,3-DPG at the expense of ATP and no improvement in storage function. The composition of a representative group of second-generation additive solutions are shown in Table 4-3. The pH of red cells during storage is very important. Above pH 7.2, the bifunctional enzyme diphosphoglycerate mutase/phosphatase converts almost all 1,3-DPG into 2,3-DPG, depriving the cell of new ATP.54 Below a pH of about 6.4, the activities of the initial enzymes of glycolysis, hexosekinase and phosphofructokinase, are too low to support ATP production. In this narrow pH range between 7.2 and 6.4, hemoglobin, the mineral salts in the suspension, and bicarbonate all serve to buffer the protons produced by glycolysis. The approximately 60 g of hemoglobin present in an RBC unit can buffer about 8 mEq of protons in that pH range.55 However, conventional first-generation acidic additive solutions, which result in an initial pH of 7.0, fail to take advantage of a quarter of that pH range and buffer capacity. Adding 20 mM/L of phosphate delivers 2 mM in the 100 mL of additive solution and buffers about 1 mM of additional protons. Adding 20 mM/L of bicarbonate again delivers 2 mM to the final RBC suspension, which will be protonated to make carbonic acid and converted to carbon dioxide and water by red cell carbonic anhydrase. The resulting solution will buffer 2 mM of protons as the carbon dioxide diffuses out of the plastic bag. Attention to formulation and pH balance in the design of second-generation additive solutions can almost double the amount of ATP energy available to stored red cells by depressing diphosphglycerate mutase activity while sustaining glycolysis. Finding additional critical nutrients has been less successful. The only approved second-generation solution is phosphate, adenine, glucose, guanosine, and saline (PAGGS)-mannitol.56 Guanosine was added because GTP was detected in red cells and known to decrease during storage. However, guanosine nucleotides play only a minimal role in critical events in red

Table 4-3. Composition and Properties (in mM/L) of Some of the Proposed Second-Generation RBC Additive Solutions

NaCl Na gluconate Bicarbonate Phosphate Adenine Glucose Mannitol Guanosine Na3 citrate pH of solution pH of red cell suspension Volume

BAGP-M

PAGGS-M

PAGGG-M

ErythroSol-1

ErythroSol-2 EAS-81

— — 115 1 1 55 27 — — — — 100

72 — — 32 2 52 55 1.5 — 6.3 6.9 110

— 72 — 32 2 52 40 1.5 — 6.3 6.9 110

— — — 20 1.5 45 40 — 25 7.4 7.2 114

— — — 18 1.3 38 50 — 21 8.8 7.3 150

— — 26 12 2 80 55 — — 8.4 7.2 110

BAGP-M 5 bicarbonate-adenine-glucose-phosphate-mannitol, the original additive solution developed by Beutler; PAGGS-M 5 phosphate-adenine-glucose-guanosine-saline-mannitol, licensed in Germany; PAGGG-M 5 phosphateadenine-glucose-guanosine-(sodium) gluconate-mannitol, developed by de Korte as a chloride-free variant of PAGGS-M; ErythroSol-1 and -2 developed by Hogman; EAS-81 5 developed by Hess and Greenwalt.

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cell storage (inhibiting the primative coagulation enzyme transglutaminase) and the solution worked only modestly better than first-generation additive solutions with a 74.6% 24-hour in-vivo recovery after 7 weeks in the only published series. As ingredients were added to advanced additive solutions, the salt concentration was generally reduced to maintain osmotic balance. Reducing the chloride concentration in the suspending solutions caused intracellular chloride to passively leave the red cells, but this can occur only if other anions countered the flow, and phosphate, bicarbonate, and hydroxyl ions are the only available anions.57 The influx of these anions initially increases the intracellular pH, but eventually the pH falls. PAGGG-M, in which the sodium chloride is replaced by sodium gluconate, is an example of an attempt to take advantage of the “chloride shift” phenomenon to maintain red cell 2,3-DPG.58 The ErythroSol solutions were developed by Hogman based on the ideas of using the “chloride shift” to increase the intracellular pH while using a more alkaline final suspending solution and additional phosphate to simultaneously maintain ATP.59 ErythroSol-1 used half-strength citrate (0.5 CPD) and had problems with incomplete anticoagulation in blood collected from donors with low (but acceptable) hematocrit levels. ErythroSol- 2 uses full strength CPD and an alkaline additive solution made with disodium phosphate. In both systems the glucose was placed in a separate bag from the rest of the additive solution during manufacture and sterilization and added only at the time of RBC component production. The original description of ErythroSol-2 suggested that the optimal pH had been determined to preserve both ATP and 2,3DPG.60 However, the study needs confirmation because only 10 units were stored in the ErythroSol-2. The natural variability in donor hematocrit means that some units with low red cell content have low buffer capacity and, therefore, become overly alkaline. The solution can achieve successful 7-week storage. Merely increasing the pH of conventional additive solutions to increase the 2,3-DPG causes the ATP concentrations to fall.61 The experimental additive solution-81 (EAS-81) was the end result of work by Hess and Greenwalt that reexamined the use of bicarbonate as a buffer to prevent erythro-apoptosis by maintaining high concentrations of red cell ATP and limited hemolysis with mannitol and hypotonic conditions.54 Formulating the solutions to get the starting pH as close as possible to 7.2 preserves the 2,3-DPG concentrations for 2 weeks.62 The buffering provided by the bicarbonate probably allows RBC storage for at least 8 weeks based on six recovery measurements.62 Finally, increasing the volume of these advanced additive solutions increases the buffering capacity and allows greater storage time. With 300 mL of additive solutions, Hess and colleagues were able to achieve storage times of 10 to 12 weeks, but the higher volume and lower storage hematocrit probably make such solutions inappropriate for clinical use, especially in infants and massively transfused trauma patients.63

Additional Factors Influencing RBC Quality Temperature and Time Lapses during Collection and Component Preparation Whole blood is collected at body temperature and must be maintained at room temperature if platelets are to be prepared from the collection. By present US regulations, whole blood must be separated into its components in 8 hours if platelets and Fresh Frozen Plasma are to be manufactured. If only RBCs and frozen plasma are to be made, the whole blood can be held on ice for up to 24 hours. The problems with holding blood at room temperature are twofold. Red cells metabolize glucose at higher rates when warm, producing more lactate and protons that reduce the pH and further slow metabolism. In addition, labile coagulation factors are lost. Attempts to validate the 24-hour warm hold for 6-week RBC storage using first-generation additive solutions have not been successful. However, in Israel and Finland, where this system is used, RBCs are rarely stored for that long because the national blood services are efficient and supply lines are short. As noted above, fresh whole blood is occasionally collected in emergencies on the battlefield or in isolated locations such as Pacific Island nations and stored at room temperature. Based on the above experience and experimental data, blood probably maintains reasonable functionality for 24 to 72 hours if maintained between 19 and 25ºC.52 However, such use can be recommended only in the most urgent of situations. Temperature and Time Lapses during Storage RBCs that have been allowed to rewarm to greater than 10ºC during storage are considered unfit for transfusion and are destroyed per FDA regulations. This has led to the wide use of the “30minute rule,” meaning that units are discarded if they have been off ice for more than 30 minutes. Experimental work has shown that glucose is metabolized about 10 times as fast at 25ºC as at 4ºC and that RBCs stored at 25ºC lose viability 10 times as fast. Thus, a day of storage at room temperature would reduce the invivo recovery equivalent to 10 days of 4ºC storage.64,65 The regulation is very conservative with regard to temperature but it is also intended to prevent bacterial overgrowth in contaminated units. Storage Containers Polyvinyl chloride bags plasticized with DEHP are the standard RBC storage containers. The presence of the DEHP reduces hemolysis by 4-fold during storage by intercalating into the red cell membrane.66 Other plasticizers, such as butyryl-n-trihexyl citrate, work almost as well, but are more expensive (⬃5% more for a multibag set with leukocyte filter) and have an unusual smell when initially unwrapped. While questions about the safety of DEHP have been raised, they are based on very limited animal testing and must be balanced against the obvious safety value of being able to visually inspect the contents of blood bags and reduce RBC losses by extending shelf-life. The general trend in Europe to avoid the slowly degradable plasticizer, DEHP, has resulted in the

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introduction of butyryl-n-trihexyl citrate plasticized multibag systems in Sweden, Spain, and Norway.

Leukocyte Reduction Leukocyte reduction improves RBC storage by removing a highly metabolically active blood component that would make the bag more acidic sooner. White cells also secrete cytokines, and when they die after exposure to the cold, they release proteolytic, glycolytic, and lipolytic enzymes that damage the red cell surface. On the other hand, some red cells are damaged in the filters, and typically 15 to 35 mL of blood is lost in the filters, depending on filter size and whether whole blood or concentrated red cells are being leukocyte reduced. Red cells from donors with sickle cell trait and mild thalassemia may also clog filters. Leukocyte reduction improves the red cell 24-hour in-vivo recovery by several percent according to the best estimates.46 Washing Washing RBCs in saline to remove the plasma causes small losses of red cells in the bag transfers, as well as loss of the supporting nutrients, glucose, phosphate, and adenine. Because the cells are used soon after washing, generally within 6 to 24 hours, only the glucose loss is physiologically important. There is still a small amount of glucose inside the washed red cells, but it is metabolized quickly especially if the cells are not promptly refrigerated. In the absence of glucose, metabolism stops and the cells are very susceptible to oxidative stress and erythro-apoptotic changes. Irradiation During storage at 4ºC, red cells lose potassium. This potassium collects in the supernatant fluid in the closed storage bag at a rate of about 1 mEq/day until equilibrium is reached between intraand extracellular concentrations, usually at about 60 to 70 mEq/L depending on the storage hematocrit (Table 4-4). Gamma irradiation in doses of 2500 cGy, given to prevent graft-vs-host disease, damages the red cell membranes and increases this rate of potassium loss to approximately 1.5 mEq/day; however the reinfused red cells have normal in-vivo survival.67 The current FDA regulation that irradiated RBCs expire 28 days after irradiation limits the potential maximum potassium concentration by limiting the period of potassium loss. Nevertheless, care must be taken in all situations when large volumes of older, high-potassium RBC units are used to prime cardiopulmonary bypass, dialysis, or apheresis circuits and then administered at high flow rates into the central circulation. Pathogen Reduction Pathogen reduction systems developed so far require additional manipulation of and stresses on the stored red cells that are system specific. Examples from two such proposed systems are illustrative. Diethanolamine (DEA) was proposed as a red cell permeable nucleic acid cross-linker with broad pathogen killing potential and minimal direct red cell toxicity. However,

64

Table 4-4. Effect of Storage Duration on Characteristics of RBC Concentrates in AS-1

% recovery % hemolysis ATP µM/g Hb [K] meq/L in plasma

35 Days (n ⴝ 25)

42 Days (n ⴝ 10)

49 Days (n ⴝ 10)

56 Days (n ⴝ 10)

86 0.28 3.1 45

82 0.32 2.7 50

76 0.51 2.3 52

71 0.68 2.3 60

Unpublished data obtained from the US Food and Drug Administration under the Freedom of Information Act. ATP
adenosine triphosphate; Hb
hemoglobin.

the system required exposure of the cells to DEA for 20 hours at room temperature and extensive secondary washing to reduce the remaining amounts of this carcinogenic chemical. When the time- and temperature-related decrease in red cell pH was added to the effects of washing with large volumes of acidic solutions and storage in an acidic additive solution, red cell recovery suffered.68 Riboflavin, a photoactive oxidizer, has also been proposed as a highly red cell permeable molecule for pathogen reduction in conjunction with ultraviolet light. However, red cells are so optically dense that the units must be diluted and transferred to large bags only a few millimeters thick for photo-treatment, and the photo-treatment must be done above a critical temperature. The bag transfers all involve losses of red cells, the choice of diluent fluid is important, and the hemoglobin and other optically active molecules in the red cells are also damaged by the light exposure. The red cells need additional energy to attempt to correct the damage. Finally, the reconcentrated and treated red cells need to be stored in an additive solution that is balanced to maximize their life span.

Functionality Since the discovery of the effect of 2,3-DPG on the oxygen affinity of hemoglobin, it has been common to discuss the functionality of stored red cells in terms of their 2,3-DPG content.69 This is an oversimplification for several reasons. First, while it is clear that low 2,3-DPG can affect oxygen delivery in animal models at critically low hemoglobin concentrations, these concentrations are well below standard transfusion triggers.70 Second, 2,3-DPG works in red cells by binding and stabilizing deoxyhemoglobin. This moves the base of the oxygen-hemoglobin dissociation curve to the right and increases the P50, but most oxygen transport occurs at the top of the binding curve which is relatively unaffected, and even in critical situations, the arterial PO2 is higher than the P50. However, 2,3-DPG may play an important role in red cell membrane transport and cytoskeleton architecture because of the its interaction with attachment points between the cell membrane and the cytoskeleton, and the interaction of deoxyhemoglobin with band 3. These interactions, the critical importance of oxygen transport, and the retrospective data that

Chapter 4: Red Cell Metabolism and Preservation

suggest an association between adverse patient outcomes and increased storage time of banked RBCs, drives continuing efforts to reduce 2,3-DPG depletion during storage with modern additive solutions. Until that is achieved, it is recommended in some countries, such as Norway, to use RBCs in SAG-M, not older than 10 days, for the transfusion of intensive care patients. Probably more important for normal red cell function is the loss of membrane, cell deformability, and ATP secretion that occurs with storage. Direct observation of the flow characteristics of fresh, stored, and stored rejuvenated RBCs suggests that ATP secretion in response to decreased shear, by which red cells dilate small vessels to maintain their forward flow, is most important.71 Membrane deformability, which is regulated by the ATP-modulated cytoskeleton, is very important for the passage of red cells through capillaries half their diameter. Maintaining high ATP concentrations is, therefore, an important function of modern additive solutions.

Rejuvenation Red cell loss of viability during storage is different from red cell senescence in the body. In the body, red cells undergo cumulative oxidative damage that leads to reduced enzyme activities and cross-linking of cytoskeletal components. These are essentially irreversible processes and are ultimately associated with macrophage clearance of old red cells, possibly mediated by phosphatidyl serine exposure or neoantigen formation. The changes that lead to loss of viability during storage are largely reversible by a process called “rejuvenation.” Hogman showed that rejuvenating red cells at the end of 6 weeks of storage in SAG-M increased their 24-hour in-vivo recovery from 77% to 89%.72 This rejuvenation is a strictly metabolic recharging of red cells at the end of their storage period. Such cells have a low pH as well as low ATP and 2,3-DPG concentrations. They can be rejuvenated by incubation in a high pH solution of phosphate, inosine, pyruvate, and adenine (PIPA, Rejuvesol, Cytosol Laboratories, Braintree, MA) for 2 hours. Such incubation increases their ATP and 2,3-DPG concentrations and increases their in-vivo recovery, probably by allowing them to internalize negatively charged membrane phospholipids that would otherwise signal clearance by macrophages. Return of the normal distribution of phospholipids also prevents red cells from participating in plasma coagulation reactions. Rejuvenation does not reverse the oxidative damage to band 3 of the cell membrane. However, the fact that RBC can be rejuvenated at the end of storage by increasing their pH and ATP suggests that improved storage could be achieved by a method designed to maintain pH and ATP. This is the fundamental idea behind second-generation additive solutions.

Frozen Storage of RBCs RBCs can be frozen, and in the frozen state they are stable for long periods—Valeri has reported storage of 37 years.73 Four methods of freezing RBCs have been extensively tested, two have been developed for practical use, and one remains in common use.74

During red cell freezing, water turns to ice and the salt concentration of the remaining intracellular water increases, drawing in more water and expanding the cell. Under normal circumstances, this leads to cell rupture. Rupture can be prevented by freezing the cells so rapidly that water does not have time to enter, or by diluting the total cellular water with a cryoprotectant so that not enough water enters to rupture the cell. While several cryoprotectants can work, glycerol is the standard material used because of its cost and safety. The two systems for RBC cryopreservation that have been developed for clinical use both use glycerol. One system uses a “low” glycerol concentration of about 20% and rapid cooling by plunge freezing in liquid nitrogen; the other uses a “high” glycerol concentration of about 40% and slow cooling in 80ºC freezers. The low glycerol frozen RBCs must be maintained in the vapor phase of liquid nitrogen whereas the high glycerol frozen RBCs are stable at temperatures below 65ºC. The high cost of maintaining liquid nitrogen freezers and the difficulty of transporting products frozen in liquid nitrogen limit the utility of the low glycerol system. High glycerol frozen RBCs can be transported on dry ice. When the RBCs are thawed, the glycerol must be removed promptly to prevent it from poisoning the red cell metabolism and to protect the recipient because the glycerol-loaded red cells would swell and rupture if placed directly into the bloodstream. Thus, once thawed, the RBCs must be deglycerolized by washing in a set of graded salt solutions. In the past, the glycerolization and deglycerolization of RBCs for freezing were open manual processes, so the thawed RBCs had to be used within 24 hours or discarded. The recent partial automation of a closed system for glycerolizing and deglycerolizing RBCs now allows them to be kept after thawing in the liquid state for 2 weeks.75 RBCs collected in any of the standard licensed systems can be frozen up to 6 days after collection and stored for up to 10 years. The deglycerolized RBCs are then stored in AS-3. Net losses of red cells in the freeze-thawwash process are approximately 5% to 15%, and the 24-hour in-vivo recovery of infused red cells is about 78% after 2 weeks. Frozen RBCs entail substantial costs for processing and storage, probably four times that of a standard liquid unit, so only for the rarest blood units is there an advantage for frozen inventory. Other systems of freezing and freeze-drying have been demonstrated, but are associated with greater than 1% hemolysis and so would require a washing step before administration. This limitation prevents their use in emergency medicine, and they are too costly and labor intensive to compete with glycerol frozen RBCs.74

Validation of RBC Storage Systems RBC storage systems have historically been validated by demonstrating that the stored cells do not hemolyze during storage and that they circulate normally after reinfusion. Measures of normal circulation have included increments in recipient hemoglobin, and determining the recovery and in-vivo half life of the transfused red cells. Transfused red cells have been counted using differential agglutination, radioactive tracer labeling, and flowcytometric differential counting.

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Chromium-51 labeling has been the standard method used in the United States for 50 years. There is a published standard method, but only a handful of laboratories perform the measurement, and a recent attempt to gather a decade’s experience identified only 900 measurements.76 Chromium-51 labeling persists in the United States because it is the only validated method compatible with autologous red cell reinfusion, and the infectious disease risks of allogeneic RBCs are considered unacceptable for storage system development work. The methods of red cell labeling have been the subject of an excellent review.77 When performing chromium-51 labeling, it is important to recognize that red cells from different donors may have very different 24-hour in-vivo recovery values. In a typical study of a modern additive solution with an 84% mean recovery, individual RBC units have recoveries as high as 95% and as low as 65%.48 These differences in viability correlate with differences in the red cell ATP concentrations, but poorly (r2
0.4). There have been attempts to measure the functionality of red cells, as opposed to their survival. Presumably, the functionality of red cells is related to their ability to deliver oxygen to tissues and to flow in the microcirculation. However, none of the red cell rigidity or microcirculatory flow measures is widely available or validated. The level of 2,3-DPG is just a surrogate marker for red cell function and is limited by the difficulty of performing the test. In a recent shared sample exercise, 12 of the world’s premier laboratories could not consistently measure 2,3-DPG.78 Finally, in countries that do not perform chromium-51 recovery measures, there has been a trend to use the ATP level as a red cell quality measure. While the ATP is a poor surrogate for recovery in small clinical trials, its central role in the inhibition of erythro-apoptosis probably gives it special importance. Unfortunately the measure is not very reproducible from laboratory to laboratory, so a wide margin of safety is required.78

Summary RBC storage systems work remarkably well, making red cells for transfusion available, safe, effective, and cheap for the populations who can organize effective national blood systems. They are a product of slow and empiric development, and our present systems do not reflect our current understanding. They can be expected to improve in the future.

Acknowledgment The authors would like to thank Stein Holme, PhD, for valuable discussions and comments on this chapter.

Disclaimer The authors have disclosed no conflicts of interest.

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References 1. Usry RT, Moore GL, Manalo FW. Morphology of stored, rejuvenated human erythrocytes. Vox Sang 1975;28:176-83. 2. De Rosa MC, Alinovi CC, Galtieri A, et al. The plasma membrane of erythrocytes plays a fundamental role in the transport of oxygen, carbon dioxide and nitric oxide and in the maintenance of the reduced state of the heme iron. Gene 2007;398:162-71. 3. Bruce LJ, Beckmann R, Ribeiro ML, et al. A band 3-based macrocomplex of integral and peripheral proteins in the RBC membrane. Blood 2003;101:4180-8. 4. Beutler E. The red cell: A tiny dynamo. In: Wintrobe MM, ed. Blood pure and eloquent. New York: McGraw-Hill, 1980:141-68. 5. Beutler E. Red blood cell metabolism. In: Simon TL, Dzik WH, Snyder EL, et al, eds. Rossi’s principles of transfusion medicine. 3rd ed. Philadelphia: Lippincott Williams & Wilkins, 2002:43-9. 6. Chiba H, Ikura K, Narita H, et al. Regulation of 2,3 bisphosphoglycerate metabolism in erythrocytes by a multifunctional enzyme. Acta Biol Med Ger 1977;36:491-505. 7. Bunn HF, Ransil BJ, Chao A. The interaction between erythrocyte organic phosphates, magnesium ion, and hemoglobin. J Biol Chem 1971;246:5273-9. 8. Moriyama R, Lombardo CR, Workman RF, Low PS. Regulation of linkages between the erythrocyte membrane and its skeleton by 2,3diphosphoglycerate. J Biol Chem 1993;268:10990-6. 9. Sheetz MP, Casaly J. 2,3-diphosphoglycerate and ATP dissociate erythrocyte membrane skeletons. J Biol Chem 1980;255:9955-60. 10. Cohen CM, Foley SF. Biochemical characterization of complex formation by human erythrocyte spectrin, protein 4.1, and actin. Biochemistry 1984;23:6091-8. 11. Schindler M, Koppel DE, Sheetz MP. Modulation of membrane protein lateral mobility by polyphosphates and polyamines. Proc Natl Acad Sci U S A 1980;77:1457-61. 12. Beutler E. Liquid preservation of red blood cells. In: Simon TL, Dzik WH, Snyder EL, et al, eds. Rossi’s principles of transfusion medicine. 3rd ed. Philadelphia: Lippincott Williams & Wilkins, 2002:50-61. 13. Campanella ME, Chu H, Low PS. Assembly and regulation of a glycolytic enzyme complex on the human erythrocyte membrane. Proc Natl Acad Sci U S A 2005;102:2402-7. 14. Low PS, Rathinavelu P, Harrison ML. Regulation of glycolysis via reversible enzyme binding to the membrane protein, band 3. J Biol Chem 1993;268:14627-31. 15. The transfusion of red cells. In: Klein HG, Anstee D. Mollison’s blood transfusion in clinical medicine. 11th ed. Oxford: Blackwell, 2005:352-405. 16. Olearczyk JJ, Stephenson AH, Lonigro AJ, Sprague RS. NO inhibits signal transduction pathway for ATP release from erythrocytes via its action on heterotrimeric G protein Gi. Am J Physiol Heart Circ Physiol 2004;287:H748-54. 17. Gutierrez E, Sung LA. Interactions of recombinant mouse transglutaminase with membrane skeletal proteins. J Membr Biol 2007; 219:93-104. 18. Gallagher PG, Forget BG. The red cell membrane. In: Beutler E, Lichtman MA, Coller BS, et al, eds. Williams’ hematology. New York: McGraw-Hill, 2001:333-43. 19. Bessos H, Seghatchian J. Red cell storage lesion: The potential impact of storage-induced CD47 decline on immunomodulation and survival of leucofiltered red cells. Transfus Apher Sci 2005;32:227-32.

Chapter 4: Red Cell Metabolism and Preservation

20. Verhoeven AJ, Hilarius PM, Dekkers DWC, et al. Prolonged storage of red blood cells affects aminophospholipid translocase activity. Vox Sang 2006;91:244-51. 21. Hilarius PM, Ebbing IG, Dekkers DW, et al. Generation of singlet oxygen induces phospholipid scrambling in human erythrocytes. Biochemistry 2004;43:4012-9. 22. Artz CP, Howard JM, Davis JH, et al. Plastic bags for intravenous infusions: Observations in Korea with saline, dextran and blood. In: Howard JM, ed. Battle casualties in Korea: Studies of the surgical research team. Washington, DC: Walter Reed Army Medical Center, 1952: 219-24. 23. AuBuchon JP, Estep TN, Davey RJ. The effect of the plasticizer di-2-ethylhexyl phthalate on the survival of stored RBCs. Blood 1988;71:448-52. 24. Collins JA. The surgeon’s point of view. In: Chaplin H Jr, Jaffe ER, Lenfant C, Valeri CR. Preservation of red blood cells. Washington, DC: National Academy of Sciences,1973:339-44. 25. Fratanoni JC. Safety of DEHP: Role of the bureau of biologics. Environ Health Perspect 1982;45:143-4. 26. Hess JR. An update on solutions for red cell storage. Vox Sang 2006;91:13-9. 27. Wallace EL, Surgenor DM, In: Chaplin H Jr, Jaffe ER, Lenfant C, Valeri CR. Preservation of red blood cells. Washington, DC: National Academy of Sciences, 1973:9-19. 28. Rous P, Turner JW. The preservation of living red blood cells in vitro. J Exp Med 1916;23:219-37. 29. Rous P, Turner JW. The transfusion of kept cells. J Exp Med 1916;23:239-47. 30. Robertson OH. Transfusion with preserved red blood cells. Br Med J 1918;1:691-5. 31. Loutit JF, Mollison PL. Advantages of a disodium-citrate-glucose mixture as a blood preservative. Br Med J 1943;2:744-5. 32. Gibson JG, Rees SB, McManus TJ, Scheitlin WA. A citrate phosphate dextrose solution for the preservation of human blood. Am J Clin Pathol 1957;28:569-78. 33. Orlina AR, Josephson AM. Comparitive viability of RBC stored in ACD and CPD. Transfusion 1969;9:62-9. 34. Gabrio BW, Donohue DM, Finch CA. Erythrocyte preservation. V. Relationship between chemical changes and viability of stored blood treated with adenosine. J Clin Invest 1955;34:1509-12. 35. Nakao K, Wada T, Kamiyama T, et al. A direct relationship between adenosine triphosphate level and in vivo viability of erythrocytes. Nature 1962;194:877-8. 36. Simon ER, Chapman RG, Finch CA. Adenine in red cell preservation. J Clin Invest 1962;41:351-9. 37. Shields CE. Effect of adenine on stored erythrocytes evaluated by autologous and homologous transfusions. Transfusion 1969;9:115-9. 38. Zuck TF, Bensinger TA, Peck CC, et al. The in vivo survival of red blood cells stored in modified CPD with adenine: Report of a multiinstitutional cooperative effort. Transfusion 1977;17:374-82. 39. Beutler E, West C. The storage of hard-packed red blood cells in citrate-phosphate-dextrose (CPD) and CPD-adenine (CPDA-1). Blood 1979;54:280-4. 40. Beutler E, Wood LA. Preservation of RBC 2.3-DPG and viability in bicarbonate containing medium: The effect of blood-bag permeability. J Lab Clin Med 1972;80:723-80. 41. Hogman CF, Hedlund K, Zetterstrom H. Clinical usefulness of red cells preserved in protein-poor media. N Engl J Med 1978;299:1377-82.

42. Hogman CF, Hedlund K, Sahlestrom Y. Red cell preservation in protein-poor media. III. Protection against in vitro hemolysis. Vox Sang 1981;41:274-81. 43. Hogman CF, Knudson F. Standard units of RBCs: Is it time for implementation? Transfusion 2000;40:263-5. 44. Valeri CR, Pivacek LE, Cassidy GP, Rango G. Volume of RBCs, 24and 48-hour posttransfusion survivals, and the lifespan of 51Cr and biotin-X-N-hydroxysuccinimide (NHS)-labeled autologous baboon RBCs: Effect of the anticoagulant and blood pH on 51Cr and biotinX-NHS elution in vivo. Transfusion 2002;42:343-8. 45. Holme S, Elfath MD, Whitley P. Evaluation of in vivo and in vitro quality of apheresis-collected RBC stored for 42 days. Vox Sang 1998;75:212-7. 46. Heaton WA, Holme S, Smith K, et al. Effects of 3-5 log10 prestorage leucocyte depletion on red cell storage and metabolism. Br J Haematol 1994;87:363-8. 47. Hornsey VS, McColl K, Drummond O, Prowse CV. Separation of whole blood into plasma and red cells by using a hollow-fibre filtration system. Vox Sang 2005;89:81-5. 48. Simon TL, Marcus CS, Myhre BA, Nelson EJ. Effects of AS-3 nutrient-additive solution on 42 and 49 days of storage of red cells. Transfusion 1987;27:178-82. 49. Moore GL. Additive solutions for better blood preservation. CRC Crit Rev Clin Lab Sci 1987;25:211-28. 50. Shinar E, Prober G, Yahalom V, Michlin H. WBC filtration of whole blood after prolonged storage at ambient temperature by use of an in-line filter collection system. Transfusion 2002;42: 734-7. 51. Sanz C, Pereira A, Faundez AI, et al. Prolonged holding of whole blood at 22 degrees C does not increase activation in platelet concentrates. Vox Sang 1997;72:225-8. 52. Hughes JD, Macdonald VM, Hess JR. Warm storage of whole blood for 72 hours. Transfusion 2007;47:2050-6. 53. Hirayama J, Abe H, Azuma H, Ikeda H. Leakage of potassium from red blood cells following gamma ray irradiation in the presence of dipyridamole, trolox, human plasma or mannitol. Biol Pharm Bull 2005;28:1318-20. 54. Rose ZB. Enzymes controlling 2,3-diphosphoglycerate in human erythrocytes. Fed Proc 1970;29:1105-11. 55. Hess JR, Greenwalt TJ. Storage of red blood cells: New approaches. Transfus Med Rev 2002;16:283-95. 56. Walker WH, Netz M, Ganshirt KH. 49 day storage of erythrocyte concentrates in blood bags with the PAGGS-mannitol. Beitr Infusionsther 1990;26:55-9. 57. Meryman HT, Hornblower M. Manipulating red cell intra- and extracellular pH by washing. Vox Sang 1991;60:99-104. 58. de Korte D, Kleine M, Korsten HGH, Verhoeven AJ. Prolonged maintenance of 2,3-DPG and ATP in red blood cells during storage. Transfusion 2008;48: 1081–9. 59. Hogman CF, Knutson F, Loof H, Payrat JM. Improved maintenance of 2,3-DPG and ATP in RBC stored in a modified additive solution. Transfusion 2002;42:824-9. 60. Hogman CF, Lof H, Meryman HT. Storage of red blood cells with improved maintenance of 2,3-bisphosphoglycerate. Transfusion 2006;46:1543-52. 61. Kurup PA, Arun, Gayathri NS, et al. Modified formulation of CPDA for storage of whole blood, and of SAGM for storage of red blood cells, to maintain the concentration of 2,3-diphosphoglycerate. Vox Sang 2003;85:253-61.

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62. Hess JR, Rugg N, Knapp AD, et al. The role of electrolytes and pH in RBC additive solutions. Transfusion 2001;41:1045-51. 63. Hess JR, Rugg N, Joines AD, et al. Buffering and dilution in red blood cell storage. Transfusion 2005:45:50-4. 64. Ruddell JP, Babcock JG, Lippert LE, Hess JR. Effect of 24 hours of storage at 25ºC on the in vitro storage characteristics of CPDA-1 packed red blood cells. Transfusion 1998; 38:424-8. 65. Reid TJ, Babcock JG, Derse-Anthony CP, et al. The viability of autologous human red blood cells stored in additive solution-5 and exposed to 25ºC for 24 hours. Transfusion 1999;39:991-7. 66. Hill HR, Oliver CK, Lippert LE, et al. The effects of polyvinyl chloride and polyolefin bags on red blood cells stored in a new additive solution. Vox Sang 2001;81:161-6. 67. Davey RJ, McCoy NC, Yu M, et al. The effect of prestorage irradiation on posttransfusion red cell survival. Transfusion 1992;32:525-8. 68. AuBuchon JP, Pickard CA, Herschel LH, et al. Production of pathogen-inactivated RBC concentrates using PEN110 chemistry: A Phase I clinical study. Transfusion 2002;42:146-52. 69. Benesch R, Benesch RE. Effect of organic phosphate from human erythrocytes on allosteric properties of haemoglobin. Biochem Biophys Res Commun 1967;26:162-7. 70. d’Almeida MS, Gray D, Martin C, et al. Effect of prophylactic transfusion of stored RBCs on oxygen reserve in response to acute isovolemic hemorrhage in a rodent model. Transfusion 2001;41: 950-6.

68

71. Raat NJ, Ince C. Oxygenating the microcirculation: The perspective from blood transfusion and blood storage. Vox Sang 2007;93:12-18. 72. Hogman CF, de Verdier CH, Ericson A, et al. Studies of the mechanism of human red cell loss of viability during storage at 4 degrees C in vitro. I. Cell shape and total adenylate concentration as determinant factors for posttransfusion survival. Vox Sang 1985;48:257-68. 73. Valeri CR, Ragno G, Pivacek LE, et al. An experiment with glycerolfrozen red blood cells stored at 80 degrees C for up to 37 years. Vox Sang 2000;79:168-74. 74. Hess JR. RBC Freezing and its impact on the supply chain. Transfus Med 2004;14:1-8. 75. Valeri CR, Ragno G, Pivacek LE, et al. A multicenter study of in vitro and in vivo parameters of human red blood cells frozen with 40 percent w/v glycerol and stored after deglyceroliztion for 15 days at 4ºC in AS-3: Assessment of RBC processing in the Haemonetics model 215. Transfusion 2001;41:933-9. 76. Dumont LJ, AuBuchon JP, for the Biomedical Excellence for Safer Transfusion (BEST) Collaborative. Evaluation of proposed FDA criteria for the evaluation of red cell recovery trials. Transfusion 2008;48:1053–60. 77. Davey RJ. The uses of radiolabeled red cells in transfusion medicine. Transfus Med Rev 1988;2:151-60. 78. Hess JR, Kagen LR, van der Meer PF, et al. Inter-laboratory comparison of measuring red cell ATP, DPG, and haemolysis. Vox Sang 2005;89:44-8.

5

Red Cell Immunology and Compatibility Testing W. John Judd Emeritus Professor of Immunohematology, Department of Pathology, University Hospitals, University of Michigan, Ann Arbor, Michigan, USA

Immunohematology is the study of immune aspects of blood. This chapter begins with a review of the basic concepts of immunity as they apply to the formation of antibodies to foreign antigens, also called alloantibodies. Other areas of immunohematology such as autoantibodies and drug-induced antibodies are presented elsewhere. The relevance of immunohematology in transfusion medicine is addressed through detailed discussion of the principles of serologic tests and their application to donor-recipient compatibility testing. Identification of the blood group specificity of alloantibodies (alloantibody identification) encountered during such testing is presented in a step-by-step manner such that a limited number of possible specificities remain. Additional testing may be required to obtain a definitive answer. The chapter concludes with discussion of computer-based programs to prevent release of ABO-incompatible units from the blood bank, as well as new technologies, including nanotechnology, that prevent transfusion errors caused by patient misidentification.

Red Cell Immunology The Immune Response Immune responses can be divided into two categories: humoral immunity and cell-mediated events. Humoral immunity is B-cell mediated and entails the generation of antibodies. Cell-mediated immunity is a reflection of the action of T cells, which regulate the immune response through the production of cytokines and direct interactions with other elements/cells of the immune system. An individual’s genetic constitution determines the characteristics of that person’s cells and fluid-phase molecules; this is

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

referred to as the “self ” of the individual. All other (“non-self ”) molecules are considered foreign. During fetal development, and continuing through the neonatal period, B and T cells produce receptors that enable each lymphocyte to recognize a single specific antigen.1

Antigen Receptors of T Cells and B Cells The antigen receptors of both T and B cells consist of two polypeptide chains synthesized under the direction of two different chromosomal loci. The loci for B-cell receptors are on chromosome 14 and either on chromosome 2 or 22; the loci for T-cell receptors are on chromosome 7 and 14 (but not the same locus as for B-cell receptors). In the early stages of lymphocyte development, the genetic material at each of these loci undergoes a process of rearrangement that is unique to each cell.2 Germline DNA at these loci contain numerous base-pair sequences, each capable of encoding different amino acid sequences. The gene rearrangement process entails selection of single base-pair sequences for each portion of the immunoglobulin molecule from the assortment (library) of base pairs available (Fig 5-1), and also involves elimination of unused genetic material.3 Each T-cell/B-cell receptor chain consists of a constant portion and a variable portion. Amino acid sequences of the variable portion determine the idiotypic specificity of the immunoglobulin molecule. For both receptors, one chain contains selections from three libraries (V, D, J), while the other chain contains selections from V and J libraries. Given that the heavy chain locus has as many as 400 exons in the V library, 20 in the J library, and six in the D library, the number of possible combinations for different protein configurations is astronomic. At the light chain loci there are some 300 possible selections in the V library, and four in the J library. Selection from each library is random, and further amino acid sequence variation is introduced by inaccurate splicing or when different exons are joined together. Consequently, 109 different receptor configurations can be generated.1

69

Section I: Part I

Unrearranged heavy chain gene VH1 VH2 VH3 VHn D1 D2 D3 Dn

J1 J2 J3 J4 J5 J6

Cµ

Cδ Cγ3 Cγ1 Cχ1 Cγ2 Cγ4 Cε Cγ4

D/J Joining Cµ

VH1 VH2 VH3 VHn

Cδ

V/D/J Joining

V/D/J

Cµ

Cδ Rearranged heavy chain gene

Transcription Primary transcript Alternative RNA splicing µ/δ mRNA V DJ Cµ

V DJ Cδ

Exposure to Foreign Protein Antigens If foreign protein antigens enter the host, antigen-presenting cells (APCs) isolate the antigen and display the molecular configurations as epitopes on their cell surfaces. T and B lymphocytes with the appropriate receptor for the unique molecular configuration can then establish intimate contact with the APC. This contact initiates cellular events that generate clones of activated lymphocytes specific for the foreign antigen. Cytokines produced by APCs and by activated T cells cause B cells to evolve into immunoglobulinsecreting plasma cells. In turn, B cells capture antigen and present it back to T cells in a manner that causes the T cells to secrete cytokines that promote more focused antibody production. Most plasma cells have a short but active life of antibody production. Some, however, become memory cells that retain intracellular changes resulting from exposure to APCs; these persist in the circulation long after their initial activation.

Primary Response The first immunoglobulin class to appear in the bloodstream is always IgM; the lag phase (time between sensitizing event and appearance of antibody) can vary from days to months. Shortly after the appearance of IgM antibody, IgG antibody of the same specificity usually becomes detectable. The IgM component disappears over time, while the IgG component persists indefinitely. Cytokines from activated T cells are essential for this isotype switching and for generation of memory cells.

T-Cell Independent Response It is of note that antibodies to carbohydrate antigens are invariably IgM. This is because B-cell APCs can interact directly with complex polysaccharides such as those that carry blood group A or B activity. Structures that have multiple identical repeat carbohydrate chains can initiate B-cell proliferation and antibody secretion without the action of T cells. In the absence of the

70

Figure 5-1. Diagram of the immunoglobulin heavy chain locus on chromosome 14. In the unrearranged locus (top) V, D, and J libraries are on the left; sequences for the constant regions are on the right. Gene rearrangement occurs by selection of one D and one J exon to form a D/J unit. This is then joined by one V exon. The V/D/J unit is adjacent to the exons for µ and δ constant regions. Through alternative splicing, the primary transcript generates either µ or δ heavy chains with the same variable region configuration (idiotype). In later generations, clonal progeny can attach the V/D/J transcript to other sequences from the constant region (isotype switch).

immunomodulatory effects of T cells, isotype switching does not occur, and no memory cells develop.4

Secondary (Anamnestic) Response Within a few hours or days after subsequent exposure to T-dependent antigen, there is a sharp rise in the level of IgG antibody. Because there has been prior clonal expansion, the number of participating B cells will be high, resulting in production of significantly (eg, 100-fold) more IgG than was produced in the primary response. Cytokines secreted by T cells promote more focused antibody production.

Affinity Maturation The affinity of IgG antibody for specific antigen increases progressively during the secondary immune response, particularly after stimulation with low doses of antigen. This affinity maturation is most pronounced after secondary challenge with antigen. One explanation for affinity maturation is clonal selection. A second explanation for affinity maturation is that, after a class switch has occurred in the immune response, somatic mutations occur that fine-tune the antibodies to be of higher affinity. There is experimental evidence for this mechanism, although it is not known how the somatic mutation mechanism is activated after exposure to antigen.

Clonality Only antigen-specific B cells proliferate and mature into antibodysecreting plasma cells. The number of progenitor B cells that are initially activated will determine the heterogeneity (clonality) of the immune response. The response will range from polyclonal (when many different B cells are activated simultaneously) to monoclonal (when the antibody-producing cells originate from a single ancestral B cell). Monoclonal antibodies for reagent use can also be produced in vitro using hybridoma technology.5

Chapter 5: Red Cell Immunology and Compatibility Testing

Immunoglobulin Molecules Figure 5-2 portrays the basic structure of an antibody molecule, which consists of four polypeptide chains: two identical light chains of 214 amino acids, and two identical heavy chains of 440 or more amino acids. Antigenic specificity is conferred by the 134 N-terminal amino acids of the light chains and the 144 N-terminal amino acids of the heavy chains; these are described as variable regions. The remaining portions of both chains are referred to as constant regions.6

Light Chains The constant region of the light chains can have one of two different amino acid sequences, designated kappa (κ) and lambda (λ). κ chains are encoded by a locus on chromosome 2, λ chains by a locus on chromosome 22. A single cell will synthesize an immunoglobulin molecule containing either κ or λ chains, but never both.

five types of immunoglobulin classes termed IgA, IgD, IgE, IgG, and IgM, respectively. The site on chromosome 14 that governs the manufacture of heavy chains contains DNA sequences for all five types of heavy chain.

Antibody Diversity Serologic studies have revealed a number of antigenic markers on the heavy and light chains of immunoglobulin molecules. The results of such studies are strongly supported by molecular analysis of the immunoglobulin genes.3,6 Differences between these antigenic markers are illustrated in Fig 5-3.

Isotypes Isotypes result from structural changes to the constant region heavy and light chains. Genes encoding the various isotypes (IgA, IgD, IgE, IgG, IgM, κ, and λ) are all present in the human genome. Antibodies for isotype determination can be prepared by heterologous immunization (eg, mouse immunization with human serum).

Heavy Chains The constant region of heavy chains can manifest one of five amino acid sequences, designated by the Greek letters α (alpha), δ (delta), ε (epsilon), γ (gamma), and µ (mu). These give rise to

VL Var iab le

Con

sta

nt

Carbohydrate

SS

VH

Disulphide bond

CL

Idiotypes Idiotypic variation is the result of differences in amino acid sequence of the variable regions of either light or heavy chains. Antibodies to idiotypes may recognize sequences either within or outside the antigen-combining site, and can be prepared by either homologous or heterologous immunization.

SS

Hinge region

S S

CH 1 CH2

CH3

SS

Antigen binding sites

Heavy chain Light chain

Blood Group Antibodies Blood group antibodies are immunoglobulins that react with antigens on the surface of red cells. They can either be acquired

Figure 5-2. Basic structure of an immunoglobulin molecule.

Figure 5-3. Diversity of antibody molecules.

Allotypes Allotypic distinctions between antibodies are the result of genetic polymorphisms, and each marker is not present in every individual. This genetic variation results from different alleles at structural loci. Antibodies to allotypic variants can be raised by same-species (homologous, allogeneic) immunization, and are often found in the sera of women who have given birth to infants with different allotypic markers. These markers usually represent amino acid changes in the constant regions of antibody molecules.

Isotype

Allotype

Idiotype

Ig class present in all members of species: IgG, IgA, IgM, IgG1, etc.

Amino acid sequence variations, not present in all members of species: Gm and Km allotypes.

Antigen binding sites, not present in all molecules within individual members of species; Fab regions.

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Section I: Part I

Table 5-1. Characteristics of Blood Group Active Immunoglobulins Immunoglobulin Characteristic

IgM

IgG

IgA

H-chain isotype Subclasses L-chain types Sedimentation constant Molecular weight Electrophoretic mobility Serum concentration (mg/dL) Antigen binding sites Fixes complement Placental transfer Direct agglutinin Hemolytic in vitro Example

µ 2 κλ 19 S 900-1000 kD Between β and γ 85-205 10 (5) Often No Yes Often Anti-A, anti-B

γ 4 κλ 7S 150 kD γ 1000-1500 2 Some Yes Usually not No Anti-D

α 1 κλ 11 S 180-500 kD γ 200-350 4 No No Usually not No Anti-Lua

naturally or through immunization with foreign red cells.7 Most blood group antibodies are either IgG or IgM; occasionally they may be IgA. The IgD and IgE immunoglobulins have not been implicated as blood group antibodies,8 and will not be discussed further.

Physical Properties The physical properties and serologic characteristics of the three immunoglobulin classes involved with blood group specificity are summarized in Table 5-1. IgM antibodies have 10 available antigen binding sites, have a wide spanning distance, and often fix complement to red cells. Antibodies to A and B antigens are predominantly IgM; they are found normally in persons whose red cells lack the corresponding antigen. They are stimulated by antigens present in the environment. Bacteria constituting normal intestinal flora carry blood group A- and B-like polysaccharides. As these flora establish themselves in the gut, they provide the immune stimulus for anti-A and anti-B.7 Anti-A and anti-B formed in this manner are often referred as “natural” antibodies. They are also called “expected” antibodies, because in adults with a normal immune system these antibodies are almost always present when the corresponding antigens are absent on the red cells. In contrast to anti-A and anti-B, most other blood group antibodies are IgG, which are immune in origin and do not appear in plasma/serum unless the host is exposed directly to foreign red cell antigens. The stimulating event is usually blood transfusion or pregnancy. IgG antibodies are smaller than IgM, and have only two sites for antigen binding. All antibodies to red cell antigens, other than naturally occurring anti-A and anti-B, are considered unexpected. They can be either alloantibodies, directed toward non-ABO-system antigens absent on the red cells of the antibody producer, or autoantibodies, directed toward self-antigens. The latter may cause autoimmune hemolytic anemia. Unexpected antibodies in donor plasma may destroy recipient red cells, while antibodies in the

72

recipient may cause accelerated destruction of transfused red cells. In pregnant women, such antibodies may cross the placenta and cause hemolytic disease of the fetus and newborn.8 Close to 300 different blood group alloantibodies have been described.9 Each antibody reacts with its specific antigen on the surface of red cells. The immunoglobulin class and clinical relevance of some of the antibodies encountered during compatibility testing are shown in Table 5-2.

Red Cell Antigen-Antibody Interactions Red cells normally repel one another. The force of repulsion (zeta potential) that exists at the red cell surface depends not only on the electronegative surface charge, but also on the ionic cloud that normally surrounds it.10,11 The electronegative charge is imparted primarily by the carboxyl (C00
) group of N-acetyl-neuraminic acid,12 which is a constituent of alkali-labile tetrasaccharides that are attached to MN and Ss sialoglycoproteins (glycophorins A and B, respectively).9 At a minimum, there are 3.2  107 of these charged groups per red cell. The magnitude of this charge is modified by the formation of an ionic cloud of positively charged () sodium ions and negatively charged (
) chloride ions that align in alternating sequence extending from the red cell membrane surface (Fig 5-4). The net effect of this ionic cloud is to decrease the electrostatic repulsion between red cells. In considering the interaction of red cell antigens with blood group antibodies, three variables must be considered: the surface charge of the red cells, the dielectric constant of the medium (a relative measure of its charge dissipation), and the mutual attraction between antigen and antibody. The latter involves electrostatic (coulombic) and hydrogen bonds, and van der Waals interactions, which hold antigen and antibody together.10 Further, there must be structural complementarity between antigen and its binding site on antibody molecules.13 There are two phases of red cell antigen-antibody interactions; these often occur simultaneously. The first phase is one of association, involving binding of antibody to antigens on the red cell membrane. The second phase involves the formation of an agglutination lattice of antibody-coated cells. For the latter to occur, antibody molecules must be able to span the distance between adjacent red cells. Interaction between red cells and blood group antibodies can be observed either directly by examination of red cell and antibody mixtures for agglutination (or clumping) and/or hemolysis, or indirectly by use of the antiglobulin test. These two serologic techniques are summarized in Table 5-3.

Direct Agglutination IgM antibody molecules can span the distance that exists between red cells when suspended in saline, and cause direct agglutination of those cells if they carry the corresponding antigen. This direct agglutination can be observed when red cells are mixed with IgM antibody, briefly incubated at room temperature, and centrifuged. Examination for hemagglutination may be performed either macroscopically or microscopically, but the latter is not

Chapter 5: Red Cell Immunology and Compatibility Testing

Table 5-2. Characteristics of Blood Group Alloantibodies Antibody

Agglutinating

Coating*

Ig Class†

C3-binding

Effect of Ficin

HDFN‡

HTR§

% Compatible

Comments

A A1 Ata B Bg C c Ch Cw Coa Cob Cra Csa D Dia Dib Doa Dob E e f(ce) Fya Fyb Ge H Hy I Jka Jkb JMH Jsa Jsb K k Kna Kpa Kpb Lan Lea LebL LebH Lua Lub M McCa N P1 PP1Pk Rg S s Sc1 Sc2

✓ ✓

✓ Some ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Some ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

M, G M G M, G G G, M G, M G G, M G G G G G, M G G G G G, M G, M G, M G G G, M M, G G M, G G G G G G G, M G G G G G M M M G, A G, A M, G G M, G M M, G G G G G G

✓ ✗ ✗ ✓ ✗ ✗ ✗ ✗ ✗ ✗ Rare ✗ ✗ ✗ ✗ ✗ ✓ ✓ ✗ ✗ ✗ Rare Rare ✓ ✓ ✗ ✓ ✓ ✓ ✗ ✗ ✗ Rare ✗ ✗ ✗ ✗ Some ✓ ✓ ✓ Rare Rare Rare ✗ ✗ Some ✓ ✗ Some Rare ✓ ✗

↑ ↑

✓ ✗ ✗ ✓ ✗ ✓ ✓ ✗ ✓ ✓ ✓ ✗ ✗ ✓ ✓ ✓ ✗ ✗ ✓ Rare ✓ ✓ ✓ ✗ ✓ ✗ ✗ ✓ ✓ ✗ ✓ ✓ ✓ ✓ ✗ ✓ ✓ ✓ ✗ ✗ ✗ Rare ✓ Rare ✗ Rare ✗ ✓ ✗ ✓ ✓ ✗ ✓

✓ Rare ✓ ✓ ✗ ✓ ✓ ✗ ✓ ✓ ✓ ✓ ✗ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

56 64 Rare 85

No dosage In 2% A2, 25% A2B In Blacks No dosage To HLA||

✓ Some Some Some

Some Some

Some Some Some Rare Some ✓ ✓ Some Some

Some

✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Rare Rare

Some ✓ Some ✓ Some Some ✓ ✓ ✓ ✓ ✓ ✓

↑ ↑ ↑ ↓ ↑

↑

↑ ↑ ↑ ↑ ↑ ↓ ↓ ↓Some ↑ ↑ ↑ ↑ ↓

↓

↑ ↑ ↑

↓ ↓ ↓ ↑ ↑ ↓ ↓ ↓

✓ ✓ ✗ ✓ ✓ ✓ ✓ ✗ ✓ ✓ ✓ Rare ✗ ✗ ✗ ✓ Rare ✗ Rare Rare ✓ ✗ ✓ ✓ ✗ ✗

30 0 2 98 Rare 91 Rare 2 15 99 Rare 33 13 70 3 36 33 20 Rare Rare Rare Rare 25 25 Rare 99 Rare 90 Rare 2 98 Rare Rare 78 28 48 92 Rare 22 1.5 28 21 Rare 3 45 11 Rare 99

Often with anti-E HTLA¶; to C4d#

In Blacks HTLA No dosage **

Often with anti-c Auto in WAIHA†† Compatible with c–

In Oh blood In Blacks In i adults

HTLA In Blacks

HTLA

True anti-Leb In A1 and A1B

HTLA Potent in N–U– No dosage HTLA; to C4d

(continued)

73

Section I: Part I

Table 5-2. (continued) Antibody

Agglutinating

Sla U V Vel Wra Xga Yka Yta Ytb

Some Some Some

Coating*

Ig Class†

C3-binding

Effect of Ficin

HDFN‡

HTR§

% Compatible

Comments

✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓

G G G M, G M, G G G G G

✗ ✗ ✗ ✓ ✗ ✗ ✗ ✗ ✗

↓

✗ ✓ ✓ ✗ ✓ ✗ ✗ ✗ ✗

✗ ✓ ✓ ✓ ✓ ✗ ✗ Some ✗

2 Rare 99 Rare 99 23 8 Rare 92

HTLA In Blacks

↑ ↑ ↓ ↓ ↓Some

HTLA

* Coating antibodies detected primarily through the use of the indirect antiglobulin test. † Predominant immunoglobulin class shown first. ‡ HDFN  reported to cause hemolytic disease of the fetus and newborn. § HTR  reported to cause hemolytic transfusion reactions. || HLA  antibody to antigen present on white blood cells that is variably expressed on red cells. ¶ HTLA  antibody, usually of high titer, to antigen of low site density on red cells. Incorrectly called high-titer, low avidity antibody. # C4d  antibody to epitopes found on the fourth component of human complement (C4). ** Rare in Whites and Blacks. Seen almost exclusively in individuals of Mongolian ancestry (South American Indians, Chippewa Indians, Chinese, Japanese). †† WAIHA  seen as an autoantibody in some cases of warm autoimmune hemolytic anemia.

Table 5-3. Two Types of Red Cell Serologic Tests

(A)

(B)

Direct Tests

Indirect Tests

Mix serum and red cells Incubate (room temperature)† Centrifuge Examine for agglutination and hemolysis

Mix serum and red cells* Incubate (37ºC) Centrifuge Examine for agglutination and hemolysis† Wash, to remove unbound globulins Add antihuman globulin reagent Centrifuge Examine for agglutination‡

* An enhancement reagent, to promote antibody uptake, may be incorporated here. † This step is optional. ‡ Confirm negative test results with IgG-coated red cells.

(C)

(D)

Figure 5-4. (A) Red cells carry a strong negative electric charge, imparted primarily by the carboxyl (C00
) group of N-acetyl-neuraminic acid. (B) When suspended in a saline medium, red cells are kept apart by virtue of their negative surface charge. The magnitude of this charge is modified by the formation of an ionic cloud of positively charged () sodium ions and negatively charged (
) chloride ions that align in alternating sequence extending from red cell membrane surface. The force of repulsion that exists between red cells in suspension is referred as the zeta potential. (C) In order for antibody molecules to cause agglutination of adjacent red cells, the antigen binding sites must be able to span the intercellular distance. Pentameric IgM antibody molecules can readily bridge the distance between adjacent red cells. (D) IgG molecules (black) cannot readily effect direct agglutination. However, they are able to coat red cells. This antibody coating can be detected by a second IgG antibody (eg, rabbit antihuman IgG), depicted here in gray.

74

encouraged because it leads to the detection of nonspecific reactivity. The strength of the observed reactions can be graded, and numerical values can be assigned based on a scoring system in common use.14

Hemolysis IgM antibodies, in particular anti-A and anti-B, can also cause direct lysis of antigen-positive red cells, especially if tests are incubated at 37ºC. This hemolysis results from the action of complement, a series of α and β globulins that act in sequence as enzymes (eg, esterases) to form a complex (membrane attack complex) that causes holes to be formed in the red cell membrane through which the intracellular hemoglobin can escape (see Fig 5-5). For initiation of the complement cascade, the “tails” (Fc portions) of two

Chapter 5: Red Cell Immunology and Compatibility Testing

C1qr2s2

Antigen  Antibody

Ca

C4a

Activated C1qr2s2

C4

C2

C2a C3 Convertase

C5 Convertase

C4b2a

C4b2a3b

C4b

C3a

C3

C3b

C5

C5a

C3bINA C3d

Figure 5-5. Classical pathway for complement activation.

pairs of immunoglobulin heavy chains must be in close proximity to each other on the red cell surface. Initiation of the complement cascade readily occurs when a single pentameric IgM antibody molecule is bound; for it to occur with IgG antibodies there must be closely adjacent heavy chain regions of two IgG molecules at the cell surface. Further, not all IgM antibodies bind complement to red cells and complement binding does not always proceed to complete red cell lysis by membrane attack complex (intravascular hemolysis). Rather, activated C3b may be cleaved to C3d, which does not initiate the binding of C5; hence, no membrane attack complex is formed. However, C3d remains bound to red cells and can be detected by the antiglobulin test (see below).8,14 Red cells coated with C3b are also removed by cells of the reticuloendothelial system (extravascular hemolysis). However, there are no receptors on macrophages for C3d. Thus, inactivators of C3b serve to limit the degree of both intravascular and extravascular hemolysis.

Antiglobulin Test IgG antibodies do not readily cause direct agglutination of red cells, because intercellular distances are wider than the spanning distance of the antigen-binding sites of the IgG molecule. However, some IgG antibodies will cause direct agglutination if the red cell zeta potential is reduced. This can be accomplished by treating the cells with proteolytic enzymes such as papain or ficin, which remove the proteins that carry the negatively charged neuraminic acid residues. Alternatively, zeta potential can be decreased by raising the dielectric constant of the red cell suspending medium by the addition of colloids such as bovine serum albumin.8 IgG antibodies, as well as complement components bound to red cells by either IgM or IgG antibodies, are best detected by the antiglobulin test. This test, once called the Coombs’ test, entails the use of antibodies raised in animals (usually rabbit or sheep), or prepared by hybridoma technology,5 to detect human IgG and

Membrane attack complex

C3c

C5b6789

C5b C9

C8

C7

C6

complement bound to red cells. The principles of this test are as follows: 1. Antibody molecules and complement components are globulins. 2. The injection of human globulins, either purified or in whole serum, into an animal stimulates the animal to produce antibodies against the foreign globulins. These antibodies are referred to as antihuman globulins (AHGs). The antihuman globulins that are important for blood group serologic work are anti-IgG and anticomplement (anti-C3d). 3. Antihuman globulin will react with human globulins, either bound to red cells or free in serum. Thus, red cells must be washed free of unbound globulins before testing with AHG. This is crucial to the avoidance of false-negative tests caused by neutralization of AHG by unbound globulins. 4. Washed red cells coated with human globulin are agglutinated by AHG. Antiglobulin tests can be performed either indirectly following in-vitro incubation of red cells with serum, or directly to demonstrate that red cells are coated with globulins in vivo. An indirect antiglobulin test (IAT) is used to detect and identify unexpected antibodies in the serum of blood donors, prospective transfusion recipients, and prenatal patients. The direct antiglobulin test (DAT) is used to detect antibodies bound to red cells in vivo; such antibodies may be seen in patients with hemolysis caused by autoantibodies or drugs, infants with hemolytic disease of the fetus and newborn, and patients manifesting an alloimmune response to a recent transfusion.14

Compatibility Testing Pretransfusion compatibility testing comprises a series of policies and procedures, including laboratory tests, the goals of which are to provide blood for transfusion that will have the

75

Section I: Part I

Table 5-4. Required Elements of Pretransfusion Testing16 Element

Requirement

AABB Standards

Positive identification of intended recipient and blood sample at time of collection Mechanism to identify phlebotomist

5.11 5.11.2.2

Label

Two independent identifiers (eg, first and last names, patient unique number) Date of collection Attached to tube before leaving side of intended recipient

5.11.2 5.11.2 5.11.2.1

Timing

Within 3 days of red cell transfusion if patient was transfused or pregnant within previous 3 months, or if patient history is uncertain or unavailable

5.13.3.2

From an attached segment, after the ABO and Rh label has been affixed Anti-A and anti-B on units labeled group A or group B Anti-A,B on units labeled group O Direct tests with anti-D on units labeled Rh-negative

5.9.5, 5.12

Repeat donors

A computer system (validated to prevent release of ABO and Rh mislabeled units) may be used

5.9.5.1

Unexpected antibodies

Confirmatory testing not required

Patient Sample Collection Identification

Donor Unit Confirmatory Testing First-time donors or if electronic crossmatch is used

Patient Sample Testing ABO

Red cells with anti-A and anti-B; serum or plasma with A1 and B red cells; concordance between red cell and serum results

5.13.1

Rh

Test with anti-D; test for weak D not required

5.13.2

Unexpected antibodies

By a method that will demonstrate clinically significant antibodies; 37ºC incubation preceding an antiglobulin test (alternative methods of documented equivalency acceptable); using reagent red cells that are not pooled

5.13.3

Confirm negative tests with IgG-coated red cells, or control system appropriate to the method

5.13.3.4

Antiglobulin test; applies if clinically significant antibodies detected, currently or in past

5.15.1

Serologic tests for ABO incompatibility if no clinically significant antibodies detected, currently or in the past

5.15.1.1

Computer system validated on site to prevent release of ABO-incompatible blood may be used to detect to ABO incompatibility

5.15.2.1

Donor/Patient Testing Crossmatch

optimal clinical effect without causing undue harm to the recipient. These elements of pretransfusion testing can be placed into one of three categories: those related to donor unit testing and processing, those related to patient sample collection and testing, and those that serve as a final check between the donor unit and intended recipient (Table 5-4). Proper performance of each element will enable the right unit of blood to be transfused to the right patient.15

Donor Testing Collection Facility The initial ABO, Rh, and antibody detection tests on donor blood, tests for infectious diseases, the interpretation of these tests, and correct labeling of donor units are functions normally carried out by a regional donor center. However, some hospitalbased transfusion services continue to procure a portion of their blood needs from the population at large (allogeneic donors), and some collect blood from patients for their own use in

76

elective surgical procedures (autologous donors). In addition, some patients request that they receive blood from relatives or friends (directed donations). Regardless of the type of donor, each unit of blood must be subjected to tests for ABO, Rh, and unexpected antibodies, as well as for markers of infectious disease (see Chapter 62). The volume of testing that needs to be performed at donor centers often necessitates use of automated equipment. Instrumentation currently in use is often based on microplate technology, and test results may be interpreted electronically. ABO grouping entails testing both donor red cells and serum/ plasma. Red cells are tested with anti-A and anti-B; and the serum/plasma is tested with A1 and B red cells. Although not required, anti-A, B, and A2 red cells may also be used, because results with these reagents serve to detect subgroups of A that may be nonreactive in direct tests with anti-A alone. Rh typing is performed with anti-D. Because D is highly immunogenic,8 it is presumed that even weak expression of the

Chapter 5: Red Cell Immunology and Compatibility Testing

antigen will evoke an immune response; consequently, donor red cells that initially type as Rh-negative in direct agglutination tests are tested further for weak D, usually by an IAT (see Table 5-3). Only those units that are negative with anti-D by this method or an equivalent procedure can be labeled Rh-negative.16(p37) All straightforward D-positive and weak D-positive blood is considered Rh-positive.

Transfusing Facility The ABO group of all units of Whole Blood or Red Blood Cells, and the Rh group of those labeled Rh-negative, must be confirmed by the transfusion facility before transfusion.16(p43) Only tests with reagent antisera and donor red cells need to be performed; units labeled group O can be tested with anti-A,B alone.17 For D typing of units labeled Rh-negative, only direct tests with anti-D are necessary; testing for weak expression of D is not required, nor is repeat testing for unexpected antibodies or for markers of infectious diseases.16(p43) For transfusion facilities that also collect blood, confirmatory testing for first-time donors must be performed after the original ABO and Rh label has been affixed using a sample from a segment attached to the donor unit. For repeat donors, the ABO/Rh may be confirmed by an electronic record check of previous donations, provided that tests for ABO compatibility are to be performed serologically. However, if blood is to be released using an electronic crossmatch process (see later), the ABO/Rh of the donor unit must be confirmed serologically by testing an attached segment, even for repeat donors.

Patient Testing Sample Collection Because the major cause of fatal, hemolytic transfusion reactions is ABO-incompatible transfusion resulting from patient/sample misidentification, the importance of the following measures cannot be overemphasized. 1. Requisition. Forms requesting blood and blood components must contain the first and last names and a unique numerical identifier of the intended recipient, such as a hospital registration number.16(p41) The name of the requesting physician, gender and date of birth of the patient, clinical diagnosis, and previous transfusion or pregnancy history are additional useful pieces of information. 2. Patient Identity. The collection of a properly labeled blood sample for pretransfusion testing from the correct patient is critical to safe blood transfusion. The person collecting the sample must positively identify the patient.14 This is facilitated through use of a wristband, containing the patient’s full name and unique hospital registration number that remains attached to the patient throughout the hospitalization. The information on the requisition form should be compared with that on the wristband; blood samples should not be collected if there is a discrepancy. In the absence of a wristband, the nursing staff should follow hospital procedure for identifying and attaching a wristband to the patient before any samples are drawn. In an emergency,

a temporary identification number should be used, and crossreferenced with the patient’s name and hospital identification number once they are known. 3. Labeling. Blood samples must be drawn into correctly labeled tubes. The tubes must be clearly labeled at the bedside with the patient’s first and last names, the patient’s unique hospital identification number, and the date of collection.11 There must be a means of identifying the person who collected the sample. Conventionally, the phlebotomist signs or initials the tube and signs or initials the requisition form so that there is a permanent record of the phlebotomist’s name.14 4. Confirmation of Sample Identity. Upon receipt of blood samples for pretransfusion testing, the information on the label must be compared with that on the requisition. A new sample must be obtained whenever there are discrepancies or if there is any doubt about the identity of the sample. It is unacceptable to correct an incorrectly labeled sample. 5. Type of Sample. Either serum or plasma may be used for pretransfusion testing, but most workers performing tube testing use serum to avoid introducing small fibrin clots into serologic tests. Such clots may be mistaken for agglutination. Fibrin clots may also form when samples from heparinized patients are collected into non-anticoagulated tubes. These samples will clot properly following the addition of protamine sulfate.14 However, anticoagulated (plasma) samples are required if testing is automated. Few workers prefer to use serum rather than plasma for compatibility testing, to facilitate detection of antibodies that primarily coat red cells with the C3d component of complement. Bound C3d will not activate the lytic phase of the complement cascade but can be detected with AHG reagents containing antiC3d.8,18 EDTA, citrate and other commonly used anticoagulants chelate calcium ions that are essential for complement activation. However, as discussed later, the use of AHG reagents containing anti-C3d for compatibility testing is not mandatory.14 6. Age of Specimen. To ensure that the specimen used for compatibility testing is representative of a patient’s current immune status, serologic studies must be performed using blood collected no more than 3 days in advance of the transfusion when the patient has been transfused or pregnant within the preceding 3 months, or when such information is uncertain or unavailable.16(p44) From a practical standpoint, it is simpler to stipulate that all pretransfusion samples must be collected within 3 days before red cell transfusions, rather than ascertain whether or not each patient has been recently transfused or pregnant. 7. Storage. Blood samples used for compatibility testing, including donor red cells, must be kept at 1-6ºC for at least 1 week after each transfusion.16(p42) This ensures that appropriate samples are available for investigational purposes should adverse responses to transfusion occur.

ABO Typing Both reagent antisera and red cells for ABO typing are available commercially. Anti-A and anti-B are monoclonal antibodies prepared by hybridoma technology. Reagent red cells are

77

Section I: Part I

Table 5-5. The Expected Reactions of the Four Common ABO Phenotypes: Results of Blood Typing Tests Blood Type

Anti-A

Anti-B

A1 Red Cells

B Red Cells

O A B AB

0 4 0 4

0 0 4 4

4 0 4 0

4 4 0 0

O  no agglutination; 4  strong agglutination

usually suspended in a preservative medium containing EDTA, to prevent lysis of the red cells by complement-binding anti-A and anti-B.14 ABO grouping is performed using a direct agglutination technique (Table 5-3).19 Red cells are tested with anti-A and anti-B, and the serum or plasma with known A1 and B red cells. Use of anti-A,B and A2 red cells is optional, but generally considered unnecessary when ABO typing potential transfusion recipients.17 The expected findings for each of the four common ABO phenotype are shown in Table 5-5. When interpreting the results of ABO grouping tests, it is important to note the reciprocal relationship that exists between the absence of A and/or B antigens on red cells and the presence of the expected anti-A and/or antiB in the serum. If there is conflict between cell and serum ABO tests, group O blood should be provided for transfusion until the discrepancy is resolved and reliable conclusions of the patient’s ABO type can be made.14

Rh Typing Only tests for D are performed routinely on the red cells of prospective transfusion candidates. There are two different types of reagent anti-D available for Rh typing. High-protein reagents are prepared with human IgG anti-D diluted in bovine albumin and other substances that potentiate agglutination. Their final protein concentration may be greater than 20 g/dL. Such a high protein level is needed to potentiate IgG antibody reactivity so that positive and negative tests can be recognized almost instantaneously using a direct agglutination technique. In the United States, low-protein reagents (protein content  7 g/dL) are a blend of monoclonal IgM and monoclonal/polyclonal human IgG anti-D. With blended anti-D reagent the IgM component causes direct agglutination of Rh-positive red cells and the IgG component permits detection of the weak expression of D by application of the antiglobulin test.14 Only direct tests with blended anti-D reagents are required on patient samples; the test for weak D is not necessary.16(p43) To avoid incorrect designation of an Rh-negative recipient as Rh-positive because of autoantibodies or abnormal serum proteins, a control system appropriate to the anti-D reagent in use is required.14 For low-protein anti-D, a concurrent negative test with anti-A and/or anti-B is considered an appropriate control system. Apparent AB Rh-positive samples should be retested concurrently with anti-D and an inert Rh control reagent.19

78

Table 5-6. Acceptable Methods for Pretransfusion Antibody Detection

Saline Albumin Low-ionic-strength saline (solution) Gel Polyethylene glycol Solid-phase adherence Low-ionic polycation

Serum

Red Cells

Incubation

AHG*

2-3 drops 2-3 drops 2 drops*

1 drop, 3-5% 1 drop,3-5% 2 drops, 2%*

30-60 min; 37ºC 15-30 min; 37ºC 10-15 min; 37º

IgG/PS IgG/PS IgG/PS

25 µL 2 drops† 1 drop 100 µL

50 µL, 0.8% 1 drop

15 min; 37ºC 15-30 min; 37ºC 10-15 min; 37ºC 1 min; RT

IgG IgG IgG None

‡

1 drop, 1%

* Drop volumes should be equal. † Plus 4 volumes of 20% polyethylene glycol. ‡ Predetermined by reagent supplier. AHG  antihuman globulin; PS  polyspecific AHG; RT  room temperature.

Tests for Unexpected Antibodies Methods for detecting unexpected antibodies in the serum or plasma of prospective transfusion recipients must be those that detect clinically significant antibodies.16 (p44) An IAT after 37ºC incubation of patient’s serum or plasma with reagent red cells that are not pooled is usually required. Table 5-2 lists many of the blood group alloantibodies that can be detected during pretransfusion testing, and provides the approximate percentage of compatible units that are likely to be encountered in a predominantly Caucasian blood donor population, except as otherwise noted. A number of options exist regarding the selection of methods for pretransfusion antibody detection (see Tables 5-6 and 5-7). Decisions relative to these options are within the purview of the blood bank medical director. They should be made based on the type of patients served, the causes and frequency of previous significant antibody-mediated transfusion reactions, the availability of resources, and with the realization that no one method will detect all clinically significant antibodies. Tube Test Methods A variety of red cell suspending media or additives are used either to enhance antibody uptake or to potentiate the agglutination phase of antibody-antigen interactions (Table 5-7). Low-ionic-strength saline (LISS) solution,20 normal saline, or red cell preservatives (modified Alsever’s solution) are used as red cell suspending media. Bovine serum albumin (22% or 30% w/v), LISS-additives, or polyethylene glycol (PEG) are commonly added directly to serum-red-cell mixtures.19,21 Antibody uptake (the first stage of an antigen-antibody interaction) is accelerated when red cells are suspended in LISS or PEG. The magnitude of the ionic cloud (see Fig 5-4) that forms around negatively charged red cells suspended in LISS (approximately 0.03 M) is lower than that surrounding red cells suspended in normal saline (0.15 M NaCl). This cloud not only decreases intercellular distances by reducing the zeta potential imparted by negatively charged carboxyl groups, but also hinders

Chapter 5: Red Cell Immunology and Compatibility Testing Table 5-7. Changes in Pretransfusion Testing Practices, 2001-200430,31 Percentage of US Laboratories Performing Test Test

Specific Methods

2001

2004

ABO/Rh

Tube Gel Liquid microplate Gel LISS-tube PEG-tube Albumin-tube Saline-tube Solid-phase adherence Liquid microplate Immediate-spin Electronic (computer) Gel LISS-tube-IAT PEG-tube-IAT Albumin-tube-IAT Saline-tube-IAT Solid-phase adherence Liquid microplate

97.4 1.4 1.1 27.5 48.2 8.6 8.7 3.2 3.3 0.5 42.4

91.6 7.7 0.6 41.1 40.2 7.7 5.9 2.2 2.4 0.5 39.6 2.0 29.3 19.8 3.1 4.3 1.6 0.2 0.1

Antibody screen

Crossmatch

17.9 26.5 3.6 6.6 2.6 0.2

association between antibody and antigen; thus, reducing the magnitude of the ionic cloud promotes antibody association.10 Weakening of the ionic cloud also causes an increase in attraction between the negatively charged red cell membrane and positive charges on the antigen-binding sites of antibody molecules. In addition, PEG competes for and removes water molecules at the red cell surface. Similar to the ionic cloud of Na and Cl
ions that forms around red cells suspended in saline, these water molecules also sterically hinder antibody association. The net effect of these phenomena is that more antibody can be bound in a shorter period of time using LISS or PEG as opposed to saline. Consequently, incubation times can be reduced to 10 to 15 minutes for LISS, or 15 to 30 minutes for PEG, compared to 30 to 60 minutes for saline.22 In contrast, albumin promotes the second stage of antigenantibody interactions, namely the agglutination of antibodycoated red cells. It does so by increasing the dielectric constant of the suspending medium, thereby reducing the zeta potential (force of repulsion) between red cells.11 Because of this effect, albumin can promote the direct agglutination of IgG-coated red cells (see Judd19 for a method). However, it should be noted that albumin is not used in the United States in a manner that promotes direct agglutination of antibody-coated red cells. Rather, the enhancing effect of albumin on red cell antigen interactions can be attributed to its formulation as a low-ionic solution. In the low-ionic polycation technique, serum is incubated with red cells suspended in LISS to enhance antibody uptake.23 The second phase of the reaction is facilitated by aggregating red

50 µL 0.8% RBCs in LISS  25 µL sample

GEL Test

15' at 37C

10' Spin

4

3

2

1

0

mf

Graded reactions Anti-IgG  GEL

Figure 5-6. The gel test for detecting unexpected antibodies.

cells with the polycation, Polybrene. Aggregation is reversed with sodium citrate, but agglutinates formed by antigen-antibody interactions are not dispersed. It is common practice to examine saline, albumin, and LISS tests for direct agglutination before subjecting them to an IAT. These examinations can be made immediately after mixing cells and serum together (immediate-spin tests) and again after incubation at 37ºC. For antiglobulin testing, either anti-IgG or polyspecific AHG (containing anti-IgG and anti-C3) may be used.14 However, use of polyspecific AHG leads to the detection of an inordinate number of unwanted positive tests,24 and its use in routine antibody detection tests is not recommended.17 Because of nonspecific aggregation and uptake of complement components that can occur in PEG tests, the tests should not be examined for direct agglutination and should not be tested with antiglobulin reagents containing anti-C3.21 Column Technologies A gel test for detecting red cell antigen-antibody interactions was first described in 1990 by Lapierre and colleagues.25 Cards consisting of six microcolumns, each containing agarose gel suspended in anti-IgG, are commercially available. Atop each card is an incubation chamber in which reagent red cells and test plasma are dispensed. The cards are incubated at 37ºC, then centrifuged. As the red cells pass through the gel, they are separated from the serum/plasma and come into contact with anti-IgG. If the red cells become coated with antibody during incubation, they will be agglutinated by anti-IgG. The agglutinated red cells become trapped in the gel; unagglutinated red cells pellet to the bottom of the microcolumn. A procedure for the gel test is depicted in Fig 5-6. The gel test has proven to be equivalent to standard tube technologies (LISS; PEG) for the detection of unexpected antibodies. In one study,26 the sensitivity and specificity of gel for potentially significant antibodies was 92% to 97%, respectively. This compares to 98% (sensitivity) and 90% (specificity) for a tube LISS procedure.

79

Section I: Part I

Column technologies offer several advantages over conventional test tube procedures: 1. Simplified testing, as follows: a. Dispense measured volumes of test plasma/serum and red cells (in LISS) into gel card incubation chambers. b. Transfer cards to an incubator (15 minutes) and then to a centrifuge (10 minutes). c. Read and record results. 2. When compared to conventional tube tests, there is no centrifugation/reading for direct agglutination after incubation; no addition of antiglobulin reagent; no need to validate negative tests using IgG-coated red cells. Omission of these manipulations (especially the associated, repetitive transfer of test tubes between racks and centrifuges) provides significant “hands-on” time savings. 3. Increased time savings through batch testing; a technologist can perform 36 to 48 antibody screens in about the same amount of time it takes to process 12 samples by conventional tube techniques.26 4. Increased reproducibility of results, through use of measured volumes of reactants, elimination of the washing process, less subjective reading of tests. 5. Stability of reactions, facilitating validation of results by a second technologist. With conventional tube methods, there is only one opportunity to read reactions; with the gel test, reactions can be read as many times as needed up to 48 hours after centrifugation. 6. Reduction in the detection of cold agglutinins (anti-I/HI -M, -P1, -Le) of no or doubtful clinical significance with consequential reduction in the number of samples requiring antibody identification. 7. Can be automated, or semi-automated using liquid sample handling devices, thereby further reducing “hands-on” time. Solid-Phase Adherence Methods Two forms of solid-phase adherence assays are available for red cell serologic testing. For direct tests, antibody is fixed to wells of a microplate, and red cells are added (eg, anti-A and anti-B for direct testing of donor/recipient ABO typing). Following centrifugation, red cells expressing the corresponding antigen will efface across the well; red cells lacking the antigen will pellet to the bottom of the well. For indirect tests, (eg, for detecting unexpected antibodies to red cell antigens) red cell membranes are affixed to microplate wells; test serum or plasma are added and the plates washed to remove unbound globulins. Indicator red cells, which are coated with antiIgG, are then added and the plates are centrifuged. The indicator red cells efface across the well in a positive test, and pellet to the center of the well in a negative test.27 An overview of a solid-phase adherence assay for antibody detection is depicted in Fig 5-7. Quality Assurance for Antiglobulin Tests All negative IATs must be validated using IgG-coated red cells.14,16(p44) The agglutination of these red cells confirms that

80

Solid Phase Assays  Antibody

Antigen-coat

 AHG-coated RBCs

Negative

Positive

Figure 5-7. Solid-phase adherence assays.

negative tests for unexpected antibodies are not caused by either inadequate washing before the addition of antiglobulin reagent, or inactivation of the antiglobulin reagent by contamination with human serum. This testing is applicable to tube tests; for gel and solid-phase assays, the control system specified by the manufacturer should be used.

Automated Pretransfusion Testing In recent years, several automated systems for pretransfusion testing have have received US market approval from the Food and Drug Administration (FDA). They are particularly suited for large hospital-based transfusion services, and include: 1. Automated gel column technology (ProVue, Ortho Clinical Diagnostics, Raritan, NJ). 2. Automated liquid microplate and solid-phase adherence technologies (Galileo, Immucor, Norcross, GA). 3. Microplates containing dried antisera for ABO/Rh typing and a modified solid-phase method for antibody detection (Tango, Biotest Diagnostics, Denville, NJ). These fully automated systems perform sample and reagent pipetting and subsequent analysis of agglutination reactions. Standard features include positive sample identification, process control through automated documentation of reagent lot numbers and expiration dates, batch and random access operating modes, STAT sample interrupt, and image analysis. The test results can be transmitted via an interface into the laboratory computer system. The benefits of automation include technologist time savings, positive sample identification, standardized testing (compliance with current good manufacturing practice), increased accuracy, and increased workload capacity. These have to be weighed against the costs for the instrument, reagents, and maintenance, as well as the time required for validation and training. Reagent Red Cells for Antibody Detection The FDA28 currently mandates that sets of reagent red cell samples licensed for use in pretransfusion antibody detection tests carry C, c, D, E, e, Fya, Fyb, Jka, Jkb, K, k, Lea, Leb, P1 M, N, S, and s antigens. Such red cells must not be pooled.16(p43)

Chapter 5: Red Cell Immunology and Compatibility Testing

It is impossible to find a single donor with red cells that carry all of the above antigens because adults rarely, if ever, have strong expression of both Lea and Leb on their red cells. Thus, reagent red cells for antibody detection are available commercially as sets of either two or three samples. The Rh phenotypes of red cells used in two-sample sets are R1R1 (DCc
E
e) and R2R2 (DC
cEe
). In three-sample sets, an rr (D
C
cE
e) sample is provided, in addition to R1R1 and R2R2 red cells. Use of three red cell samples facilitates the inclusion of red cells from individuals homozygous for particular blood group genes. Such red cells tend to have a stronger expression of an antigen when compared to red cells from individuals heterozygous for the same gene; this phenomenon is known as dosage. It is easier to find double-dose expression of blood group antigens among three reagent red cell samples than two samples; however, use of three samples increases the workload for antibody detection by 50% and rarely affords detection of significant alloantibodies that are not detected with two reagent red cell samples.29

Further Options Table 5-7 shows changes in pretransfusion testing practices within the United States from 2001 to 2004.30,31 Of note is the increased use of gel technology for both ABO/Rh typing and antibody detection. Not shown (2004 survey did not include) are practices that are considered redundant, including the following: 1. Use of polyspecific AHG reagents. These contain anticomplement activity in addition to anti-IgG, and facilitate detection of some antibodies that coat red cells with complement components including, notably, anti-Jka and anti-Jkb. Failure to detect such antibodies may lead to acute intravascular destruction of antigen-positive red cells.8 However, use of polyspecific AHG also facilitates detection of IgM complement-binding autoantibodies that are of no clinical significance.24 Many of these antibodies are autoantibodies directed toward the I antigen, and are found naturally in virtually all normal adult human sera.8 2. Room temperature incubation to detect agglutinating alloantibodies, as well as the detection of IgM autoantibodies. Antibodies that do not react at body temperatures rarely, if ever, cause significant destruction of transfused incompatible red cells.8 While room temperature incubation of tests was widely used through the early 1980s, many laboratories have since abandoned the practice.30 3. The use of a microscope to examine serologic tests. Examination for agglutination may be macroscopic, performed using an illuminated concave mirror, or microscopic. The latter is rarely necessary in routine practice; indeed, such critical examination of serologic tests can result in incorrectly recording negative tests as positive.17,18 4. Inclusion of an autocontrol (AC) as part of pretransfusion testing. The AC consists of testing the patient’s serum against his or her own red cells under the same conditions as those to which screening tests for unexpected antibodies are subjected. This test, or the DAT, is performed to detect globulins bound to

the patient’s red cells in vivo. Such in-vivo coating of red cells occurs in patients with autoimmune hemolytic anemia or hemolytic disease of the fetus and newborn, and may also be seen following therapy with certain drugs or transfusion with incompatible blood.14 Moreover, a positive DAT may be the earliest manifestation of an alloimmune response to a previous, recent transfusion.32 Inclusion of the DAT/AC as part of routine pretransfusion testing is no longer advocated; in the absence of detectable serum antibodies, the predictive value of a positive DAT is so low (0.29%) that routine testing is not cost effective.32 However, the DAT/AC is a good predictive test for immune-mediated hemolysis when performed on samples from patients with clinical manifestations of hemolytic anemia.33

The Principles of Antibody Identification When unexpected antibodies are present, as indicated by positive screening tests, they must be identified. At a minimum, this involves testing the patient’s serum against a panel of fully phenotyped reagent red cell samples as well as the patient’s own cells. A typical panel is shown in Table 5-8, which also includes the results of antibody identification tests with a serum containing a mixture of anti-M and anti-K. It should be noted that the tests performed in this example do include a reading for direct agglutination after room temperature incubation and after incubation at 37ºC. The results of tests with ficin-treated red cells are also displayed. This example will be used to illustrate a process by which the antibody specificities can be ascertained whenever there are reactive and nonreactive red cell samples. A typical approach follows: 1. The reactions of the AC are examined. Alloantibodies, by definition, should not react with the red cells of the antibody producer. When the AC is reactive, autoantibodies may be present or the AC may react because alloantibodies have formed to recently transfused red cells that are still circulating in the recipient. In other cases, both auto- and alloantibodies may be present. Therapy with certain drugs such as intravenous penicillin or a cephalosporin can also cause the DAT/AC to be positive.14 The AC is negative in the case shown in Table 5-8, so autoantibodies likely are not present. 2. The graded reaction strengths are examined. If all positive tests are equally reactive, then only a single antibody may be present. If there are no negative tests with reagent red cells but the AC is nonreactive, the presence of antibody to a high-prevalence antigen should be considered. Variability in reaction strength may be an indication of dosage; ie, the antibody reacts stronger with red cells from homozygotes (double-dose) than with red cells from heterozygotes (single-dose). Except for antibodies to D, P1, and Xga antigens, most blood group antibodies manifest dosage. There is variable expression of P1 and Xga antigen on red cells from different donors, but this is unrelated to their zygosity. Given the varying degrees of reactivity of positive red cell samples in Table 5-8, more than one antibody appears to be present and MN
red cells react significantly stronger than MN red cells. 3. A process of crossing out is undertaken. This process involves evaluating the antigens present on nonreactive red cells. Only

81

Section I: Part I

Table 5-8. Results of Tests between a Panel of Reagent Red Cells and a Serum Containing Anti-M and Anti-K RH

MNS

P1 LE

KEL

JK

PANEL

D

C

c

Cw E

e

f

M

N

S

s

P1 Lea

Leb

K k

1 rr 2 R1R1 3 R1R1 4 R2R2 5 r r 6 rrV 7 rr 8 rr 9 rr 10 rr 11 Ror PATIENT

0    0 0 0 0 0 0 

   0 0 0 0 0 0 0 0

 0 0        

0 0  0 0 0 0 0 0 0 0

   0       

 0 0 0       

 0  0   0    

  0  0    0  

  0 0  0 0    0

0    0    0  0

0         0 

 0    0  0  0 

0 0  0 0 0  0 0  0

0 0 0   0 0 0 0 0 0

0  0 0 0 0 0  0  0

reagent red cell samples 2 and 4 of Table 5-8 are nonreactive. Antibodies to any of the antigens (D, C, c, E, e, N, S, s, P1, Lea, Leb, Jka, Jkb, Fya, Fyb, and Xga) present on these two cell samples can be eliminated from initial consideration. This leaves only antibodies to M, K, Cw, f, Kpa, and Jsa. However, Cw, Kpa, and Jsa are low-prevalence antigens and are not likely to have been present on the reagent red cells used for antibody detection. Similarly, f antigen will not be present on a two-sample screening set of R1R1 and R2R2 cells. Thus, a provisional specificity of anti-M and anti-K can be assigned to this serum. 4. The test phase at which reactivity is observed is evaluated. Antibodies that are usually IgM (see Table 5-2), such as anti-M, -N, -Lea, and -P1, react as direct agglutinins at room temperature. They do not react solely by an IAT, although there may be carryover of direct agglutination that can be observed in antiglobulin tests. Similarly, those antibodies that are usually IgG (eg, anti-Rh, -K, -Fy, -Jk, -S) react preferentially by an IAT. However, IgM antibodies of these specificities can be encountered, especially during the early stages of the immune response. If hemolysis of reagent red cells has been observed, a complement binding antibody such as anti-Lea or anti-Jka may be present. This complement-binding activity is often enhanced in tests with ficin-treated red cells, and in the absence of direct lysis of test red cells can best be observed through the use of polyspecific AHG. With the case under discussion, the anti-M appears to react best at room temperature and the anti-K reacts best by IAT. This is consistent with the anti-M being IgM, although many examples of antiM do have an IgG component,9 while the anti-K is most likely IgG (see Table 5-2). The anti-f does not appear to be present because f-positive cell samples 1, 6, 8, and 11 are nonreactive by IAT. 5. The results of tests with enzyme-treated red cells, if performed, are evaluated. Knowledge of the anticipated behavior of certain antibodies with enzyme-treated red cells can be very helpful when identifying antibodies—especially when dealing with

82

      0    

FY

XG

LISS

Xga

RT

37

IAT 37

IAT

3 0 4 0 4 3 0 3 4 3 3 0

1 0 3 0 3 1 0 1 3 1 1 0

0 0 3 0 3 0 4 0 3 3 0 0

0 0 3 0 0 0 4 0 0 3 0

Kpa

Jsa

Jka

Jkb

Fya Fyb

0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 

   0  0 0    

       0 0  

0    0 0   0  0

  0    0   0 0

      0 0   0

1 2 3 4 5 6 7 8 9 10 11 AC

FICIN

0 0 0 0 0 0 0 0 0 0 0

sera containing mixtures of alloantibodies. Antigens of the MNS, FY, and XG systems are cleaved by treatment of red cells with proteolytic enzymes such as papain or ficin. The high-prevalence antigens Ch, Ena, JMH, Inb, Rg, Yta, and some Ge antigens are also cleaved by proteolytic enzymes.9 Negative or weak reactions are observed when antibodies to these antigens are tested with ficin or papain treated red cells. In contrast, treatment of red cells with proteolytic enzymes enhances their reactivity with Rh antibodies, as well as complement-binding antibodies such as anti-Lea, -P1, and -Jka. With the case under discussion, the double-dose M-positive red cell samples (3, 5, 9) react in LISS tests at 37ºC and by IAT. These cells are nonreactive when pretreated with ficin, which of course is expected for anti-M. In contrast, K antigen is not affected by ficin treatment, so there is no difference in the IAT reactions of ficin and LISS tests with cell samples 3, 7, and 10. Anti-f can now be completely eliminated from consideration because if present, it should have reacted with all the K-negative ficin-treated cells from donors with r (ce) haplotype. 6. The data are reviewed to ensure that all other antibodies that must be excluded (by virtue of the FDA’s requirements for reagent red cells) are not present in the serum. In this case, this was accomplished in Step #3. The fact that antibodies to Cw, Jsa, and Kpa could be present should not be of concern; they are no more likely to be present in this case than they are in patients with negative screening tests for unexpected antibodies. Some workers believe that exclusion tests should be performed with double-dose red cells. Their rationale appears to be based on the notion that if a patient has made one alloantibody they are likely to make another—more so than a nonalloimmunized patient is likely to make their first antibody following transfusion. To detect newly forming antibodies, they advocate the use of red cells from apparent homozygotes. However, there are no data to support this notion.

Chapter 5: Red Cell Immunology and Compatibility Testing

Use of double-dose red cells for pretransfusion antibody detection is not required by either the FDA28 nor recommended by the AABB14 (see requirements for reagent red cells). Reagent manufacturers do provide red cells with double-dose expression of Rh antigens, and some workers have established institutional policies regarding the required expression of other antigens on these cells. It would seem appropriate to use the same policies for exclusion purposes whenever practical; ie, ensure absence of anti-Jka with Jk(ab
) cells if such apparent double-dose expression is required for antibody detection. It would not, however, be practical to exclude anti-E in a patient found to have anti-D using D-negative cells carrying double-dose expression of E, because this would require use of rare r r cells. 7. The data are subjected to statistical analysis. Before final conclusions can be made regarding antibody specificity, there must be sufficient negative test results with red cells that lack the corresponding antigen and sufficient positive test results with red cells that carry that antigen. To obtain a confidence level of 95% (p  0.05), there should be at least three nonreactive antigennegative red cell samples and at least three reactive antigenpositive red cells. This requirement has been met for both the anti-M and the anti-K of Table 5-8. 8. The patient’s red cells lack the corresponding antigen. As discussed earlier, this is fundamental to the formation of alloantibodies following transfusion or pregnancy. The red cells from the patient of Table 5-8 should be tested with anti-M and anti-K. They should lack both M and K antigens. There are, however, exceptional cases of alloantibody formation when the corresponding antigen appears to be present on the autologous red cells. Most notably, this is seen with Rh-positive individuals of the partial-D phenotype who make antibody to the portions of D antigen that are absent from their red cells. The above illustration represents but a basic approach to antibody identification. More complex cases involving autoantibodies, multiple alloantibodies, mixtures of both auto- and alloantibodies and antibodies to high-prevalence antigens will require the resources of an immunohematology reference laboratory. The interested reader is referred elsewhere14,19 for information regarding the investigation of complex antibody problems.

Donor-Recipient Testing Selection of Blood for Transfusion ABO and Rh Red Blood Cells (RBCs) and Whole Blood selected for transfusion must be compatible with the serum of the intended recipient. To avoid the hemolytic and often fatal consequences of an ABO-mismatched transfusion, red cells carrying A and/ or B antigens should not be transfused to a patient unless the patient’s red cells also carry those antigens. Group O individuals should receive group O red cells, but AB individuals can receive red cells of any ABO type. Rh-negative individuals, particularly females of childbearing potential, should receive Rhnegative blood. Rh-positive individuals may receive blood of either Rh type.14

Unexpected Antibodies When the identified antibodies are known to cause accelerated destruction of transfused incompatible red cells, blood selected for transfusion should be shown to lack the corresponding antigen or antigens.14,16(p45) This entails testing donor units with reagent antisera that are available commercially or prepared in-house from previously investigated samples. Examples of potentially significant antibodies include those directed toward antigens of the RH, JK, KEL, and FY systems, and the S and s antigens of the MNS system, as well as most other antibodies active at 37ºC and/or by an IAT (see Table 5-2). When antibodies with specificities directed toward M, N, P1, and LE antigens are present, particularly when the antibodies react best at or below room temperature, blood selected for transfusion need only be shown to be compatible by IAT following 37ºC incubation; demonstrating that compatible units lack the relevant antigen(s) is not required.34,35 In instances when autoantibodies are present, least incompatible units should be selected for transfusion once it has been established that the autoantibody is not masking a concomitant, clinically significant alloantibody.33

Serologic Crossmatch Before whole blood or red cells are administered, except in an emergency, a major crossmatch must be performed. This usually entails tests between donor red cells selected for transfusion and the prospective recipient’s serum or plasma sample that was used for ABO, Rh, and antibody detection tests. The methods used should be capable of detecting ABO incompatibility and include an IAT. However, in the absence of unexpected antibodies (and absence of records of prior detection of such antibodies) in the intended recipient’s serum, only testing to detect ABO incompatibility is required.16(p46) Antiglobulin Crossmatch When clinically significant unexpected antibodies are present, or a patient’s records indicate that such antibodies have been detected previously, blood selected for transfusion must be tested with the patient’s serum or plasma by an IAT. Any of the methods described earlier for antibody detection can be used. An antiglobulin crossmatch can also be performed routinely on patients with nonreactive screening tests for unexpected antibodies. This will detect ABO incompatibility and may detect unexpected antibodies that were missed in pretransfusion screening tests. Unexpected antibodies to low-incidence antigens and antibodies manifesting dosage may be detected in this manner, as may antibodies missed in screening tests because of technical error. However, the predictive value of a positive IAT crossmatch following nonreactive screening tests for unexpected antibodies is sufficiently low that many large hospital transfusion services do not perform an IAT crossmatch except as required when unexpected antibodies are present or there are records of such antibodies.30,31 Detection of ABO Incompatibility Only a procedure for detecting ABO incompatibility is required when screening tests for unexpected antibodies are negative and there is no record of the patient having had such antibodies in

83

Section I: Part I

the past.16(p46) Detection of ABO incompatibility can be done serologically or, in certain situations, electronically through the use of computers. An immediate-spin crossmatch between the prospective recipient’s serum or plasma and donor red cells suspended in EDTA-saline (to prevent false-negative results from prozone by complement-fixing high-titer anti-A and -B) is an acceptable serologic method for the detection of ABO incompatibility.36 Alternatively, the ABO groups of both the donor units and a blood sample from the intended recipient can be confirmed immediately before the units are released for transfusion.

Electronic Crossmatch With the emergence of blood bank information systems, laboratories are beginning to use the integrity of computer software to detect ABO incompatibility between the sample submitted for pretransfusion testing and the donor unit selected for transfusion.37 This computer or electronic crossmatch (EXM) replaces the immediatespin test for detecting ABO incompatibility. Currently, an EXM is performed in some 2% of North American facilities,30,31 and in other countries (Canada, Sweden, Australia, United Kingdom, Hong Kong).38-42 An EXM may be used instead of an immediate-spin crossmatch provided that: 1. The computer system has been validated on-site to ensure that only ABO-compatible red-cell-containing components have been selected for transfusion. 2. No clinically significant antibodies are detected in the recipient’s serum/plasma and there is no record of previous detection of such antibodies. 3. There are concordant results of at least two determinations of the recipient’s ABO type on record, one of which is from a current sample. 4. The system contains the donor unit number, the component name, its ABO/Rh type and the interpretation of confirmatory tests; and recipient information and ABO/Rh. 5. A method exists to verify the correct entry of data before release of blood or components. 6. The computer contains logic to alert the user to discrepancies between donor unit labeling and confirmatory test interpretation, and to ABO incompatibilities between the recipient and the donor unit. 7. The donor unit blood type has been confirmed serologically using red cells from an attached segment. These requirements are promulgated in AABB Standards,16 which should be consulted for the precise wording. Table 5-9 summarizes the FDA requirements for facilities wishing to implement an electronic crossmatch.43 Flow diagrams for EXM processes are depicted in Fig 5-8. Information on implementing an EXM is available elsewhere.44-46 Advantages of the EXM The advantages of implementing an EXM are as follows: 1. Technologist time savings. This is the major advantage, and equates to approximately 2.5 minutes per unit crossmatched.37

84

2. Reduced sample volume requirements. A 5- to 7-mL sample is more than adequate, and can be used for EXM for at least 30 days after collection provided other pretransfusion tests are performed soon after collection. The latter greatly facilitates sameday admission programs. 3. Reduced sample handling. Significant reduction in exposure to biohazardous materials results from elimination of the need to prepare donor red cell suspensions from attached segments and the need to use the patient’s sample for the immediate-spin crossmatch. 4. No unwanted “false”-positive test results. Cold agglutinins, rouleaux, etc, are a frequent cause of a positive immediate-spin crossmatch following a negative antibody screen. These unwanted positive results are avoided with use of an EXM. 5. Improved accuracy (no unwanted “false”-negative test results), unlike the immediate-spin crossmatch, which is prone to yield false-negative results between some 50% of group B sera and eclectic A2B red cells.47,48 The EXM will detect such incompatibilities provided, of course, the patient and donor ABO types are accurately entered into the computer, and the EXM system has been properly designed, validated, and implemented. Moreover, unwanted negative test results associated with potent, prozoning anti-A and anti-B36 are avoided. 6. Decreased turnaround time, which can lead to a decrease in the crossmatch-to-transfusion ratio. Disadvantages of the EXM The disadvantages of implementing an EXM are few. Barriers to implementation include availability of a laboratory information system with software for blood bank applications, the effort required to develop a system that conforms to AABB and FDA requirements, and the on-site validation process that must be undertaken to obtain FDA approval. Consideration should also be given to the fact one cannot bill for performing EXM, but given current health-care reimbursement practices and policies (managed care, diagnosis-related groups), this may not be a real issue. One can, and should, bill for the second ABO and Rh, if required. A current procedural terminology code of 909090 has been assigned to the EXM. This code can be used to track activity, but does not generate a charge. 49

Prior Records Check As part of ongoing quality assurance, and for compliance with the Standards for Blood Banks and Transfusion Services of the AABB,16 the results of current ABO, Rh, antibody detection, and compatibility tests must be checked against records of previous tests, if performed. This must be accomplished before blood is released for transfusion, preferably at the time pretransfusion tests are completed. The specific records that must be checked are those for ABO and Rh typing performed within the previous 12 months, and any difficulties in typing, unexpected

Chapter 5: Red Cell Immunology and Compatibility Testing Table 5-9. FDA Expectations of Facilities Performing an Electronic Crossmatch43 STANDARD OPERATING PROCEDURE (SOP) Standard operating procedures shall include: 1. Identification of the source(s) of the application software (name of software vendor or developer, the name and version of the software package). 2. A note to the effect that the computer crossmatch function was designed by a commercial vendor or a modification or extended usage under the control of the blood bank or transfusion service. 3. The location(s) where the system will be used. 4. Institution-specific, detailed instructions that address all facets of the procedure; including all circumstances for which the SOP will be used. SOFTWARE REQUIREMENTS The software will not permit electronic release of blood products unless there are: 1. Records of two determinations of the recipient’s ABO group, one of which is from a current sample. 2. A record of a negative test for clinically significant antibodies from a current specimen and no record of previous detection of such antibodies. INFORMATION The software contains: 1. Donor unit information: a. Unit number. b. Component name. c. ABO and Rh type. d. The interpretation of the ABO confirmatory test (for units collected, processed and labeled by a separate facility). 2. ABO and Rh type of the recipient. 3. Detection of ABO incompatibility: a. The complete decision table is defined in the software; eg, expected response for each possible combination of recipient/donor ABO combinations. b. Appropriate restrictions based on component type are defined in the software; eg, most facilities will use decision tables to approve/reject release of a number of different red cell products (decision tables must be validated for each product code). 4. Logic to alert the user to: a. Missing required information. b. Donor unit labeling and confirmatory test interruption do not match. c. ABO incompatibilities between the recipient and the donor unit. d. Any other user-defined criteria. VALIDATION 1. The validation design must cover both “automated” software decision-making and decisions made “manually” by users following the SOP. 2. The validation must be performed on site using the same hardware and software that will be employed in routine use. 3. Validation documentation includes: a. Testing strategies. b. Test acceptance and completion criteria. c. Copies of the documentation and testing results. 4. There is a method to review and validate entry of data before it can be used in the decision process. For manual entry this may be double entry of data or an affirmation response after reviewing on-screen display in addition to initial entry. 5. Validation testing must include: a. At least one case of each possible combination of decision variables. b. A reasonable number of cases where one or more required data elements are missing. c. A reasonable number of challenges for the selection of blood and/or components that do not meet specified criteria. 6. If the decision tables govern issues other than ABO (eg, donor/recipient Rh match), they must be adequately tested in the validation protocol.

antibodies, severe adverse reactions to transfusion, and any special transfusion requirements. Any discrepancies between past and present ABO and Rh typing results must be thoroughly investigated; the most likely explanation is that the present sample is not from the same individual whose blood was tested previously. Further, even in the absence of detectable unexpected antibodies in the current sample, a record of such unexpected antibodies in previous samples must be taken into consideration when selecting and crossmatching blood for present and future transfusions.

Labeling Before blood is released for transfusion the container shall have an affixed label or tie tag indicating the recipient’s first and last names, unique identification number, the donor unit number, and the interpretation of compatibility tests, if performed.16(p50) The unit must be inspected visually before release; if any abnormality in color or appearance is noted, the unit should not be issued. A record should be made of this inspection. The expiration date should also be checked to avoid issuing an outdated unit.14

85

Section I: Part I

verify against invoice

NO

verify computer interpretation

select reserved units by patient name and ID#

release only O Rh-

update to available status

YES

NO

verify patient name and ID#

valid patient ABO/Rh?

create worksheet do ABO, Rh antibody screen

confirm ABO Rh- units

ABO/Rh confirmed?

return to vendor

verify data entry

enter patient name, unique ID# from barcode label

er t!

al

er t!

check

quarantine unit

al

enter unit #, product code, ABO/Rh and outdate from barcode

YES

ABO/Rh conflict with records?

verify computer interpretation

NO

save sample

investigate

get patient sample or requisition

create worksheet from ID#

verify name, collection date, ID#

YES

two ABO/Rh? no antibodies?

select and barcode units

enter unit # from barcode

NO

YES

stop!

NO

unit ABO compatible?

YES

issue

YES

stop!

ABO mismatch?

NO

manage per SOP

print and affix labels

Figure 5-8. Flow diagrams for the electronic crossmatch. From the top left: process for donor unit entry and confirmatory ABO/Rh typing; process for initial patient ABO/Rh typing and detecting unexpected antibodies; electronic crossmatch process; dispense (release) process.

Issue At the time blood and blood components are issued, there must be a final check of records maintained in the transfusion service.16(p50-51) These records shall include: 1. The recipient’s name and unique identification number; the ABO group and Rh type of the recipient, if required. 2. The donor unit number, or pool identification number (for platelets and cryoprecipitate); and the ABO group and Rh type of the donor. 3. The interpretation of compatibility tests, if performed. 4. The date and time of issue. Documentation that this process occurred can be facilitated by use of a log book or the information system, in which the name of the person performing the release check can be recorded.14 Emergency Release When a patient’s ABO group is not known, only group O Red Blood Cells can be issued.16(p52) For female patients of childbearing potential, these should also be Rh-negative. If the ABO group and Rh type have been determined on a current sample, typespecific Whole Blood or ABO-compatible RBCs may be issued. The container label or tie tag should indicate that compatibility testing was not completed at the time the unit was released. Testing should be completed expeditiously, and the records should contain a statement, signed by the requesting physician,

86

indicating the need for transfusion before completion of compatibility testing.

Bedside Check Before administering blood, the physician’s written order should be reviewed to verify the request for transfusion.14 The transfusionist who administers the blood is responsible for this and for performing a final errors check. Verification of the following information must occur16(p53-54): 1. Recipient Identification: The name and identification number on the patient’s wristband must be identical to the name and number on the form attached to the unit. 2. Unit Identification: The unit number on the blood container must match the unit number on the transfusion form. 3. ABO/Rh: The ABO and Rh type on the donor unit primary label must agree with that recorded on the transfusion form. 4. Expiration Date: The expiration date of the unit should be checked and the unit verified as acceptable for transfusion.

Hemovigilance and Pretransfusion Testing Blood transfusion subjects a patient to significant risks, ranging in severity from febrile nonhemolytic reactions and red cell alloimmunization to disease transmission and death from bacterial contamination or transfusion of grossly incompatible blood. The latter can prove fatal, and while considerable resources have been spent

Chapter 5: Red Cell Immunology and Compatibility Testing

in recent decades to reduce infectious disease transmission, only recently has the issue of “errors in transfusion” been addressed. These errors in transfusion care stem from the following causes: 1. Wrong blood in tube, or WBIT, resulting from misidentification of the patient at time of pretransfusion sample collection or mislabeling of the collection tube. 2. Inappropriate transfusion; ie, exposing patients to the hazards of transfusions that they did not need. 3. Mistransfusion; ie, transfusion of the wrong unit or transfusion to the wrong patient. Adherence to standard operating procedures mandated at an institutional level and use of dedicated phlebotomy service personnel to collect pretransfusion samples can decrease the incidence of WBIT (Friedman BA. personal communication). To eliminate errors caused by WBIT, some facilities require concordant results on two independently collected samples before release of non-group-O red cell products. Yet others have implemented locking devices (eg, Bloodloc, Novatec Medical, Effingham, IL) that require assignment of a three-letter code to any potential transfusion candidate. This code is printed on the patient’s identification bracelet and transcribed on the sample tube and requisition at time of sample collection. After completion of pretransfusion testing, units of blood are placed into plastic bags, each of which is closed with a combination lock set to the patient’s assigned three-letter code. At the bedside, the transfusion nurse can open the lock only by using the patient’s code. If there is any discrepancy, the blood is returned to the blood bank. More recently, transfusion services have begun to apply new technologies widely available in commerce to prevent these errors in transfusion.50 Such technologies include: 1. Barcodes and radio-frequency identification (RFID) technologies, both of which serve to improve the accuracy of sample labeling and the bedside checking process. RFIDs are also capable of constant monitoring of blood product location and storage temperature, thereby improving compliance with good manufacturing practice regulations. 2. Computerized order entry and support systems to improve the decision-making process, including use of nanotechnology sensors that act as barriers to inappropriate transfusions. Technologies now being implemented in Europe place the entire transfusion process under computerized control.51 Features of such a process include: 1. Direct order entry, requiring documentation of the reason to transfuse and notification of the ordering physician if the request does not meet established guidelines. 2. Barcode scanning of both phlebotomist and patient identification bracelets at time of sample collection, using a personal digital assistant (PDA) device to produce accurate sample labels. 3. Following processing and compatibility testing, scanning and placement of blood into a computer controlled blood issue refrigerator. 4. Tracking of all movements in and out of the refrigerator. Access to patient-assigned products requires entry of operator and patient identification information.

Following FDA approval and successful validation, it is anticipated that such systems will gain acceptance within the United States.

Conclusion Compatibility testing constitutes a quality assurance program designed to detect serologic incompatibility between donor unit and the intended recipient and to prevent both clerical and technical errors that may have serious if not fatal consequences. Assurance of quality requires proper performance of each individual task. There can be no substitute for proper patient identification, proper sample labeling, and proper performance of serologic tests.

Disclaimer The author has disclosed no conflicts of interest.

References 1. Alt F, Blackwell TK, Yancopoulos GD. Development of the primary antibody repertoire. Science 1987;248:1079-87. 2. Kuby TJ, Kindt TJ, Osborne BA, Goldsby RA. Immunology, 6th ed. New York: WH Freeman & Company, 2006. 3. Parslow TC. Immunoglobulins and immunoglobulin genes. In: Parslow TC, Stites DP, Terr AL, Imboden JB, eds. Medical immunology, 10th ed. New York: McGraw-Hill, 2001:95-114. 4. Chestnut RW, Grey HM. Antigen presentation by B cells and its significance in T-B interactions. Adv Immunol 1986;39:51-64. 5. Kohler G, Milstein C. Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur J Immunol 1976; 6:511-19. 6. Silberstein LE. The antibody response to antigen. In: Nance S, ed. Alloimmunity: 1993 and beyond. Bethesda: AABB, 1993:25-47. 7. Race RR, Sanger R. Blood groups in man, 6th ed. Oxford: Blackwell Scientific Publications, 1965. 8. Klein HG, Anstee D. Mollison’s blood transfusion in clinical medicine. 11th ed. Oxford: Blackwell Scientific Publications, 2005. 9. Reid ME, Lomas-Francis C. The blood group antigen FactsBook, 2nd ed. London: Academic Press, 2004. 10. Pollack W. Some physicochemical aspects of hemagglutination. Ann NY Acad Sci 1965;127:892-900. 11. Pollack W, Hager HJ, Reckel R, et al. A study of the forces involved in the second stage of hemagglutination. Transfusion 1965;5:158-83. 12. Cook GMW, Heard DH, Seamen GVF. A sialomucopeptide liberated by trypsin from the human erythrocyte. Nature (Lond) 1960;188:1011-12. 13. Judd WJ. Antibody elution from red cells. In: Bell CA, ed. Antigenantibody reactions revisited. Arlington, VA: AABB, 1982:175-221. 14. Roback JD, Combs MR, Grossman BJ, Hillyer CD, eds. Technical manual, 16th ed. Bethesda, MD: AABB, 2008. 15. Shulman IA. Controversies in red cell compatibility testing. In: Nance SJ, ed. Immune destruction of red cells. Arlington, VA: AABB, 1989:171-99. 16. Price TH, ed. Standards for blood banks and transfusion services, 25th ed. Bethesda, MD: AABB, 2008.

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17. Oberman HA, Judd WJ. Cost containment in transfusion medicine: A view from the United States. In: Cash JD, ed. Progress in transfusion medicine, vol 3. Edinburgh: Churchill-Livingstone, 1988. 18. Issitt PD. Applied blood group serology, 4th ed. Durham, NC: Montgomery Scientific Publications, 1998. 19. Judd WJ. Judd’s Methods in immunohematology, 3rd ed. Bethesda, MD: AABB Press, 2008. 20. Löw B, Messeter L. Antiglobulin tests in low-ionic strength salt solution for rapid antibody screening and crossmatching. Vox Sang 1974;26:53-61. 21. Nance S, Garratty G. A new technique to enhance antibody reactions using polyethylene glycol (abstract). Transfusion 1985;25:475. 22. Fitzsimmons JM, Morel PA. The effects of red blood cell suspending media on hemagglutination and the antiglobulin test. Transfusion 1979;19:81-5. 23. Lalezari P, Jiang AF. The manual Polybrene® test: A simple and rapid procedure for detection of red cell antibodies. Transfusion 1980;20:206-11. 24. Garratty G. The role of complement in blood group serology. CRC Crit Rev Clin Lab Sci 1985;20:25-56. 25. Lapierre Y, Rigal D, Adam J, et al. The gel test: A new way to detect red cell antigen-antibody reactions. Transfusion 1990;30:109-13. 26. Judd WJ, Steiner EA, Knafl PC. The gel test: sensitivity and specificity for unexpected antibodies to blood group antigens. Immunohematology 1997;13:132-5. 27. Judd WJ. ‘New’ blood bank technologies. Clin Lab Sci 1998;11:106-13. 28. Code of federal regulations Title 21 CFR, parts 606 and 660. Washington DC: US Government Printing Office, 2008 (revised annually). 29. Judd WJ. Testing for unexpected red cell antibodies—two or three reagent red cell samples. Immunohematology 1997;13:90-2. 30. Maffei LM, Johnson ST, Shulman IA, Steiner EA. Survey on pretransfusion testing. Transfusion 1998;38:343-49. 31. Shulman IA, Maffei LM, Downes KA. North American pretransfusion testing practices, 2001-2004: Results from the College of American Pathologists Interlaboratory Comparison Program survey data, 2001-2004. Arch Pathol Lab Med 2005;129:984-9. 32. Judd WJ. Barnes BA, Steiner EA, et al. The evaluation of a positive direct antiglobulin test (autocontrol) revisited. Transfusion 1986;26:220-4. 33. Judd WJ. Investigation and management of immune hemolysis— autoantibodies and drugs. In: Wallace ME, Levitt J, eds. Current applications and interpretation of the direct antiglobulin test. Arlington, VA: AABB, 1988:47-103. 34. Cronin CA, Pohl BA, Miller WV. Crossmatch-compatible blood for patients with anti-P1. Transfusion 1978;18:728-30. 35. Issitt PD. Antibodies reactive at 30 centigrade, room temperature and below. In: Butch SH, ed. Clinically significant and insignificant antibodies: A technical workshop. Washington, DC: AABB, 1979:13-28.

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36. Judd WJ, Steiner EA, O’Donnell DB, Oberman HA. Discrepancies in ABO typing due to prozone: How safe is the immediate-spin crossmatch? Transfusion 1988;28:334-8. 37. Butch SH, Judd WJ, Steiner EA, et al. Electronic verification of donorrecipient compatibility: The computer crossmatch. Transfusion 1994; 34:105-9. 38. Sawfenberg J, Hogman CF, Cassemar B. Computerized delivery control: A useful and safe complement to the type and screen compatibility testing. Vox Sang 1997;72:162-8. 39. Cox C, Enno A, Deveridge S, et al. Remote electronic release blood system. Transfusion 1997;37:960-4. 40. Chan A, Chan JC, Wong LY, Cheng G. From maximum surgical blood ordering schedule to unlimited computer crossmatching: Evolution of blood transfusion for surgical patients at a tertiary hospital in Hong Kong. Transfus Med 1996;6:121-4. 41. British Committee for Standardization in Haematology, Blood Transfusion Task Force. Guidelines for pre-transfusion compatibility procedures in blood transfusion laboratories. Transfus Med 1996;6:273-83. 42. Georgsen J, Jensen F, Jeppesen S, et al. Transfusion service of the county of Funen. Organizational and economic aspects of restructuring. Ugeskr Laeger 1997;159:1758-62. 43. Food and Drug Administration. Computer crossmatch reviewer’s checklist. Rockville, MD: CBER Office of Communication, Training, and Manufacturers Assistance, 1997. 44. Butch SH, Judd WJ. Requirements for the computer crossmatch (letter). Transfusion 1994;34:187. 45. Judd WJ. Requirements for the electronic crossmatch. Vox Sang 1998;74(S2):409-17. 46. Judd WJ. The electronic crossmatch: An alternative method to the immediate-spin crossmatch to detect ABO incompatibility. Advance 1998;10(15):16-23. 47. Steane EA, Steane SM, Montgomery SR, Pearson JR. A proposal for compatibility testing incorporating the manual hexadimethrine bromide (Polybrene®) test. Transfusion 1985;25:176-8. 48. Berry-Dortch S, Woodside C, Boral LI. Limitations of the immediate spin crossmatch when used for detecting ABO incompatibility. Transfusion 1985;25:540-4. 49. Physician’s current procedural terminology: Chicago: American Medical Association, 2007. 50. Dzik WH. New technology for transfusion safety. Br J Haematol 2007;136:181-90. 51. New blood tracking system used across Europe. E-Health Europe Newsletter, December 17, 2007. [Available at http://ehealtheurope. net/news/3315 (accessed January 9, 2008).]

6

Carbohydrate Blood Groups Eldad A. Hod,1 Patrice F. Spitalnik,2 & Steven L. Spitalnik3 1

Transfusion Medicine Fellow, Department of Pathology and Cell Biology, Columbia University Medical Center, New York, New York, USA 2 Assistant Professor of Clinical Pathology, Department of Pathology and Cell Biology, Columbia University Medical Center, New York, New York, USA 3 Professor of Pathology and Vice Chairman of Laboratory Medicine, Department of Pathology and Cell Biology, Columbia University Medical Center, New York, New York, USA

Many important human blood group antigens are glycoconjugates. These antigens are grouped together in this chapter because there are similarities in their biosynthesis, in the human immune response to these antigens, and in the outcome in vivo after transfusion of incompatible blood. The antigens in the ABH, Lewis, P, and I blood group systems are synthesized by interrelated pathways. These oligosaccharide antigens may exist free in solution. In addition, they may be covalently attached to lipids (ie, ceramides) to form glycosphingolipids, or to polypeptides to form mucins, integral membrane glycoproteins, or soluble glycoproteins. Specific glycosyltransferase enzymes catalyze formation of the relevant glycosidic linkages (ie, the bonds between monosaccharides). Some glycosyltransferases, found in all individuals, form framework structures. The genes encoding other glycosyltransferases are allelically inherited and the resulting enzymes specify the synthesis of variable structures. Because of their variable inheritance and expression, the latter may form immunologically recognized blood group antigens. The absence of particular blood group antigens in certain individuals may result in specific antibody production after stimulation by the foreign antigen. As described below, antigens in the ABH, Lewis, P, and I blood group systems are synthesized on common precursor framework molecules. Competition between genetically inherited blood-group-specific glycosyltransferases results in a rich mixture of antigenic molecules. In addition, a single oligosaccharide may encode several different blood-group antigens. The immune response to carbohydrate antigens, particularly when presented as repetitive epitopes, is typically thymus independent (for review, see Mond et al1). That is, the repetitive, multivalent antigens can directly stimulate B cells to synthesize antibodies without the aid of helper T cells. Thymus-independent immune responses classically produce IgM antibodies, and most antibodies to carbohydrate blood group antigens are of the IgM

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

class. Individuals lacking a particular carbohydrate blood group antigen on their red cells often have “naturally occurring” IgM antibodies to that antigen in their sera, even if they were not previously exposed to human blood components. Most evidence suggests that these antibodies did not arise spontaneously without prior antigenic stimulation, but rather that cross-reacting antigens in the environment, such as on gut bacteria, stimulate specific IgM production.2 For example, a particular serotype of Escherichia coli synthesizes the blood group B antigen as part of its lipopolysaccharide.3 In contrast, high-titered IgG antibodies to carbohydrate antigens are found in certain individuals. These IgG antibodies may be induced by a thymus-dependent form of these oligosaccharides, perhaps as individual epitopes on glycoproteins, in which T-cell help leads to an isotype switch from IgM to IgG. Because antibodies to carbohydrate blood group antigens are predominantly IgM, these decavalent molecules directly agglutinate antigen-positive human red cells without the aid of an antiglobulin reagent. Agglutination by these antibodies in vitro typically improves at temperatures ⬍37ºC. Because most IgM molecules directly fix complement, these antibodies can cause immediate intravascular hemolysis after transfusion of incompatible, antigen-positive red cells. In unusual cases, carbohydrate-specific IgG antibodies coat red cells in vivo, leading to extravascular hemolysis after incompatible transfusion. In addition, the latter may cross the placenta, resulting in hemolytic disease of the fetus and newborn.4,5

ABH, Secretor, and Lewis Systems ABH Antigens: Introduction The ABO blood group system is the most important one with respect to blood transfusion, hematopoietic stem cell transplantation, and solid organ transplantation. Karl Landsteiner was the first to discover human alloantigens by using a conceptually simple experiment (Table 6-1). The red cells of each individual were found either to lack or to have one or both of two antigens, A and B. In addition, the serum of each subject contained “naturally

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Section I: Part I

Table 6-1. ABO Blood Group Antigens and Antibodies

H

Red Cell Antigens

Serum Antibodies

Blood Group

A

B

Anti-A

Anti-B

A B AB O

⫹ ⫺ ⫹ ⫺

⫺ ⫹ ⫹ ⫺

⫺ ⫹ ⫺ ⫹

⫹ ⫺ ⫺ ⫹

Gal-R / Fuc(α1-2)

occurring” directly agglutinating antibodies that recognized the antigens absent from their own red cells. The modern explanation of this experiment is that cross-reacting carbohydrate structures on environmental agents stimulate thymus-independent production of IgM anti-A and/or anti-B in individuals not tolerant of these antigens. The IgM antibodies then directly agglutinate the corresponding antigen-positive red cells. Although the ABH antigens (the H antigen is the relevant carbohydrate structure present on group O red cells) are typically described as “blood group antigens” because of their presence on red cells, they are also found on other tissues, and are more appropriately termed “histo-blood group antigens.”6 In blood they are both membrane bound (eg, on platelets7) and soluble (eg, as blood-group-active glycosphingolipids inserted in plasma lipoprotein particles). They also are membrane antigens on such diverse cells as vascular endothelial cells and intestinal, cervical, urothelial, and mammary epithelial cells. Soluble forms are found in various secretions and excretions, such as saliva, milk, urine, meconium, and feces. Their expression in some tissues is developmentally regulated.8 Despite their wide distribution, genetic inheritance, developmental regulation, and importance in transfusion and transplantation, their normal physiologic function, if any, remains a mystery.

ABH Antigens: Biochemistry To appreciate the structure and antigenicity of ABH antigens, and their relationship to other blood group systems, it is necessary to understand the underlying biochemistry. Early studies indicated that anti-A, anti-B, and anti-H specifically recognize epitopes composed of the terminal trisaccharides or disaccharides illustrated in Figure 6-1. From these results it was possible to conclude that the A, B, and H antigens are not directly encoded by the corresponding genes; rather, the genes encode glycosyltransferases, commonly called the A, B, and H transferases, or equivalently, the A, B, and H enzymes. The H enzyme is a fucosyltransferase that specifically adds fucose in an α1-2 linkage to a terminal galactose. The A and B enzymes then add N-acetylgalactosamine or galactose, respectively, in an α1-3 linkage to the same terminal galactose. In addition, the substrate for the A and B enzymes is the H antigen sequence; these enzymes do not transfer the relevant sugar to galactose in the absence of (α1-2)-linked fucose. Similarly, the H enzyme does not function if this galactose is

90

A

GalNAc(α1-3) \ Gal-R / Fuc(α1-2)

B

Gal(α1-3) \ Gal-R / Fuc(α1-2)

Figure 6-1. Biochemical structures of the immunodominant oligosaccharides of the A, B, and H antigens. Gal ⫽ D-galactose; GalNAc ⫽ D-N-acetylgalactosamine; Fuc ⫽ L-fucose.

already substituted with a different sugar. Thus, the biosynthetic pathway is as follows: 1 2 Gal-R → H → A or B where reaction 1 is catalyzed by the H enzyme and reaction 2 by the A or B enzyme. The finding that the A and B genes encode glycosyltransferases explains some classical results from the analysis of family pedigrees. In particular, the A and B genes are inherited in a strict mendelian fashion and are dominant compared to O, but A and B are codominant with each other. That is, an individual with the genotype AO (or BO) is phenotypically A (or B), an individual of genotype AB is phenotypically AB, and an individual of genotype OO is phenotypically O. Because the A and B enzymes both use the H antigen as substrate, even the presence of only approximately 50% of these enzyme proteins in an AO (or BO) heterozygote is sufficient to convert the red cells to the corresponding A (or B) phenotype. Similarly, if both the A and B enzymes are present, they each convert approximately 50% of the available H antigen substrate, yielding red cells expressing both the A and B antigens. Most of the molecular biology of the ABH system has been elucidated over the last 15 years. The allelic A and B genes are located on chromosome 99,10 (see Table 6-2). They encode membranebound glycosyltransferases containing 354 amino acids (Fig 6-2) and are members of the Glycosyltransferase 6 family cataloged in the CAZY database (http://www.cazy.org). Homologues of the A and B genes can be identified throughout evolution, beginning with amphibians.21 In addition, the A and B enzymes exhibit a great degree of homology and differ at only four amino acid residues, at positions 176, 235, 266, and 268.15,22 The two adjacent residues near the C-terminus of the protein are critically important in determining their substrate specificity: amino acids 266

Chapter 6: Carbohydrate Blood Groups

and 26823,24 (Fig 6-2, Table 6-3). A valuable Web site, maintained by the National Center for Biotechnology Information, contains a current catalog of the sequences of the known human blood group antigens, including the glycosyltransferases encoding the ABH system (Blood Group Antigen Gene Mutation Database; http://www.ncbi.nlm.nih.gov/projects/mhc/xslcgi.fcgi?cmd= bgmut/home; Blumenfeld and Patraik25). The current chapter uses the nomenclature defined in that database. The explosion of readily available sequence data led to the identification of many variant alleles in the ABH system. For example, to date, more than 100 alleles of the A and B genes have been identified. Several illustrative variants are described below

and in Tables 6-3 through 6-5. For example, in addition to identifying the original (ie, wild type) A and B genes (herein denoted A101 and B101; see Table 6-3), molecular methods also identified several fully functional variants of each (ie, A102, A103, A104, B102, and B103) (Tables 6-4 and 6-5). These variants contain silent nucleotide polymorphisms that do not affect the function of these enzymes.15,26-30 A common allele corresponding to blood group O, O01, is highly homologous to the A and B alleles, but contains a single nucleotide deletion near the N-terminus of the protein. This leads to a shift in reading frame and a prematurely terminated translation product that lacks enzymatic activity (Table 6-3).15 More than 50 other O alleles have been identified that do not encode functional glycosyltransferases,27,28,31-38 including some with missense mutations that abolish enzymatic activity.31,32,36

Table 6-2. Chromosomal Assignment of Cloned Genes in the ABH, Se, Le, I, and P Blood Group Systems Gene

Gene Product

Location

Ref.

H (FUT1) Se (FUT2) Sec1

(α1-2) fucosyltransferase (Fuc-TI) (α1-2) fucosyltransferase (Fuc-TII) Pseudogene (nonfunctional; homologue of FUT1 and FUT2) (α1-3/4) fucosyltransferase (Fuc-TIII) (α1-3) N-acetylgalactosaminyl transferase (α1-3) galactosyltransferase (ß1-6) N-acetylglucosaminyl transferase (α1-4) galactosyltransferase (α1-4) galactosyltransferase (β1-3) N-acetylgalactosaminyl transferase

19q13.3 19q13.3 19q13.3

11 11 12,13

19p13.3 9q34.1-q34.2

11,14 9, 15

9q34.1-q34.2 9q21

9, 15 16

22q13.2 22q12.3-q13.1 3q25

17 18,19 20

Le (FUT3) A B I k

P P1 P

N-glycans

NH2 -

- COOH

Motif I Cytoplasmic Domain

Motif II

Transmembrane Domain

Figure 6-2. Structure of a generic blood group-active glycosyltransferase. The enzymes involved in the synthesis of blood-group-active carbohydrate antigens all share a similar overall structure. That is, they are Type II membrane glycoproteins that contain a single N-terminal cytoplasmic domain, a single transmembrane domain, a single large catalytic domain, a variable number of N-glycans, and a variable number of structurally important motifs. In particular, they all share a “DXD” motif involved in binding to a catalytically critical divalent cation (eg, a Mn⫹2 atom).

The A and B genes are allelic. The H, Se, and Sec1 genes are encoded at distinct, but closely linked, loci and are not alleles of each other. Although the H, Se, Sec1, Le, A, B, P, Pk, and I cDNA sequences and some genomic sequences have been determined, neither the P1 gene nor its cDNA have yet been cloned.

Table 6-3. Nucleotide and Amino Acid Sequences of the cDNA Derived from the A, B, and O Genes Nucleotide Residues 188 Alleles A101 B101 O01 O02 Mutation

189

220

261

297

526

646

G

c

C

G

a g

C G

T

A

t

T

63R→F

silent

74P→S

657

681

703

c t

g

G A

771

796

803

829

930

c

C A

G C

G

g a

∆G g frameshift silent

A 176R→G 216F→I

a silent

silent

t 235G→S

silent

A 266L→M

268G→A

277V→M

silent

The A1 and B transferases are 354 amino acid, Type II, membrane glycoproteins. All sequences in this figure are compared with the A1 sequence encoded by the A101 allele; blank boxes represent nucleotides identical to the A101 allele. An uppercase nucleotide indicates that there is an amino acid change induced by the mutation, whereas a lowercase letter indicates that the nucleotide polymorphism is silent at the amino acid level. The frameshift mutation induced by the deletion at nucleotide 261 in O01 allows for the translation of a significant length of amino acid sequence that differs from the A101 sequence before an early termination signal at a new stop codon.

91

Section I: Part I

92

Table 6-4. Weak Subgroups of A: cDNA Sequences Nucleotide Residues

Alleles A101 A102 A103 A104 A201 A202 A204 A205 A206 A301 A302 AX01 AX02 AX03 Ael01 Ael02 B101 Mutation

297

467

526

564

646

657

681

703

771

796

803

829

871

930

1009

1054

1059

a

C T T

C

c

T

c

g

G

c

C

G

G

G

g

A

C

C

t

g ∆C

T T g

G

t

A

t

A

T

G ∆C A ∆C

A g

A A A

a a

A

a

t t

A A ins G

T g silent 156P→L

G 176R→G

silent

216F→I

t silent

silent

A 235G→S

silent

A 266L→M

C 268G→A

277V→M

291D→N

a silent

337R→G

352R→W

frameshift

All sequences in this table are compared with the A101 allele. The B101 allele is included for comparison. The frameshift mutation induced by the deletion at nucleotide 1059 in alleles A201, A206, and A302 allows for the translation of several additional amino acids beyond the original stop codon, until the translation process encounters a new stop codon. The insertion at nucleotide 803 in the Ael01 allele induces a frameshift that allows for the translation of a significant length of amino acid sequence that differs from the A101 sequence before a termination signal at a new stop codon.

Chapter 6: Carbohydrate Blood Groups

Table 6-5. Weak Subgroups of B and the B(A) and cis-AB Phenotypes: cDNA Sequences Nucleotide Residues

Alleles A101 B101 B102 B103 B301 Bx01 Bel01 Bel02 ABO1cs B(A)01 B(A)02 Mutation

297

467

526

641

657

669

700

703

796

803

871

930

1054

a g g g g g

C

C G G G G G G G

T

c t t

G

C

G A A A A A A A

C A A A A A A A

G

g a

C

A 235G→S

A A 266L→M

G C C C C C C C C C C 268G→A

G

t t t t

T

T g g silent

G G 156P→L 176R→G

214M→R

t silent

223E→D

G 234P→A

A

291D→N

a a a a a a a silent

T

352R→W

All sequences in this table are compared with the A101 allele. The B101 allele is included for comparison. The critical amino acids that distinguish the A and B enzymes are at positions 176, 235, 266, and 268.

These findings using molecular biology not only confirm and extend the results obtained from carbohydrate biochemistry and enzymology, but they also suggest new ways of blood typing.38-41

Antigenic Variants in the ABH System Some relatively common variations in the ABH system relate to the “strength” of the A antigen on group A red cells. Multiple subgroups of A that have weak expression of the A antigen are known. The red cells of most group A individuals (eg, 80% of Whites) type as A1 due to their inheritance of one of the A1 alleles. Most of the remaining group A individuals have weaker expression of this antigen and are denoted A2. Other less common subgroups of A (eg, A3, Ax, Am, Ael) have progressively weaker A antigen expression. Many individuals with weak A expression produce an antibody, anti-A1, which does not agglutinate their own red cells but does agglutinate A1 red cells. This phenomenon is explained by quantitative and qualitative differences in antigen expression. For example, the number of A antigen sites per red cell varies from approximately 800,000 sites for A1 cells to 250,000 sites for A2 cells to 700 sites for Am cells.42 The finding that A2 individuals can produce A1-specific antibodies also implies the existence of qualitative differences. Indeed, biochemical investigations demonstrate that A antigens on A1 red cells differ from those on cells of the various subgroups of A.43,44 Many different classes of ABH-active glycosphingolipids have been isolated from human red cells (for example, see Clausen et al43) and novel structures continue to be discovered. Examples of relevant structures, provided in Fig 6-3, illustrate that complex oligosaccharides (ie, on Type 3 chains) can contain the A epitope not only as a terminal trisaccharide, but also as an “internal” structure. Thus, one hypothesis is that individuals whose red cells type as A1 can express all of these classes of structures, whereas those whose red cells type as A2 lack the

structures built on Type 3 and Type 4 chains; this implies that A1 antibodies specifically recognize these structures. The presence of Type 3 chains on A1 human red cells has another practical implication related to the goal of producing “universal donor” red cells by enzymatically removing the immunodominant GalNAc residues from A-active oligosaccharides.45 Thus, although many α-N-acetylgalactosaminidases can remove terminal GalNAc residues, all α-N-acetylgalactosaminidases identified to date are unable to remove the internal GalNAc residues found on Type 3 chains.46 Therefore, although it is now possible to perform the enzymatic A2 → O conversion, the goal of performing the A1 → O conversion to prepare large numbers of universal donor units currently remains elusive.46 More recently, the molecular biological approach has identified mutations in the cDNA and genomic DNA sequences of the A alleles of individuals with red cells expressing weak A activity (Table 6-4).28,29,47-53 For example, the A transferase in A2 individuals encoded by the A201 allele has a 156 P→ L missense mutation and a frame shift mutation near the 3⬘-end of the coding sequence.49 In addition, some A3 individuals have an allele, A301, containing a 291 D→ N mutation in the coding sequence.48 These types of findings are summarized in Table 6-4. In some cases, transfection studies prove that these mutations result in weakened or variant enzyme activity of the encoded glycosyltransferase (for example, see Yamamoto et al49). When the coding sequence is evaluated alone, there is not always a one-to-one correspondence between genotype and phenotype. For example, even within a given family, the red cells of individuals inheriting the A206 allele can have either the A2 or A3 phenotype.53 In another family, the red cells of individuals inheriting the A201 allele typed as either A2 or Ax.50 A complete understanding of these results is not yet available, but it may be important to investigate the regulation of expression of

93

Section I: Part I

Type 1 A

GalNAc(α1-3) \ Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc(β1-1′)cer / Fuc(α1-2)

Type 2 A

GalNAc(α1-3) \ Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc(β1-1′)cer / Fuc(α1-2)

GalNAc(α1-3) \ Gal(β1-4)GalNAc(α1-3) Type 3 A / \ Fuc(α1-2) Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc(β1-1′)cer / Fuc(α1-2) GalNAc(α1-3) \ Gal(β1-3)GalNAc(β1-3)Gal(α1-4)Glc(β1-1′ )cer /

Type 4 A Fuc(α1-2)

these genes in greater detail, particularly by focusing on the promoter and enhancer regulatory regions.54-57 Finally, the identification of individuals with weak subgroups of A allowed the first successful attempts to transplant “incompatible” A2 renal allografts into group O recipients.58 By analogy with the subgroups of A, “weak” subgroups of B also exist (eg, B3, Bx, Bel). For example, the gene encoding the B301 allele responsible for the B3 subgroup has a 352 R → W mutation in the coding sequence that is not found in the wild type B101 allele (Table 6-5).48 The B(A) and cis AB phenotypes are interesting and unusual variations in the ABH system.59 In each case, one chromosome in the affected individual apparently encodes for a gene (or genes) that leads to the synthesis of both the A and B antigens. In the B(A) phenotype, the red cells predominantly have B antigens with a small amount of A antigens.47 In the cis AB phenotype, the red cells have equivalent amounts of A and B antigens.60 Molecular studies demonstrated that the coding sequence of one B(A) transferase, B(A)01, is virtually identical to the wild type B101 allele, except that it lacks the 235 G → S mutation.47 Thus, at three of the relevant amino acid positions the B(A)01 allele is identical to B101 and at one position it is identical to A101; this allows the resulting glycosyltransferase to create small amounts of A antigen in addition to large amounts of B antigen.47 In contrast, the sequence of the B(A)02 allele is identical to B101 except for the addition of a 234 P → A mutation (Table 6-5); this mutation is immediately adjacent to one of the four key amino acid residues that confer A or B transferase activity,61 thereby presumably modifying their function. In one case of cis AB, the AB01cs allele has mutations resulting in two amino acid substitutions (Table 6-5).62 The 156 P→ L mutation is identical to that found

94

Figure 6-3. Biochemical structures of the A antigen-active terminal trisaccharide carried on various types of backbone structures (ie, Types 1-4) attached to a glycosphingolipid. Glc ⫽ D-glucose; GlcNAc ⫽ D-N-acetylglucosamine; cer ⫽ ceramide; Gal ⫽ D-galactose; GalNAc ⫽ D-Nacetylgalactosamine; Fuc ⫽ fucose.

in the weak A allele, A201; the 268 G → A mutation is a change that is important for conferring B transferase activity to the resulting enzyme.23,24 Thus, these changes result in an enzyme that functions as an AB transferase chimera. No evidence for an alternative model, in which crossing-over results in one chromosome that contains both an intact A gene and a separate, intact B gene, has been found. A major recent advance in our understanding of the structure and function of the wild type and variant A and B transferases resulted from the determination of the x-ray crystal structure of the blood group B transferase.63 This initial structure provided great insight to the mechanism of binding of the nucleotide sugar donor (ie, UDP-Gal) and the oligosaccharide acceptor substrate (ie, the blood group H-active disaccharide). In addition, the availability of this structure allowed for models to be constructed of the A transferase.64 More recently, mechanistic insight has been gained regarding why the 214 M → R mutation encoded by the Bel01 gene (Table 6-5) leads to markedly weakened B transferase activity.65 In particular, the amino acid at position 214 is immediately adjacent to the “DXD” motif at D211-V212-D213 that is critically involved in coordinating the Mn⫹2 atom required for enzyme activity. Therefore, this single mutation prevents binding of Mn⫹2 and, thereby, markedly diminishes transferase function, an instructive and remarkably beautiful result. Similar types of studies will no doubt yield increasingly important and useful information.66

Secretion of ABH Antigens The ABH antigens are found not only on red cells, but also in secretions, particularly saliva and plasma. The ability to secrete ABH antigens is genetically inherited: approximately 80% of Whites are

Chapter 6: Carbohydrate Blood Groups

Table 6-6. Biochemical Structures of ABH Antigens Blood Group

Secretions (Type 1 Chain)

H

A

Red Cells (Type 2 Chain)

Gal(ß1-3)GlcNAc-R

Gal(ß1-4)GlcNAc-R

Fuc(α1-2)

Fuc(α1-2)

GalNAc(α1-3)

GalNAc(α1-3) Gal(ß1-3)GlcNAc-R

B

Gal(ß1-4)GlcNAc-R

Fuc(α1-2)

Fuc(α1-2)

Gal(α1-3)

Gal(α1-3) Gal(ß1-3)GlcNAc-R

Fuc(α1-2)

secretors and 20% are non-secretors. This trait is inherited as a single locus gene in simple mendelian fashion. The secretor gene (Se) is dominant; non-secretor (se) is recessive. The terminal carbohydrate sequences of the ABH antigens in saliva and plasma are identical to those on red cells. However, the backbone or framework carbohydrate structures are different. ABH antigens on glycosphingolipids and glycoproteins synthesized by red cell precursors are primarily coupled to framework Type 2 chains (ie, Gal(ß1-4)GlcNAc-R); the same antigens on plasma glycosphingolipids and salivary mucins are coupled to framework Type 1 chains (ie, Gal(ß1-3)GlcNAc-R) (Table 6-6). Because ABH blood-group-active glycosphingolipids on plasma lipoproteins are also passively transferred onto red cells, red cells of secretors have ABH antigens not only on Type 2 precursor chains, but also on small numbers of Type 1 chains. In contrast, red cells of non-secretors have ABH antigens only on Type 2 chains. Initially, it was thought that the H gene was a structural gene coding for the H enzyme and the secretor locus encoded a regulatory gene that permitted expression of the H gene in the relevant tissues. This hypothesis suggested that a single H enzyme transferred fucose in an α1-2 linkage to the terminal galactose residue on either Type 1 or Type 2 chains. In this model, the H enzyme is always expressed in red cell precursors, but expression in “secretory” tissues (eg, salivary epithelium) is controlled by the Se gene. However, this model did not explain all the available data, and multiple biochemical, immunologic, and genetic studies supported an alternative model.67,68 The latter postulated two different H transferases: one adding fucose to Type 1 chains (an H Type 1 enzyme) and one acting on Type 2 chains (an H Type 2 enzyme). In this model, the H Type 1 enzyme is the structural protein encoded by the secretor gene (Se) and is expressed only in secretory tissues.

Se and H Genes The cloning of genes and cDNAs encoding multiple mammalian fucosyltransferases (for review, see Oriol et al69) show that Se is

Gal(ß1-4)GlcNAc-R Fuc(α1-2)

the FUT2 gene and that the product of the Se gene, the H Type 1 enzyme, is the Se (or Fuc-TII) enzyme.12,70 At least one copy of the Se gene is found in approximately 80% of individuals and leads to ABH antigen expression in secretions. Indeed, FUT2 mRNA expression is found in both normal epithelial cells12 and cell lines derived from carcinomas,71 but not in cell lines derived from hematopoietic neoplasia.71 By contrast, the traditional H locus encodes the H Type 2 enzyme; the latter is equivalent to the H (or Fuc-TI) fucosyltransferase.72 This gene (H or FUT1) is active in virtually all individuals (for exceptions, see below) and leads to ABH antigen expression on red cells and other tissues. The FUT1 (or H) and FUT2 (or Se) genes are closely linked on chromosome 19 (Table 6-2).11 In addition, a third gene in this region, Sec1, has been cloned.12 Sec1 is a pseudogene of FUT212 and, although actively transcribed in human cell lines,71 but perhaps not in normal human tissues,12 it has no intrinsic enzymatic activity. These genes are arranged on chromosome 19 in the following sequence: Centromere → Sec1 → FUT2 → FUT1 → Telomere. In addition, the high homology of these three genes, and the proximity of many Alu sequences, make this region a hot spot for mutation and genetic recombination71 (see below).

The Se (Fuc-TII) Glycosyltransferase The Se enzyme is a Type II membrane glycoprotein containing 332 amino acids, a short cytosolic domain, and a large lumenal domain (see Figure 6-2).70 There are three potential Nglycosylation sites in the lumenal domain at N177, N271, and N297. Its overall structure is similar to that of other mammalian glycosyltransferases, including the A and B enzymes. By analyzing the sequences of many eukaryotic and prokaryotic (α12)fucosyltransferases, three highly conserved sequence motifs were identified.69 Motifs I, II, and III encompass amino acid residues 184-204, 226-239, and 278-288, respectively; Motif I may be responsible for binding the nucleotide sugar substrate, GDPfucose, required for the catalytic action of this enzyme.

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Section I: Part I

Table 6-7. Functional Alleles of the Secretor (Se or FUT2) Gene: cDNA Sequences Nucleotide Residues

Alleles Se Se1 Se2 Se3 Se4 Se5 Se6 Se7 Se8 Se9 Se10 Sew Mutation

40

210

278

357

375

379

385

480

481

855

A

a

C

c t

a

C

A

c

G

a

G A A

G g

a T 14I→V

silent

93A→V

t t t t t t silent

t t

T

c T silent

127R→C

129I→F

silent

161D→N

silent

The H type 1 transferase (FUT2) is a 365 amino acid, Type II membrane glycoprotein. All sequences in this table are compared with a “parental” Se sequence. There is no generally agreed upon nomenclature for naming variant alleles of the Se gene; the system chosen here is based on the literature, but is otherwise arbitrary.

Following cloning of Se,12,70 multiple functional and nonfunctional variants were described. For example, many individuals have a normally functioning variant, Se1, which contains a silent mutation: 357 c → t (Table 6-7).73-77 In other individuals, normally functioning genes are found that contain only missense mutations (eg, Se2, Se3, Se4) or combinations of silent and missense mutations (eg, Se6 and Se7).76,77 In addition, the Sew mutant encodes a functional, but significantly less active, enzyme.74,78,79 This allele contains a missense mutation that leads to partial expression of the secretor phenotype; individuals with this allele can have red cells with the unusual Le(a⫹b⫹) Lewis blood group phenotype (see below). None of the mutations found in functional Se variants occur in any of the three highly conserved (α1-2)fucosyltransferase motifs (Table 6-7). In contrast, at least 10 nonenzymatically active variants of the Se gene have been described (Table 6-8). Most of these are caused by nonsense (ie, se1, se2, se3, se4, se5, se8) or small deletion (ie, se9) mutations that yield premature stop codons, resulting in the expression of truncated, nonfunctional enzyme proteins.70,73,75-77,80-82 However, the se7 allele contains one missense mutation at amino acid 101 that presumably encodes a full-length nonfunctional enzyme; nonetheless the effect of this mutation on enzyme function was not directly tested with recombinant protein.83 Similarly, the se10 allele contains a three base-pair deletion resulting in the deletion of a single amino acid, V230; although the encoded protein is presumably nonfunctional, this was not verified by transfection studies.84,85 The mutation in the se10 allele occurs in the highly conserved (α1-2)fucosyltransferase Motif II (Fig 6-5 and 6-7). One nonfunctional allele, se6, is the result of a fusion gene between FUT2 (ie, Se) and the adjacent upstream Sec1 pseudogene.75,80 Although the recombinant se6 protein has low, but measurable, enzymatic

96

activity using model substrates in vitro, it did not produce H antigen when expressed in transfected cells. Finally, the complete deletion of the entire FUT2 gene is seen in individuals with the classical Bombay phenotype (see below); this allele is denoted as sedel.86,87 This large deletion is mediated by the presence of Alu sequences,88 many of which are present in this chromosomal region.71 Given the abundance of sequencing data, it is possible to speculate on the temporal sequence during which the various mutations in the Se gene developed. One possible sequence is provided below: se10 ↑ Se → Se1 → Se6 → Se7 ↓ Se

↓ w

se8

This model supports the “out of Africa” hypothesis for the evolution of human populations. The Se and Se1 genes are commonly found in Black, White, and Asian populations. In contrast, se10 and Sew have been found only in Asian populations.74,78,79,84 In addition, Se6 has been found in Black and White, but not Asian, populations.76 Finally, se8 and Se7 have been found only in Black and White populations, respectively.76

The H (Fuc-TI) Glycosyltransferase The H enzyme contains 365 amino acids and is highly homologous to the Se enzyme (Fig 6-2).70 In particular, it also contains the three highly conserved (α1-2)fucosyltransferase sequence motifs: Motifs I, II, and III comprise amino acids 214-224, 256-269, and 308-318,

Chapter 6: Carbohydrate Blood Groups

Table 6-8. Nonfunctional Alleles of the Secretor (Se or FUT2) Gene: cDNA Sequences Nucleotide Residues 171

216

302

Alleles Se a c C g t se1 se2 se3 se4 se5 se6 se7 T se8 se9 se10 Mutation silent silent 101P→L

357

428

480

571

628

658

685

739

778

849

960

c

G A

c

C

C

C

G

G S

C

G

a g

T T

t

A T

t

∆C

t T

t ∆GTG silent 143W→stop silent 191R→stop 210R→stop 220R→stop 230V→∆V 247G→S 260P→stop 283W→stop silent

All sequences in this table are compared with a “parental” Se sequence. There is no generally agreed upon nomenclature for naming variant alleles of the Se gene; the system chosen here is based on the literature, but is otherwise arbitrary. In the se1 allele, the amino acid change at position 247 is downstream of the stop codon at position 143. The se6 allele is a fusion gene that encodes an unusual chimeric protein where the N-terminus is encoded by the Sec1 pseudogene, which is homolgous to Se, and the C-terminus is encoded by the Se gene.75 In the se8 allele, the frameshift mutation induced by the deletion at nucleotide 778 allows for the translation of a length of amino acid sequence that differs from the Se sequence before an early termination signal at a new stop codon. In the se10 allele, the three nucleotide deletion (ie, GTG) at position 685 does not lead to a shift in the reading frame, but just leads to a deletion of a single amino acid (V230). The sedel allele, which is not included in this table, represents a complete deletion of the Se gene and is found in individuals with the Bombay phenotype.

respectively.69 Based on the premise that the H gene codes for the H Type 2 glycosyltransferase (FUT1), then the allelic h gene codes for a nonfunctional enzyme. Since the cloning and sequencing of the H gene,72 various groups have investigated the molecular nature of the defective h alleles.86,89-95 More than 25 different h alleles have been identified, variously containing missense, nonsense, and frameshift mutations (Table 6-9). It is noteworthy that most families examined in these studies each had a different mutation leading to inactivation of Fuc-TI activity. In addition, several missense mutations occurred in the (α1-2)fucosyltransferase motifs; for example, the amino acid substitution 220 R → C occurs in Motif I, 259 V → E and 262 S → K occur in Motif II, and 315 A → V occurs in Motif III. The mutation at position 220 is particularly interesting in that the arginine at this position, in the putative nucleotide sugar binding domain, is absolutely conserved in (α1-2) and (α1-6)fucosyltransferases throughout evolution from bacteria to mammals.97 Although the 327 N → T mutation does not occur in an (α1-2)fucosyltransferase motif, it does destroy a potential Nglycosylation site, suggesting that this posttranslational modification is important in the activity of this enzyme. In addition to the variants described above, one individual had a completely normal coding sequence, but no enzyme activity,92 suggesting a defect in transcriptional regulation rather than in protein structure. This latter result shows that caution is required when molecular approaches for blood typing based on genotype are used, rather than classical methods, which directly determine blood group phenotype.

H-Deficient Phenotypes and Genotypes Based on the information described above, it should be clear that there are multiple molecular mechanisms by which an individual could express low amounts of H antigens on their red cells and/ or in their secretions. The most common example of an H-deficient individual is a “classical non-secretor,” that is, an individual with the general genotype Hh sese or HH sese (see Table 6-10). This individual has normal amounts of H Type 2 antigens on red cells, but does not express H Type 1 antigens in secretions. In this case, the individual would carry two se alleles, which could theoretically be combinations of any of the following: se1, se2, se3, se4, se5, se6, se7, se8, se9, se10, or sedel. “Classical weak secretors” are those individuals who are either homozygous or heterozygous for wild-type H and are either SewSew or Sewse at the secretor locus. Sew is a weak secretor allele (see above) that can synthesize small amounts of H Type 1 antigens and allows expression of the unusual Le(a⫹b⫹) phenotype (see below). The most striking example of an H-deficient phenotype is the “classical Bombay phenotype,” often denoted as Oh.98 These individuals were originally identified in Bombay, India, their red cells type as group O, and they are non-secretors of ABH antigens. In addition, they do not express any H antigens on their red cells and their sera contain high-titered, IgM, hemolytic H antibodies that induce complement-mediated lysis of red cells of individuals

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Section I: Part I

Table 6-9. h Alleles: Weak or Inactive Alleles of FUT1 Nucleotide Change

Amino Acid Change Enzymatic Activity

Ref.

Missense Mutations 349 C → T 442 G → T 460 T → C 460 T → C 1042 G → A 461 A → G 491 T → A 513 G → C 658 C → T 721 T → C 725 T → G 776 T → A 785 G → A 786 C → A 944 C → T 980 A → C 1042 G → A 1047 G → C

117 H → Y 148 D → Y 154 Y → H 154 Y → H 348 E → K 154 Y → C 164 L → H 171 W → C 220 R → C 241 Y → H 242 L → R 259 V → E 262 S → K

Weak Weak Weak Weak or absent

86 90, 96 90, 93, 96 90, 93

None None None None Weak None None None

92 89 92 96 90 86-88 92 94

315 A → V 327 N → T 348 E → K 349 W → C

None None Weak None

92 96 90, 93 92

180 Q → stop silent 182 R → frameshift 232 W → stop 276 S → stop 294 F → frameshift 316 Y → stop 323 V → frameshift 330 L → frameshift

None

95

None None None None None None Weak

96 90 89 96 89 92 90

Nonsense Mutations 538 C → T 1089 t → g 547 ∆AG 695 G → A 826 C → T 880 ∆TT 948 C → G 969 ∆CT 990 ∆G

active A and/or B enzymes, the A (or B) antigen is not detected because the appropriate substrate for these enzymes (ie, H Type 1 or H Type 2 chains) is not synthesized (Table 6-6). Nonetheless, the functional A (or B) genes can be transmitted to the next generation, yielding the apparently paradoxical pedigree O ⫻ O → A (or B). On rare occasions, individuals are identified with ABHdeficient red cells but with ABH antigens in their secretions; this is often referred to as the “para-Bombay phenotype.”89-91,93,99 However, the literature can be confusing on this point and this broad phenotypic description can, at least theoretically, encompass individuals with functional Se alleles and nonfunctional H alleles, or with weakly functioning H alleles and nonfunctional Se alleles (see Table 6-10). Thus, individuals with the “RBC Hdeficient, secretor,” “RBC H-deficient, weak secretor,” and “RBC weak H, non-secretor” phenotypes could all be described as having the para-Bombay phenotype. Therefore, confusion can be avoided by using more specific terms that describe the types of alleles an individual has at the Se and H loci (Table 6-10).

Medical Implications of the ABH and Secretor Systems In transfusion medicine, the major implications of these systems relate to the occurrence of serious, acute hemolytic transfusion reactions in patients receiving incompatible red cells (eg, red cells from a group A donor transfused to a group O recipient, or red cells from a regular donor transfused to a Bombay O recipient). The incidence, pathophysiology, prevention, and treatment of such reactions are discussed elsewhere in this book. However, these blood group systems are also relevant in several other situations, examples of which are described below.

This table summarizes results published in peer-reviewed papers; several additional mutations have been reported in abstract form. The activity of each h allele was determined by transfection and expression of the recombinant protein and/or inferred on the basis of serologic studies of the red cells of the corresponding propositus. The h allele containing the 327 (N → T) amino acid mutation also contains an amino acid mutation at position 12 (A → V). However, because the latter is a conservative change in the putative transmembrane domain, it probably is not involved in the function of this enzyme. The h allele containing the 180 (Q → stop) mutation in the amino acid sequence also contains a silent nucleotide change (1089 t → g), which does not change the amino acid code, and is downstream of the stop codon. One h allele has been identified in which there are no mutations in the coding sequence; the lack of enzymatic activity is presumably due to a mutation(s) in a regulatory sequence.92

Biological Relevance of the ABH Antigens in Coagulation The normal physiologic functions of the ABO histo-blood group system are still unknown. However, the level of expression of the A/B antigens on the N-glycans of the von Willebrand factor (vWF) procoagulant glycoprotein affects circulating plasma levels of both vWF and Factor VIII, the latter of which is protected from degradation by its association with vWF.100 Thus, group AB individuals have the highest levels of vWF followed by those of group B, A, and then O, who have the lowest. This finding correlates well with the increased incidence of thrombotic diseases in non-group-O individuals as opposed to the increased bleeding tendency in group O individuals. Nonetheless, the precise mechanisms underlying how the presence of terminal ABH structures on vWF N-glycans may affect overall thrombotic risk are still unclear,101 although their contribution to the resistance of vWF to degradation by ADAMTS13 may be critically important.102

of any ABO group, except those from another individual with a similar type of global deficiency of H antigens. Individuals with the classical Bombay phenotype are homozygous for an h allele with the amino acid substitution 242 L → R; they are also homozygous for a deletion of the entire FUT2 gene, that is, the sedel allele of Se.8688 In this way, they are incapable of synthesizing either H Type 1 or H Type 2 structures. Although some of these individuals do express

The Role of the ABH Antigens in Malaria: A New Hypothesis A fascinating new hypothesis may provide a comprehensive explanation of the distribution of the ABO blood groups in various human populations.103 This unifying hypothesis may also explain variations in the Secretor system and the Lewis blood group system (see below). In brief, this hypothesis addresses the pathophysiology of adherence of red cells infected with

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Chapter 6: Carbohydrate Blood Groups

Table 6-10. Phenotypes and Genotypes Involving FUT1 and FUT2 Phenotype

RBC ABH Antigen Level

Salivary ABH Antigen Level

H (FUT1) Gene

Se (FUT2) Gene

Classical secretor Classical non-secretor Classical weak secretor Classical Reunion phenotype RBC weak H, non-secretor RBC weak H, weak secretor RBC weak H, secretor Classical Bombay phenotype Other “Bombay” phenotype H-deficient, weak secretor H-deficient, secretor

Normal Normal Normal Weak Weak Weak Weak Absent Absent Absent Absent

Normal Absent Weak Absent Absent Weak Normal Absent Absent Weak Normal

HH or Hh HH or Hh HH or Hh hh: 117 H → Y hh: eg, 241 Y → H hh: eg, 241 Y → H hh: eg, 241 Y → H hh: 242 L → R hh hh hh

SeSe or Sese sese SewSew or Sewse se1se1 sese SewSew or Sewse SeSe or Sese sedelsedel sese SewSew or Sewse SeSe or Sese

Where the type of Se, se, or h allele is not specified, various alleles described in Tables 6-7 through 6-9 could theoretically be used. Most, if not all, of the phenotypes in this table have been described in the literature using serologic and/or genetic methods. The “RBC H-deficient, weak secretor” and the “RBC H-deficient, secretor” phenotypes are often described as para-Bombay phenotypes; however, on occasion, the “RBC weak H, non-secretor” phenotype can also be described as a “para-Bombay” phenotype, which can lead to confusion.

Plasmodium falciparum to capillary endothelium, which is particularly relevant for the severe, and often fatal, complication of cerebral malaria. Thus, the surface of infected red cells expresses the malarial PfEMP-1 protein, which has lectin-like properties with specificity for the A (and B) oligosaccharide structures. In this way, infected red cells could adhere directly to endothelial cells bearing A (or B) antigens. Alternatively, infected red cells could indirectly adhere to endothelial cells by first binding to either platelets or vWF expressing A (or B) structures. If correct, this hypothesis would predict that individuals who did not express A or B antigens (ie, group O individuals) would be at least partially protected from the severe consequences of P. falciparum malaria. Similarly, the presence of the Secretor phenotype would be somewhat protective in group A or B individuals, because the A/B antigens on plasma lipoproteins would block endothelial adherence of infected red cells. This protection would be enhanced if these particular individuals also lacked Lewis antigens (see below); that is, if they had the Le(a⫺b⫺) phenotype. In summary, this hypothesis would explain the relative abundance of the group O and Le(a⫺b⫺) phenotypes in populations that have a long history of exposure to P. falciparum malaria. It will be of great interest to see whether future studies confirm this novel hypothesis.

ABO-Incompatible Solid Organ Transplantation The increasing number of patients needing organ transplantation and the shortage of available organs led to studies of the feasibility of ABO-incompatible (ABOI) transplantation. Because of naturally occurring IgM and IgG anti-A/B found in “unimmunized” naïve individuals, early attempts at ABOI transplantation resulted in hyperacute or acute rejection.104,105 However, protocols allowing for successful ABOI transplantation were subsequently developed. These typically involve removal of anti-donor,

anti-A/B by plasmapheresis before transplantation along with pharmacologic immunosuppressive agents, often combined with splenectomy.106,107 Using these approaches, the overall 1-year graft survival is approximately 50% to 60% following ABOI kidney, liver, or heart transplantation, compared with 70% to 80% for ABO-compatible transplantation.108 ABOI kidney transplants in adults have been performed in significant numbers and with quite good success, especially in Japan where religious beliefs preclude the use of cadaveric organs.109 In particular, the reduced antigen expression in blood group A2 donors results in protection from antibody-mediated injury; thus, group A2 kidneys can be transplanted into group B or O individuals without prior depletion of anti-A.110 ABOI liver transplants in adults tend to have a poor outcome because of major complications associated with high preoperative IgM and IgG anti-A/B titers.111 Graft survival of ABOI liver transplants is still substantially lower than for ABO-matched transplants; therefore, ABOI liver transplants are reserved for emergent situations when a compatible donor cannot be found.112 In addition, in ABO-compatible but nonidentical transplants (ie, “minor” mismatch), graft-derived anti-A/B can be produced by passenger lymphocytes, leading to often mild, but occasionally serious, hemolysis of the recipient’s red cells.113 ABOI heart transplants in adults are associated with high mortality.114 In contrast, following preoperative antibody reduction, ABOI heart transplants in newborns and infants have been relatively successful, especially considering the high mortality associated with extended stays on the transplant waiting list.115

ABOI Hematopoietic Stem Cell Transplantation Because of the distribution of ABO blood types in human populations, many allogeneic hematopoietic stem cell transplants are ABO incompatible.108 These may result from a major incompatibility

99

Section I: Part I

Table 6-11. The Lewis Blood Group System

Gal(β1-3) \ GlcNAc(β1-3)Gal(β1-4)Glc(β1-1⬘)ceramide /

Lea

Leb

Red Cell Antigens

Serum Antibodies

Fuc(α1-4)

Blood Group

Lea

Leb

Anti-Lea

Anti-Leb

Gal(β1-3) /

Le(a⫹b⫺) Le(a⫺b⫹) Le(a⫺b⫺)

⫹ ⫺ ⫺

⫺ ⫹ ⫺

— Very Rarely Occasionally

Very Rarely — Occasionally

Fuc(α1-2)

\ GlcNAc(β1-3)Gal(β1-4)Glc(β1-1⬘)ceramide /

Fuc(α1-4)

Figure 6-4. Biochemical structures of the Lewis-active glycosphingolipids. Glc ⫽ D-glucose; GlcNAc ⫽ D-N-acetylglucosamine; cer ⫽ ceramide; Gal ⫽ D-galactose; Fuc ⫽ fucose.

(eg, group A or B donor cells transplanted into a group O recipient),116 where the primary immediate risk from the procedure is the transfusion of ABO-incompatible red cells present in the hematopoietic stem cell product. This type of risk can be ameliorated by removing red cells in the donor product and/or removing anti-A/B in the recipient by plasmapheresis. In contrast, transplants involving a minor ABO mismatch (eg, group O donor cells transplanted into a group A or B recipient) may produce severe delayed hemolysis resulting from anti-A/B production by passenger lymphocytes in the graft;117 various methods can be employed to address this issue (for example, see Worel et al118).

The Lewis System The two Lewis blood group antigens Lea (Lewis a) and Leb (Lewis b) were discovered in the 1940s (for review, see Henry et al119). Virtually all individuals fall into one of three different Lewis types: Le(a⫹b⫺), Le(a⫺b⫹), and Le(a⫺b⫺) (Table 6-11). These molecules are not intrinsic red cell antigens; they are synthesized in another tissue (probably intestinal epithelium), circulate in plasma attached to lipoproteins, and then passively transfer onto red cells.120 Biochemically these are carbohydrate antigens on glycosphingolipids (Fig 6-4). They are structurally similar to the Type 1 ABH antigens found on plasma glycosphingolipids, which can also transfer onto red cells. The Lewis gene (Le) is on chromosome 1911,14 and is distantly linked to the H and Se loci (Table 6-2). The gene encodes an (α1-4)fucosyltransferase, denoted Fuc-TIII, and thus behaves in a dominant fashion. A human cDNA derived from the Le gene (equivalently, the FUT3 gene) was cloned and encodes a 361 amino acid Type II membrane-bound glycoprotein (Fig 6-2).121 In addition to adding fucose in an α1-4 linkage to the GlcNAc residue on a Type 1 chain, it can also add fucose in an α1-3 linkage to the GlcNAc on a Type 2 chain (Table 6-6). Although the latter reaction is relatively inefficient, it forms the Lewis x antigen, Lex, an important tumor-associated antigen. Thus, FucTIII is a very unusual glycosyltransferase in that it can transfer a monosaccharide to two very different substrates using different glycosidic linkages; therefore, it is technically an (α1-3/4)fucosyltransferase.121 The sequence near its NH2-terminus is important

100

for determining its acceptor specificity.122-126 Thus, mutating the sequence of Fuc-TIII at residues 111 and 112 to that which is found in the (α1-3)fucosyltransferase Fuc-TVI (ie, from WD in Fuc-TIII to RE in Fuc-TVI), significantly improves its ability to use Type 2 chains as substrates.122 By sequence analysis, Fuc-TIII is a member of a family of (α1-3)fucosyltransferases that is conserved from bacteria to mammals.69,97 As such, there are two highly conserved sequence motifs: Motif I comprising amino acids 152-171 and Motif II comprising amino acids 239272. Fuc-TIII also contains two potential N-glycosylation sites; the site at N154 is in sequence Motif I. Although FUT-TIII has not yet been crystallized, molecular modeling has provided some insight into its three-dimensional structure.127 The transfer of fucose to a Type 1 chain by Fuc-TIII results in formation of the Lea antigen; the addition of α1-4 linked fucose to the H Type 1 structure leads to formation of Leb. Thus, the latter is formed by the cooperation of two glycosyltransferases encoded by two genes, one from the Lewis system (Le on chromosome 19) and one from the ABH system (Se at a different locus on chromosome 19). The biosynthetic pathways connecting the ABH, Secretor, and Lewis systems (encoded by genes at three distinct loci on chromosomes 9 and 19) are shown in Fig 6-5. Cooperation of the Le, Se, and A (or B) genes leads to the formation of a minor antigen, ALeb (or BLeb), recognized by both anti-A (or anti-B) and anti-Leb. Because the Se enzyme (ie, Fuc-TII) converts virtually all Type 1 chains into H Type 1, whether or not Fuc-TIII is present, Lewis-positive secretors have virtually no Lea antigen and their red cells type as Le(a⫺b⫹). By contrast, Lewis-positive non-secretors have Le(a⫹b⫺) red cells. This is summarized in Table 6-11. Individuals whose red cells type as Le(a⫹b⫹) are very unusual among Whites, but are relatively common in Asian populations. In these individuals the Le gene is normal but the Se (Fuc-TII) enzyme is partially defective due to a variant allele (Sew) containing the 129 I → F mutation 74,77-79 (Table 6-7). Because the Sew enzyme has partial activity, some Type 1 chains are converted to H Type 1, which are then converted to Leb by the normal Lewis enzyme, Fuc-TIII (Fig 6-5). In addition, some Type 1 chains avoid α1-2 fucosylation by the partially defective Sew enzyme and are converted into Lea by the normal Lewis Fuc-TIII enzyme (Fig 6-5). Thus, the red cells of these individuals contain both the Lea and Leb antigens and type as Le(a⫹b⫹).

Chapter 6: Carbohydrate Blood Groups

Gal(β1-3) \ GlcNAc(β1-3)-R H type 1 gene (Se,Fuc-TII)

Type1 H

Gal(β1-3) / \ Fuc(α1-2) GlcNAc(β1-3)-R

Type 1

Le gene (Fuc-TIII) Gal(β1-3) \ GlcNAc(β1-3)-R / Fuc(α1-4)

Lea

Gal(β1-3) / \ Fuc(α1-2) GlcNAc(β1-3)-R / Fuc(α1-4)

Leb

A gene

Type1 A

Le gene (Fuc-TIII) GalNAc(α1-3) \ Gal(β1-3) / \ Fuc(α1-2) GlcNAc(β1-3)-R

Le gene (Fuc-TIII) GalNAc(α1-3) \ Gal(α1-3) / \ Fuc(α1-2) GlcNAc(β1-3)-R / Fuc(α1-4)

ALeb

Figure 6-5. Biosynthesis of blood group antigens with Type 1 chains. The genes encoding the relevant glycosyltransferases are denoted in italics and the reactions catalyzed are indicated by arrows. The names of the individual blood group antigens are shown in boxes next to the relevant structures. GlcNAc ⫽ D-N-acetylglucosamine; Gal ⫽ D-galactose; GalNAc ⫽ D-N-acetylgalactosamine; Fuc ⫽ fucose.

The presence of defective Le alleles is relatively common and approximately 5% of Whites and 25% of Blacks type as Le(a⫺b⫺). The increased prevalence of the Le(a⫺b⫺) phenotype in Blacks may have evolved from selective pressure due to infection with P. falciparum malaria (see above).103 To date, at least 10 different defective Le alleles (le1 through le10) have been identified (Table 6-12).128-135 Based both on family studies and expression of transfected chimeric cDNAs in vitro, the mutations at amino acids 68,134 170,129,130,132 223,136 270,136 and 356129-131 severely inhibit or abolish enzyme activity. One of these mutations occurs in (α1-3)fucosyltransferase Motif I and one in Motif II. The mutation at amino acid 20, which is in the transmembrane domain, affects Golgi localization of this enzyme and its activity with glycosphingolipid and glycoprotein substrates, thus yielding a paradoxical “nongenuine” Lewis-negative phenotype.129-132 That is, although red cells of individuals homozygous for le3 lack Lewis-active glycosphingolipids and type as Le(a⫺b⫺), their salivary mucins do contain Lewis antigens.131,137 Although hemolytic transfusion reactions and hemolytic disease of the fetus and newborn are rarely caused by antibodies to the Lewis blood group antigens, this blood group system may be important in renal transplantation,138 coronary heart

disease,139,140 gastrointestinal cancer,141 infection by Helicobacter pylori,142,143 and P. falciparum malaria (see above).103

Ii Blood Group System The Ii antigens are oligosaccharides that form the Type 2 chain precursors for the ABH antigens (Fig 6-6; see also Table 6-6). The best available evidence indicates that the difference between the I and i antigens relates to branching of the oligosaccharide chain; i antibodies recognize an unbranched oligosaccharide chain, and I antibodies recognize a similar chain that is also branched. Fetal and cord red cells contain mostly i antigen with small amounts of I; adult red cells demonstrate the opposite pattern. This suggests that the glycosyltransferase necessary for synthesis of the branched structure, a (β1-6)N-acetylglucosaminyltransferase, is developmentally regulated. The availability of the cloned cDNA encoding both this enzyme16 and the “i-extension” enzyme144 (Table 6-2) allowed this hypothesis to be tested. Rare adults have red cells that type as i; these individuals have mutations in a particular isoform of (β1-6)N-acetylglucosaminyltransferase and their red cell precursors lack the relevant branching glycosyltransferase activity.145-147

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Section I: Part I

Table 6-12. Functional and Nonfunctional Alleles of the Lewis (Le or FUT3) Gene: cDNA Sequences Nucleotide Sequence

Alleles Le Le1 Le2 le1 le2 le3 le4 le5 le6 le7 le8 le9 le10 Mutation

59

202

304

314

370

484

508

667

808

1067

T

T

C A

C

T

G

G

G

G

T

G G G G G

20L→R

A A A A

C C C C

68W→R

T T

102Q→K

105T→M

124S→A

A A A 162D→N

170G→S

A A 223G→R

A 270V→M

356I→K

The Lewis transferase (FUT3) is a 361 amino acid, Type II membrane glycoprotein. All sequences in this table are compared with a “parental” Le sequence. There is no generally agreed upon nomenclature for naming variant alleles of the Le gene; the system chosen here is based on the literature, but is otherwise arbitrary.

Gal(β1-4) GlcNAc(β1-3) i

\ Gal(β1-4)GlcNAc(β1-)R

P Blood Group System

Gal(β1-4) GlcNAc(β1-3) \ Gal(β1-4)GlcNAc(β1-)R /

I Gal(β1-4) GlcNAc(β1-6)

Figure 6-6. Ii blood group antigens. GlcNAc ⫽ D-N-acetylglucosamine; Gal ⫽ D-galactose; GalNAc ⫽ D-N-acetylgalactosamine.

Antibodies specific for the Ii antigens are clinically relevant in the setting of cold type autoimmune hemolytic anemia. The sera of these patients typically contain high titers of a monoclonal antibody, usually with anti-I specificity. In patients demonstrating hemolysis in vivo, the antibodies bind to red cells at or near 37ºC in vitro and have the ability to fix complement. Surprisingly, almost all normal individuals have low titers of anti-I; these autoantibodies agglutinate red cells only at room temperature or below and do not cause accelerated red cell destruction in vivo. Patients with particular infectious diseases such as infectious mononucleosis and mycoplasmal pneumonia often develop cold agglutinins, typically with anti-i and anti-I specificity, respectively.148 In unusual cases, this may result in immune-mediated hemolytic anemia. At present, there is not a complete understanding of the mechanisms

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underlying the differences between cold agglutinins that cause hemolysis in vivo and those that do not.149-152

The P blood group system consists of at least three well-defined glycosphingolipid antigens (Pk, P, and P1) and associated structures such as LKE, Globo-H, and Globo-A (Table 6-13) (for review, see Spitalnik133). These carbohydrate chains are related structurally and biosynthetically.154 Each has a common precursor, Gal(β1- 4)Glc-ceramide (also called lactosylceramide). The Pk antigen is the biosynthetic precursor of the P antigen and both Pk and P1 share a common disaccharide structure, Gal(α14)Gal(β1-4)-R, at the nonreducing end of the glycosphingolipid. These antigenic structures are not found on red cell membrane glycoproteins.155 The glycosyltransferase that converts lactosylceramide to the Pk antigen is denoted as Pk synthase, that which converts Pk to P is denoted as P synthase, and that which converts paragloboside to P1 is denoted as P1 synthase (Tables 6-13 and 614, Fig 6-7). Although the human Pk synthase was cloned,17,20 the gene encoding the P1 synthase has been localized to chromosome 22, but has not yet been cloned (Table 6-2).18,19 Indeed, identifying the P1 synthase gene has been quite controversial.156 Five different red cell phenotypes have been described in which various combinations of these three antigens are expressed (Table 6-14). The P1 and P2 phenotypes are common and account for almost the entire population. The serum of some P2 individuals contains anti-P1. These antibodies are usually low-titered IgM cold

Chapter 6: Carbohydrate Blood Groups

Table 6-13. Structures of P Blood Group and Related Glycosphingolipid Antigens Lactosylceramide Pk P Gal-globoside LKE Globo-H Globo-A Lacto-N-neotriaosylceramide Lacto-N-neotetraosylceramide P1 H Type 2 A Type 2

Galß1-4Glcß1-1⬘cer Galα1-4Galß1-4Glcß1-1⬘cer GalNAcß1-3Galα1-4Galß1-4Glcß1-1⬘cer Galß1-3GalNAcß1-3Galα1-4Galß1-4Glcß1-1⬘cer NeuAcα2-3Galß1-3GalNAcß1-3Galα1-4Galß1-4Glcß1-1⬘cer Fucα1-2Galß1-3GalNAcß1-3Galα1-4Galß1-4Glcß1-1⬘cer GalNAcα1-3[Fucα1-2]Galß1-3GalNAcß1-3Galα1-4Galß1-4Glcß1-1⬘cer GlcNAcß1-3Galß1-4Glcß1-1⬘cer Galß1-4GlcNAcß1-3Galß1-4Glcß1-1⬘cer Galα1-4Galß1-4GlcNAcß1-3Galß1-4Glcß1-1’cer Fucα1-2Galß1-4GlcNAcß1-3Galß1-4Glcß1-1⬘cer GalNAcα1-3[Fucα1-2]Galß1-4GlcNAcß1-3Galß1-4Glcß1-1⬘cer

Lactosylceramide

Lacto-N-neotriosylceramide

Pk

Lacto-N-neotetraosylceramide

P

P1

H Type 2 Gal(β1-3)globoside

A Type 2 Figure 6-7. Biosynthetic pathway of antigens in the P blood group system and related structures. The biochemical structures of these glycosphingolipids are shown in Table 6-13. Table 6-14. Red Cell Phenotypes in the P Blood Group System Phenotype Frequency Red Cell Enzymes Antigens Present P1 P2 P1k P2k p

75% 25% Rare Rare Rare

P1, P, Pk P, Pk P1, Pk Pk None

P1, P, Pk synthases P, Pk synthases P1, Pk synthases Pk synthase None

Serum Antibodies None Anti- P1 Anti-P Anti- P1, anti-P Anti- P1PPk (anti-Tja)

agglutinins and are rarely of clinical significance; however, rare acute hemolytic transfusion reactions have been described.157 In contrast, the unusual individuals with the P1k, P2k, and p phenotypes158 have naturally occurring high-titered IgM antibodies with specificity either for the P antigen (ie, anti-P) or for all the antigens in the P blood group system (ie, anti-P1PPk, or, equivalently, anti-Tja). These antibodies are clinically relevant in that they can cause severe hemolytic transfusion reactions. An unusual syndrome of recurrent spontaneous abortions has also been associated with these antibodies,159 presumably caused by the presence of Pk- and P-active glycosphing-olipids on trophoblastic tissue.160 In addition, the syndrome

Globo-H

LKE

Globo-A

of paroxysmal cold hemoglobinuria, originally associated with syphilis, is caused by Donath-Landsteiner antibodies. The latter are coldreacting, complement-fixing IgG antibodies with anti-P specificity that cause immune-mediated hemolysis in vivo.161 The P blood group antigens are present on multiple cell types in addition to red cells and have various functions. For example, the P antigen is the receptor for parvovirus B19 on erythropoietic precursors.162,163 This virus causes both transient aplastic crises in patients with underlying hemolysis (eg, in sickle cell disease) and anemia in immunocompromised patients.164 In addition, the Pk antigen, also denoted as CD77, is the receptor on endothelial cells for E. coli verotoxins, which are important in the pathogenesis of the hemolytic uremic syndrome.165 The LKE antigen also functions as a receptor for uropathogenic E. coli .166 Finally, the Pk antigen appears to be important in signal transduction following binding of α-interferon and also in apoptosis.167,168

Summary The carbohydrate blood group antigens that are most relevant for the practice of transfusion medicine are found in the ABH, Secretor,

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Section I: Part I

Lewis, Ii, and P human blood group systems. These may be more appropriately regarded as “histo-blood group antigens” in that their expression is not restricted to red cells. The oligosaccharide structures in these systems are related to each other biochemically and immunologically. Great progress has been made in understanding the enzymatic pathways involved in synthesizing these antigens and in the molecular biology of the genes encoding these glycosyltransferases. Although the normal biological functions of these structures are not yet fully understood, many of them play important roles in human infectious diseases.

Disclaimer The authors have disclosed no conflicts of interest.

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Chapter 6: Carbohydrate Blood Groups

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66. Hosseini-Maaf B, Letts JA, Persson M, et al. Structural basis for red cell phenotypic changes in newly identified, naturally occurring subgroup mutants of the human blood group B glycosyltransferase. Transfusion 2007;47:864-75. 67. Oriol R, Le Pendu J, Mollicone R. Genetics of ABO, H, Lewis, X, and related antigens. Vox Sang 1986;51:161-71. 68. Sarnesto A, Kohlin T, Hingsgaul O, et al. Purification of the secretor-type beta-galactoside alpha 1-2-fucosyltransferase from human serum. J Biol Chem 1992;267:2737-44. 69. Oriol R, Mollicone R, Cailleau A, et al. Divergent evolution of fucosyltransferase genes from vertebrates, invertebrates, and bacteria. Glycobiology 1999;9:323-34. 70. Kelly RJ, Rouquier S, Giorgi D, et al. Sequence and expression of a candidate for the human Secretor blood group alpha(1,2)fucosyltra nsferase gene (FUT2). J Biol Chem 1995;270:4640-9. 71. Koda Y, Soejima M, Wang B, Kimura H. Structure and expression of the gene encoding secretor-type galactoside 2-alpha-L-fucosyltransferase (FUT2). Eur J Biochem 1997;246:750-5. 72. Larsen RD, Ernst LK, Nair RP, Lowe JB. Molecular cloning, sequence, and expression of a human GDP-L-fucose: ß-D-galactoside 2-α-L-fucosyltransferase cDNA that can form the H blood group antigen. Proc Natl Acad Sci U S A 1990;87:6674-8. 73. Yu LC, Broadberry RE, Yang YH, et al. Heterogeneity of the human Secretor alpha(1,2)fucosyltransferase gene among Lewis (a⫹b⫹) non-secretors. Biochem Biophys Res Commun 1996;222:390-4. 74. Yu LC, Yang YH, Broadberry RE, et al. Correlation of a missense mutation in the human Secretor alpha1,2-fucosyltransferase gene with the Lewis (a⫹b⫹) phenotype: A potential molecular basis for the weak Secretor allele (Sew). Biochem J 1995;312:329-32. 75. Koda Y, Soejima M, Liu Y, Kimura H. Molecular basis for secretor type alpha(1,2)-fucosyltransferase gene deficiency in a Japanese population: A fusion gene generated by unequal crossover responsible for the enzyme deficiency. A J Hum Genet 1996;59:343-350. 76. Liu Y, Koda Y, Soejima M, et al. Extensive polymorphism of the FUT2 gene in an African (Xhosa) population of South Africa. Hum Genet 1998;103:204-10. 77. Yip SP, Lai SK, Wong ML. Systematic sequence analysis of the human fucosyltransferase 2 (FUT2) gene identifies novel sequence variations and alleles. Transfusion 2007;47:1369-80. 78. Henry S, Mollicone R, Fernandez P, et al. Molecular basis for erythrocyte Le(a⫹b⫹) and salivary partial-secretor phenotypes: Expression of a FUT2 secretor allele with an A→ T mutation at nucleotide 385 correlates with reduced alpha(1,2)fucosyltransferase activity. Glycoconj J 1996;13:985-93. 79. Henry S, Mollicone R, Fernandez P, et al. Homozygous expression of a missense mutation at nucleotide 385 in the FUT2 gene associates with the Le(a⫹b⫹) partial-secretor phenotype in an Indonesian family. Biochem Biophys Res Commun 1996;219:675-8. 80. Liu YH, Koda Y, Soejima M, et al. The fusion gene at the ABOsecretor locus (FUT2): Absence in Chinese populations. J Hum Genet 1999;44:181-4. 81. Peng CT, Tsai CH, Lin TP, et al. Molecular characterization of secretor type alpha(1, 2)-fucosyltransferase gene deficiency in the Philippine population. Ann Hematol 1999;78:463-7. 82. Henry S, Mollicone R, Lowe JB, et al. A second nonsecretor allele of the blood group alpha(1,2)fucosyltransferase gene (FUT2). Vox Sang 1996;70:21-5.

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83. Chang, JG, Yang TY, Liu TC, et al. Molecular analysis of secretor type alpha(1,2)-fucosyltransferase gene mutations in the Chinese and Thai populations. Transfusion 1999;39:1013-17. 84. Yu LC, Lee HL, Chu CC, et al. A newly identified nonsecretor allele of the human histo-blood group alpha(1,2)fucosyltransferase gene (FUT2). Vox Sang 1999;76:115-9. 85. Svensson L, Petersson A, Henry SM. Secretor genotyping for A385T, G428A, C571T, C628T, 685delTGG, G849A, and other mutations from a single PCR. Transfusion 2000;40:856-60. 86. Fernandez-Mateos P, Cailleau A, Henry S, et al. Point mutations and deletion responsible for the Bombay H null and the Reunion H weak blood groups. Vox Sang 1998;75:37-46. 87. Koda Y, Soejima M, Johnson PH, et al. Missense mutation of FUT1 and deletion of FUT2 are responsible for Indian Bombay phenotype of ABO blood group system. Biochem Biophys Res Commun 1997;238:21-5. 88. Koda Y, Soejima M, Johnson PH, et al. An Alu-mediated large deletion of the FUT2 gene in individuals with the ABO-Bombay phenotype. Hum Genet 2000;106:80-5. 89. Kelly RJ, Ernst LK, Larsen RD, et al. Molecular basis for H blood group deficiency in Bombay (Oh) and para-Bombay individuals. Proc Natl Acad Sci U S A 1994;91:5843-7. 90. Kaneko M, Nishihara S, Shinya N, et al. Wide variety of point mutations in the H gene of Bombay and para-Bombay individuals that inactivate H enzyme. Blood 1997;90:839-49. 91. Yu LC, Yang YH, Broadberry RE, et al. Heterogeneity of the human H blood group alpha(1,2)fucosyltransferase gene among paraBombay individuals. Vox Sang 1997;72:36-40. 92. Wagner FF, Flegel WA. Polymorphism of the h allele and the population frequency of sporadic nonfunctional alleles. Transfusion 1997;37:284-90. 93. Wang B, Koda Y, Soejima M, Kimura H. Two missense mutations of H type alpha(1,2)fucosyltransferase gene (FUT1) responsible for para-Bombay phenotype. Vox San 1997;72:31-5. 94. Wagner T, Vadon M, Staudacher E, et al. A new h allele detected in Europe has a missense mutation in alpha(1,2)-fucosyltransferase motif II. Transfusion 2001;41:31-8. 95. Storry JR, Johannesson JS, Poole J, et al. Identification of six new alleles at the FUT1 and FUT2 loci in ethnically diverse individuals with Bombay and Para-Bombay phenotypes. Transfusion 2006;46:2149-55. 96. Yu LC, Yang YH, Broadberry RE, et al. Heterogeneity of the human H blood group alpha(1,2)fucosyltransferase gene among paraBombay individuals. Vox Sang 1997;72:36-40. 97. Breton C, Oriol R, Imberty A. Conserved structural features in eukaryotic and prokaryotic fucosyltransferases. Glycobiology 1998;8:87-94. 98. Bhatia HM, Sathe MS. Incidence of “Bombay” (Oh) phenotype and weaker variants of A and B antigen in Bombay (India). Vox Sang 1974;27:524-32. 99. Le Pendu J, Clamagirand-Mulet C, Cartron JP, et al. H-deficient blood groups of Reunion Island. III. alpha-2-L-fucosyltransferase activity in sera of homozygous and heterozygous individuals. Am J Hum Genet 1983;35:497-507. 100. O’Donnell J, Laffan MA. The relationship between ABO histoblood group, factor VIII and von Willebrand factor. Transfus Med 2001;11:343-51. 101. Jenkins, PV O’Donnell JS. ABO blood group determines plasma von Willebrand factor levels: A biologic function after all? Transfusion 2006;46:1836-44.

Chapter 6: Carbohydrate Blood Groups

102. Bowen DJ. An influence of ABO blood group on the rate of proteolysis of von Willebrand factor by ADAMTS13. J Thromb Haemost 2003;1:33-40. 103. Cserti CM, Dzik WH. The ABO blood group system and Plasmodium falciparum malaria. Blood 2007;110:2250-8. 104. Hume DM, Merrill JP, Miller BF, Thorn GW. Experiences with renal homotransplantation in the human: Report of nine cases. J Clin Invest 1955;34:327-82. 105. Starzl TE, Marchioro TL, Holmes JH, et al. Renal homografts in patients with major donor-recipient blood group incompatibilities. Surgery 1964;55:195-200. 106. Egawa H, Ohmori K, Haga H, et al. B-cell surface marker analysis for improvement of rituximab prophylaxis in ABO-incompatible adult living donor liver transplantation. Liver Transpl 2007;13:579-88. 107. Stegall MD, Dean PG, Gloor JM. ABO-incompatible kidney transplantation. Transplantation 2004;78:635-40. 108. Wu A, Buhler LH, Cooper DK. ABO-incompatible organ and bone marrow transplantation: Current status. Transpl Int 2003;16:291-9. 109. Takahashi K. Recent findings in ABO-incompatible kidney transplantation: Classification and therapeutic strategy for acute antibody-mediated rejection due to ABO-blood-group-related antigens during the critical period preceding the establishment of accommodation. Clin Exp Nephrol 2007;11:128-41. 110. Rydberg L, Breimer ME, Samuelsson BE, Brynger H. Blood group ABO-incompatible (A2 to O) kidney transplantation in human subjects: A clinical, serologic, and biochemical approach. Transpl Proc 1987;19:4528-37. 111. Gugenheim J, Samuel D, Reynes M, Bismuth H. Liver transplantation across ABO blood group barriers. Lancet 1990;336:519-23. 112. Bjoro K, Ericzon BG, Kirkegaard P, et al. Highly urgent liver transplantation: Possible impact of donor-recipient ABO matching on the outcome after transplantation. Transplantation 2003;75:347-53. 113. Angstadt J, Jarrell B, Maddrey W, et al. Hemolysis in ABO-incompatible liver transplantation. Transpl Proc 1987;19:4595-7. 114. Daebritz SH, Schmoeckel M, Mair H, et al. Blood type incompatible cardiac transplantation in young infants. Eur J Cardiothorac Surg 2007;31:339-43. 115. West LJ, Pollock-Barziv SM, Dipchand AI, et al. ABO-incompatible heart transplantation in infants. N Engl J Med 2001;344:793-800. 116. Remberger M, Watz E, Ringden O, et al. Major ABO blood group mismatch increases the risk for graft failure after unrelated donor hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2007;13:675-82. 117. Nair V, Sharma A, Ratheesh J, et al. Severe intravascular haemolysis following minor group mismatched peripheral blood stem cell transplantation. Bone Marrow Transplant 2007;39:805-6. 118. Worel N, Greinix HT, Supper V, et al. Prophylactic red blood cell exchange for prevention of severe immune hemolysis in minor ABO-mismatched allogeneic peripheral blood progenitor cell transplantation after reduced-intensity conditioning. Transfusion 2007;47:1494-502. 119. Henry S, Oriol R, Samuelsson B. Lewis histo-blood group system and associated phenotypes. Vox Sang 1995;69:166-182. 120. Marcus DM, Cass LE. Glycosphingolipids with Lewis blood group activity: Uptake by human erythrocytes. Science 1969;164:553-5. 121. Kukowska-Latallo JF, Larsen RD, Nair RP, Lowe JB. A cloned human cDNA determines expression of a mouse stage-specific

122.

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127.

128.

129.

130.

131.

132.

133.

134.

135.

136.

embryonic antigen and the Lewis blood group alpha(1,3/ 1,4)fucosyltransferase. Genes Dev 1990;4:1288-303. Dupuy F, Petit JM, Mollicone R, et al. A single amino acid in the hypervariable stem domain of vertebrate alpha1,3/1,4fucosyltransferases determines the type 1/type 2 transfer. Characterization of acceptor substrate specificity of the lewis enzyme by site-directed mutagenesis. J Biol Chem 1999;274:12257-62. de Vries T, Srnka CA, Palcic MM, et al. Acceptor specificity of different length constructs of human recombinant alpha 1,3/4fucosyltransferases. Replacement of the stem region and the transmembrane domain of fucosyltransferase V by protein A results in an enzyme with GDP-fucose hydrolyzing activity. J Biol Chem 1995;270:8712-22. Legault DJ, Kelly RJ, Natsuka Y, Lowe JB. Human alpha(1,3/1,4)fucosyltransferases discriminate between different oligosaccharide acceptor substrates through a discrete peptide fragment. J Biol Chem 1995;270:20987-96. Nguyen AT, Holmes EH, Whitaker JM, et al. Human alpha1,3/4fucosyltransferases. I. Identification of amino acids involved in acceptor substrate binding by site-directed mutagenesis. J Biol Chem 1998;273:25244-9. Xu Z, Vo L, Macher BA. Structure-function analysis of human alpha1,3-fucosyltransferase. Amino acids involved in acceptor substrate specificity. J Biol Chem 1996;271:8818-23. de Vries T, Knegtel RM, Holmes EH, Macher BA. Fucosyltransferases: Structure/function studies. Glycobiology 2001;11:119R-128R. Pang H, Liu Y, Koda Y, et al. Five novel missense mutations of the Lewis gene (FUT3) in African (Xhosa) and Caucasian populations in South Africa. Hum Genet 1998;102:675-80. Nishihara S, Yazawa S, Iwasaki H, et al. Alpha (1,3/1,4)fucosyltran sferase (FucT-III) gene is inactivated by a single amino acid substitution in Lewis histo-blood type negative individuals. Biochem Biophys Res Commun 1993;196:624-1. Nishihara S, Narimatsu H, Iwasaki H, et al. Molecular genetic analysis of the human Lewis histo-blood group system. J Biol Chem 1994;269:29271-8. Mollicone R, Reguigne I, Kelly RJ, et al. Molecular basis for Lewis alpha(1,3/1,4)-fucosyltransferase gene deficiency (FUT3) found in Lewis-negative Indonesian pedigrees. J Biol Chem 1994;269:20987-94. Koda Y, Kimura H, Mekada E. Analysis of Lewis fucosyltransferase genes from the human gastric mucosa of Lewis-positive and -negative individuals. Blood 1993;82:2915-19. Elmgren A, Borjeson C, Svensson L, et al. DNA sequencing and screening for point mutations in the human Lewis (FUT3) gene enables molecular genotyping of the human Lewis blood group system. Vox Sang 1996;70:97-103. Elmgren A, Mollicone R, Costache M, et al. Significance of individual point mutations, T202C and C314T, in the human Lewis (FUT3) gene for expression of Lewis antigens by the human alpha(1,3/1,4)-fucosyltransferase, Fuc-TIII. J Biol Chem 1997;269:21994-8. Elmgren A, Rydberg L, Larson G. Genotypic heterogeneity among Lewis negative individuals. Biochem Biophys Res Commun 1993;196:515-20. Pang H, Koda Y, Soejima M, Kimura H. Significance of each of three missense mutations, G484A, G667A, and G808A, present in

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137. 138.

139.

140.

141.

142.

143.

144.

145. 146.

147.

148.

149. 150.

151. 152. 153.

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an inactive allele of the human Lewis gene (FUT3) for alpha(1,3/ 1,4)fucosyltransferase inactivation. Glycoconj J 1998;15:961-7. Nishihara S, Hiraga T, Ikehara Y, et al. Molecular behavior of mutant Lewis enzymes in vivo. Glycobiology 1999;9:373-82. Spitalnik S, Pfaff W, Cowles J, et al. Humoral immunity to Lewis blood group antigens correlates with renal transplant rejection. Transplantation 1984;37:265-8. Salomaa V, Pankow J, Heiss G, et al. Genetic background of Lewis negative blood group phenotype and its association with atherosclerotic disease in the NHLBI family heart study. J Int Med 2000;247:689-98. Ellison RC, Zhang Y, Myers RH, et al. Lewis blood group phenotype as an independent risk factor for coronary heart disease (the NHLBI Family Heart Study). Am J Cardiol 1999;83:345-8. Weston BW, Hiller KM, Mayben JP, et al. Expression of human alpha(1,3)fucosyltransferase antisense sequences inhibits selectinmediated adhesion and liver metastasis of colon carcinoma cells. Cancer Res 1999;59:2127-35. Boren T, Falk P, Roth KA, et al. Attachment of Helicobacter pylori to human gastric epithelium mediated by blood group antigens. Science 1993;262:1892-5. Clyne M, Drumm B. Absence of effect of Lewis A and Lewis B expression on adherence of Helicobacter pylori to human gastric cells. Gastroenterology 1997;113:72-80. Sasaki K, Kurata-Miura K, Ujita M, et al. Expression cloning of cDNA encoding a human beta-1,3-N-acetylglucosaminyltransferase that is essential for poly-N-acetyllactosamine synthesis. Proc Natl Acad Sci U S A 1997;94:14294-9. Lin M, Hou MJ, Yu LC. A novel IGnT allele responsible for the adult i phenotype. Transfusion 2006;46:1982-7. Inaba N, Hiruma T, Togayachi A, et al. A novel I-branching beta1,6-N acetylglucosaminyltransferase involved in human blood group I antigen expression. Blood 2003;101:2870-6. Yu LC, Twu YC, Chou ML, et al. The molecular genetics of the human I locus and molecular background explain the partial association of the adult i phenotype with congenital cataracts. Blood 2003;101:2081-8. Feizi T, Loveless RW. Carbohydrate recognition by Mycoplasma pneumoniae and pathologic consequences. Am J Respir Crit Care Med 1996;154S:133-6. Rosse WF, Adams JP. The variability of hemolysis in the cold agglutinin syndrome. Blood 1980;56:409-16. Jefferies LC, Carchidi CM, Silberstein LE. Naturally occurring antii/I cold agglutinins may be encoded by different VH3 genes as well as the VH4.21 gene segment. J Clin Invest 1993;92:2821-33. Silberstein LE. B-cell origin of cold agglutinins. Adv Exp Med Biol 1994;347:193-205. Havouis S, Dumas G, Ave P, et al. A murine transgenic model of human cold agglutinin disease. Haematologica 1999;84:67-9. Spitalnik PF, Spitalnik SL. The P blood group system: Biochemical, serological, and clinical aspects. Transfus Med Rev 1995;9:110-22.

154. Bailly P, Piller F, Gillard B, et al. Biosynthesis of the blood group Pk and P1 antigens by human kidney microsomes. Carbohydr Res 1992;228:277-87. 155. Yang Z, Bergstrom J, Karlsson KA. Glycoproteins with Gal alpha 4Gal are absent from human erythrocyte membranes, indicating that glycolipids are the sole carriers of blood group P activities. J Biol Chem 1994;269:14620-4. 156. Hellberg A, Chester MA, Olsson ML. Two previously proposed P1/ P2-differentiating and nine novel polymorphisms at the A4GALT (Pk) locus do not correlate with the presence of the P1 blood group antigen. BMC Genet 2005;6:49. 157. Arndt PA, Garratty G, Marfoe RA, Zeger GD. An acute hemolytic transfusion reaction caused by an anti-P1 that reacted at 37 degrees C. Transfusion 1998;38:373-7. 158. Hellberg A, Steffensen R, Yahalom V, et al. Additional molecular bases of the clinically important p blood group phenotype. Transfusion 2003;43:899-907, 2003. 159. Iseki S, Masaki S, Levine P. A remarkable family with the rare human isoantibody anti-Tja in four siblings; anti-Tja and habitual abortion. Nature 1954;173:1193-4. 160. Hansson G, Wazniowska K, Rock JA, et al. The glycosphingolipid composition of the placenta of a blood group P fetus delivered by a blood group P1k woman and analysis of the anti-globoside antibodies found in maternal serum. Arch Biochem Biophys 1988;260:168-76. 161. Schwarting GA, Kundu SK, Marcus DM. Reaction of antibodies that cause paroxysmal cold hemoglobinuria (PCH) with globoside and Forssman glycosphingolipids. Blood 53:1979;186-92. 162. Brown KE, Anderson SM, Young NS. Erythrocyte P antigen: Cellular receptor for B19 parvovirus. Science 1993;62:114-17. 163. Brown KE, Hibbs JR, Gallinella G, et al. Resistance to parvovirus B19 infection due to lack of virus receptor (erythrocyte P antigen). N Engl J Med 1994;330:1192-6. 164. Brown KE, Young NS. Human parvovirus B19 infections in infants and children. Adv Pediatr Infect Dis 1997;13:101-26,. 165. Lingwood CA. Glycolipid receptors for verotoxin and Helicobacter pylori: Role in pathology. Biochim Biophys Acta 1999;1455:375-86. 166. Stroud MR, Stapleton AE, Levery SB. The P histo-blood grouprelated glycosphingolipid sialosyl galactosyl globoside as a preferred binding receptor for uropathogenic Escherichia coli: Isolation and structural characterization from human kidney. Biochemistry 1998;37:17420-8. 167. Maloney MD, Binnington-Boyd B, Lingwood CA. Globotriaosyl ceramide modulates interferon-alpha-induced growth inhibition and CD19 expression in Burkitt’s lymphoma cells. Glycoconj J 1999;16:821-8. 168. Taga S, Carlier K, Mishal Z, et al. Intracellular signaling events in CD77-mediated apoptosis of Burkitt’s lymphoma cells. Blood 1997;90:2757-67.

7

Rh and LW Blood Group Antigens Connie M. Westhoff1 & Don L. Siegel2 1

Scientific Director, Molecular Blood Group and Platelet Testing Laboratory, American Red Cross, and Adjunct Assistant Professor, Division of Transfusion Medicine, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA 2 Vice-Chair and Professor, Pathology and Laboratory Medicine, and Chief, Division of Transfusion Medicine, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA

Although it is now clear that the Rh and LW antigens are carried on entirely different proteins, they are incorporated together in this chapter on the basis of a historic serologic connection (and confusion) and, more recently, evidence that they are physically associated within the red cell membrane.

Rh Blood Group System History and Nomenclature The Rh system is second only to the ABO system in importance in transfusion medicine because Rh antigens, especially D, are highly immunogenic and cause hemolytic disease of the fetus and newborn (HDFN) and hemolytic transfusion reactions (HTRs). HDFN was first described by a French midwife in 1609 in a set of twins, one of whom was hydropic and stillborn, while the other was jaundiced and died of kernicterus.1 That a wide range of observed clinical scenarios involving red cell hemolysis were related—from severely hydropic stillborn fetuses to infants with mild or significant levels of jaundice and kernicterus—was not realized until 1932.2 The cause of red cell hemolysis remained elusive until 1939, when Levine and Stetson described a woman who delivered a stillborn fetus and also suffered a severe hemolytic reaction when transfused with blood from her husband. Levine and Stetson correctly surmised that the mother had been immunized by a fetal red cell antigen inherited from the father and suggested that the cause of the erythroblastosis fetalis was maternal antibody in the fetal circulation.3 They did not give the target blood group antigen a name. Meanwhile Landsteiner and Wiener, in an effort to discover new blood groups, injected rabbits and guinea pigs with rhesus monkey red cells. The antiserum they obtained agglutinated not only rhesus monkey red cells, but also the red cells of 85% of White subjects, whom they called “Rh positive”; the remaining 15% of individuals studied were termed “Rh negative.”4(p195) The “anti-Rhesus” serum seemed to be Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

reacting similarly to the maternal antibody in serologic testing, hence the blood group system responsible for HDFN came to be known as “Rh.” It is now clear that the anti-Rhesus serum was detecting the LW antigen (subsequently named for Landsteiner and Wiener), which is present in greater amounts on D-positive than on D-negative red cells.4(p407) Years of debate followed concerning whether the human antibodies and the Rhesus antibodies were detecting the same antigen and extended long after serologic profiles suggested they were reacting with different structures. Landsteiner and Wiener never accepted the LW terminology, because doing so would imply that they had not discovered the cause of HDFN.5 It was soon obvious that Rh was not a simple, single-antigen system. In 1941, Fisher named the C and c antigens (A and B had been used for ABO), and used the next letters of the alphabet, D and E, to define antigens recognized by additional antibodies. In 1945, the e antigen was identified.4(p195) It is important for transfusion medicine specialists to appreciate that the often confusing nomenclature used to describe Rh antigens results from the difference in opinion that existed concerning the number of genes that were involved in their expression. The Fisher-Race nomenclature suggested that three closely linked genes (C/c, E/e, and D) were responsible, while the Wiener nomenclature (Rh-Hr) was based on his belief that a single gene encoded one “agglutinogen” that carried several blood group factors. Even though neither theory was correct (there are two genes—RHD and RHCE—correctly proposed by Tippett6), for written communication the Fisher-Race designation (CDE) for haplotypes is preferred, and for spoken communication a modified version of Wiener’s nomenclature is preferred (Table 7-1). The “R” indicates that D is present and use of a lowercase “r” (or “little r”) indicates that it is not. The C or c and E or e Rh antigens carried with D are represented by subscripts: 1 for Ce (R1), 2 for cE (R2), 0 for ce (R0), and Z for CE (Rz). The CcEe antigens present without D are represented by superscript symbols: “prime” for Ce (r⬘), “double-prime” for cE (r⬙), and “y” for CE (r y). The “R” versus “r” terminology allows one to convey the common Rh antigens present on one chromosomal haplotype in a single term (a phenotype).

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of the computer era.7 With a few exceptions (Rh17, Rh29), the numeric designations are not widely used in the clinical laboratory.

Table 7-1. Nomenclature and Prevalence of Rh Haplotypes HaplotypeBased Antigens (Fisher/Race)

DCe DcE Dce DCE ce Ce cE CE

Shorthand for Haplotype (Modified Wiener) R1 R2 R0 RZ r r⬘ r⬙ ry

Occurrence (%) Whites

Blacks

Asians

Genes and Their Expressed Proteins 42 14 4 ⬍0.01 37 2 1 ⬍0.01

17 11 44 ⬍0.01 26 2 ⬍0.01 ⬍0.01

70 21 3 1 3 2 ⬍0.01 ⬍0.01

Table 7-2. Rosenfeld Numerical Terminology for Rh Antigens Numeric

Symbol

Numeric

Symbol

Numeric

Symbol

Rh1 Rh2 Rh3 Rh4 Rh5 Rh6 Rh7 Rh8 Rh9 Rh10 Rh11 Rh12 Rh17 Rh18 Rh19 Rh20 Rh21 Rh22 Rh23 Rh26

D C E c e ce or f Ce Cw Cx V Ew G Hr0* Hr hrs VS CG CE Dw c-like

Rh27 Rh28 Rh29 Rh30 Rh31 Rh32 Rh33 Rh34 Rh35 Rh36 Rh37 Rh39 Rh40 Rh41 Rh42 Rh43 Rh44 Rh45 Rh46 Rh47

cE hrH “total” Goa hrB Rh32† Rh33‡ HrB Rh35§ Bea Evans Rh39 Tar Rh41 Rh42 Crawford Nou Riv Sec Dav

Rh48 Rh49 Rh50 Rh51 Rh52 Rh53 Rh54 Rh55 Rh56 Rh57

JAL STEM FPTT MAR BARC JAHK DAK LOCR CENR CEST

Note: Rh13 through 16, 24, and 25 are obsolete. *High-frequency antigen. The antibody is made by D⫺/D⫺ and similar phenotypes. † Low-incidence antigen expressed by RN and DBT phenotypes. ‡ Original described on R0Har phenotype but also found on RN, DVIa(C)⫺, R0JoH, R1Lisa. § Low-frequency antigen on D(C)(E) cell.

The major Rh antigens are D, C, c, E, and e, but the Rh blood group system is one of the most complex because of the number of additional antigens that have been reported (Table 7-2). These additional antigens include compound antigens in cis [eg, f (ce), Ce, CE], low-incidence antigens arising from partial-D hybrid proteins (eg, Dw, Goa, Evans), and antigens arising from various point mutations in the RhCE protein (eg, Cw, Cx, VS). Table 7-2 also includes the numeric designations for Rh antigens that Rosenfeld introduced in 1962 in anticipation

110

Two genes designated RHD and RHCE encode the Rh proteins.8 They are 97% identical, each has 10 exons, and they are the result of a gene duplication on chromosome 1p34-36.9 Rh-positive individuals have both genes, while most Rh-negative individuals have only the RHCE gene (see below). RhD and RhCE are 417-amino acid, nonglycosylated proteins. One protein carries the D antigen, and the other carries various combinations of the CE antigens (ce, cE, Ce, or CE). RhD differs from RhCE by 32 to 35 amino acids, depending on which form of RhCE is present (Fig 7-1).10-13 This degree of difference explains why D is the most immunogenic of all the blood group proteins, because most other blood group antigen polymorphisms result from single amino acid changes in the respective protein. The Rh proteins migrate in sodium dodecyl sulphate polyacrylamide gels with an approximate Mr of 30 to 32 kD and hence are sometimes referred to as the Rh30 proteins. They are predicted to span the membrane 12 times and are covalently linked to fatty acids (palmitate) in the lipid bilayer (Fig 7-1).14

Molecular Basis for Antigen Expression D Antigen “Rh positive” and “Rh negative” refer to the presence or absence of the D antigen, respectively. The Rh-negative phenotype occurs in 15% to 17% of Whites, but is not as common in other ethnic populations.4(p195) The absence of D in people of European ancestry was caused by a complete deletion of the RHD gene15 and probably occurred on a Dce (Ro) haplotype because the allele most often carried with the deletion is ce. In contrast, inactive or silent RHD rather than a complete gene deletion causes D-negative phenotypes in Asians or Africans. D-negative phenotypes in Asians occur with a frequency of ⬍1%,4(p195) and most carry mutant RHD genes associated with Ce, indicating that they probably originated on a DCe (R1) haplotype. Only 3% to 7% of South African Blacks are D-negative, but 66% have RHD genes that contain a 37-bp internal duplication, which results in a premature stop codon. The 37-bp insert RHD-pseudogene was also found in 24% of D-negative African-Americans. Additionally, 15% of D-negative phenotypes in Africans result from a hybrid RHD-CE-D gene that does not encode D epitopes. In total, only 18% of D-negative Africans and 54% of D-negative AfricanAmericans completely lack RHD.16 This is important when designing polymerase chain reaction (PCR)-based methods to predict the D status of the fetus and the possibility of HDFN. The population being tested and the different molecular events responsible for D-negative phenotypes must be considered. Even among D-negative Whites, rare cases of an RHD gene that is not expressed because of mutation or bp-insertions have been reported.17

Chapter 7: Rh and LW Blood Group Antigens

RhD

NH2

Cx Thr36

COOH Cw C Arg41 Ser103

e Ala226

RhCe

NH2

COOH c Pro103

Rhce

e Ala226

VS

NH2

COOH c Pro103

E Pro226

RhcE

NH2

COOH

Figure 7-1. Predicted membrane topology of RhD and the major RhCE proteins. The amino and carboxy termini are cytoplasmic and the proteins are predicted to transverse the membrane 12 times. The location of the amino acid residues that differ between D and CE are represented by open circles, only nine of which are predicted to be extracellular. The C/c (Ser103Pro) polymorphism located on the second extracellular loop and the e/E (Ala226Pro) polymorphism on the fourth extracellular loop are shown. The shared region of RhD and RhCe responsible for expression of the G antigen is shown in black. Amino acid changes responsible for Cx and Cw are located on the first extracellular loop and the VS antigen, common in Blacks, is located in the eighth transmembrane domain of Rhce. The zigzag lines represent covalent linkage to fatty acid in the lipid bilayer.

Weak D An estimated 0.2% to 1% of Whites (and a greater number of Blacks) have reduced expression of the D antigen,4(p407) which is characterized serologically as failure of such red cells to agglutinate directly with anti-D typing reagents, requiring the use of an indirect antiglobulin test for detection. The molecular basis

of weak-D expression is heterogeneous and is associated with the presence of point mutations in RHD. The mutations cause amino acid changes predicted to be intracellular or in the transmembrane regions of RhD rather than on the outer surface of the red cell (Fig 7-2).18 This suggests that these mutations affect the efficiency of insertion and, therefore, the quantity of protein in the membrane, and may not affect the expression of D epitopes. This explains why most individuals with a weak-D phenotype can safely receive D-positive blood and do not make anti-D. Exceptions occur, however, and include individuals who make D antibodies that cross-react with their own red cells and appear to be autoantibodies, others that make alloanti-D, or both. There are over 50 different mutations known to cause weak-D expression (Type 1 through Type 54). The long history of transfusing patients who have weak-D red cells with D-positive blood suggests that weak-D Types 1, 2, and 3 (which represent the majority of Whites with weak D) are unlikely to make anti-D. Nevertheless, some have made anti-D, suggesting they have altered D epitopes. Predicting which mutations alter D epitopes is an active area of investigation.19,20 It is important that donor center typing procedures detect and label weak-D Red Blood Cell (RBC) units as D-positive, because they can stimulate D-negative recipients. However, weak-D typing is not required for transfusion recipients, who would then receive D-negative units without untoward effects. A very weak form of D expression (Del), which cannot be detected by routine serology methods but can be demonstrated by adsorbing and eluting anti-D, is not uncommon in Asians (10-30% of apparent D-negative). Red cells with very low levels of D are primarily of concern for donor testing, because they have stimulated anti-D in D-negative recipients.21 Partial D The D antigen has long been described as a “mosaic” because of the observation that some Rh-positive individuals produce anti-D when exposed to D antigen. It was hypothesized that the red cells of these individuals lack some part of D and that they can produce antibodies to the missing portion. Molecular analysis has shown that this is correct, but what was not predicted is that the missing portions of RHD are replaced by corresponding portions of RHCE in the great majority of cases (Fig 7-3).17,20 The novel sequences of amino acids and the conformational changes that result from segments of RhD joined to segments of RhCE can generate new antigens (eg, BARC, Dw, FPTT, DAK, Goa, Evans, Rh32) (Fig 7-3 and Table 7-2). The replacements are the result of gene conversion, the hallmark being that the donor gene is unchanged. Replacements involve single or multiple exons, while others involve short stretches of amino acids (Fig 7-3). Some also result from only single amino acid changes (eg, DMH, DFW, DII) (Fig 7-2). In contrast to the single amino acid changes that cause weak D (above), which are predicted to be cytoplasmic or transmembrane in location, those that cause partial-D phenotypes are predicted to be located on the extracellular loops of the protein (Fig 7-2). The extracellular location

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G385A Type 2

Weak D Point mutations

NH2 S3C Type 3

V270G Type 1

COOH

G282D weak D Type 15 E233Q/K H166P L54P L110P DFW DVa type 4/5 R229L DMH DVII DHR

G353R DNU

G354D DII T283I G355S DHMI DNB C285Y M358T DIM DWI

Partial D Point mutations

NH2

of the changes explains why these individuals recognize conventional D as foreign. From a clinical standpoint, individuals with partial-D antigens ideally should receive D-negative blood, especially females of childbearing potential, but in practice most will type as D-positive (especially if a weak-D test is performed) and will be recognized only after they have made anti-D following a transfusion with D-positive cells. Elevated D Several phenotypes, including D--, Dc-, and DCw-, have enhanced expression of D antigen and no, weak, or variant CE antigens, respectively.4(p407) These phenotypes are analogous to the partial-D rearrangements described above, only they involve the opposite situation—that is, replacement of portions of RHCE by RHD (Fig 7-3). The additional RHD sequences in RHCE along with a normal RHD explain the enhanced D and account for the reduced or missing CE antigens. Although these represent altered RHCE genes (see below), they are included here because of their elevated D phenotype. Individuals with such altered CE phenotypes can make anti-Rh17 when immunized.

C/c and E/e Antigens The four major forms of the RHCE gene encode four different proteins: RhCe, -ce, -cE and -CE (Fig 7-1).12 C and c differ by four amino acids: Cys16Trp encoded by exon 1, and Ile60Leu, Ser68Asn, and Ser103Pro encoded by exon 2 (Fig 7-1, open circles

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COOH

Figure 7-2. Predicted location of point mutations that cause weak D and partial-D phenotypes. Weak D phenotypes carry mutations in RHD that primarily cause amino acid changes predicted to be intracellular or in the transmembrane regions of D (upper panel). The specific amino acid mutation and the location in the protein are indicated. The partial D phenotypes that carry mutations in RHD are predicted to be located on extracellular loops of RhD (lower panel). The specific amino acid mutation and location in the protein, as well as the common name for each are indicated.

on RhCe). Of those four amino acids, only the residue at 103 is predicted to be extracellular and is located on the second loop. All the amino acids encoded by exon 2 of RHCe are identical to those encoded by exon 2 of RHD (Fig 7-1, black on RhCe). This suggests that RHCe arose from the transfer of exon 2 from an RHD into an RHce gene, respectively. The sharing of exon 2 encoded amino acids by RHD, RHCe, and RHCE accounts for the expression of the G antigen on red cells that are D or C positive. E and e differ by one amino acid, Pro226Ala, predicted to reside on the fourth extracellular loop of the protein (Fig 7-1, solid circle). The E antigen arose from a single point mutation that occurred in exon 5 of a RHce gene, giving rise to RHcE. Altered CE Cw and Cx are low-incidence antigens that result from single amino acid changes (Gln41Arg and Ala36Thr, respectively) predicted to be located on the first extracellular loop of RhCE (Fig 7-1, gray circles).22 These antigens are more common in Finns (4%), and are most often present on RhCe. Cw is also associated with the deletion phenotype DCw- (Fig 7-3). V and VS antigens, which are expressed on red cells of more than 30% of Blacks, result from a Leu245Val substitution located in the predicted eighth transmembrane segment of Rhce (Fig 7-1).23 The V⫺VS⫹ phenotype results from a Gly336Cys change on the 245Val background.24 V⫹ and VS⫹ are associated with weak and altered expression of e, indicating that Leu245Val probably

Chapter 7: Rh and LW Blood Group Antigens

RHCE

RHD

Partial D More prevalent in those of European ancestry DVI Type 1 DVI Type 2 DVa DFR

BARC⫺ BARC⫹ Dw FPTT More prevalent in African ethnic groups

DIIIa DIVa DAR DOL

DAK⫹ Go(a⫹)

Elevated D D-- (SH) D-- (LM) DC w- (AM) D .. (DAV)

Evans

Altered CE More prevalent in those of European ancestry DHar ceSL

no D no D More prevalent in African ethnic groups

RN ceAR ceEK ceS ceMO

Rh32⫹; DAK⫹ often linked to DAR (above) often linked to DAR (above) often linked to D-CE-D hybrid often linked to DAU-0

Figure 7-3. Gene conversion events between RHCE and RHD produce chimeric Rh proteins. Gray (RHCE) and black boxes (RHD) represent the 10 exons that encode Rh polypeptides. Gene conversion events involve amino acids (vertical bars) or whole exons (filled boxes). Replacement of portions of RHD by RHCE (upper set of panels) causes many of the partial-D phenotypes, some of which are shown here. Replacement of portions of RHCE by RHD cause elevated D phenotypes with concurrent loss of expression of CE antigens (middle panel) or altered CE expression (lower set of panels).

causes a local conformation change on the fourth extracellular loop where the e-specific amino acid resides. Most individuals have e-positive red cells, but the e antigen is considered to be second in complexity to D because variant expression has frequently been observed.25 The e antigen is altered on red cells with the following alleles—DHar, ceSL, RN, ceAR, ceEK (hrS⫺), ceS (hrB⫺), and ceMO (hrS⫺, hrB⫺) (Fig 7-3). Extensive discussion is beyond the scope of this chapter, but the majority are found in African ethnic groups, and consequently, are prevalent in patients with sickle cell disease (SCD).20,22,26 These patients not infrequently produce anti-e following transfusion, despite having an e-positive red cell phenotype. Altered expression of e also results from loss of the codon for Arg229,17 and is also associated with the presence of a 16Cys residue in Rhce, which occurs frequently on the R0 haplotype common in African-Americans.27 E variants are not common and include EI, EII, and EIII, which result from a point mutation (EI) or gene conversion replacement

of RhcE amino acids with RhD residues (EII and EIII) with concurrent loss of some E epitope expression. Category EIV red cells, which have an amino acid substitution in an intracellular domain, do not lack E epitopes but have reduced E expression.28 Variants of c are infrequent. The very rare RH:-26 results from a Gly96Ser transmembrane amino acid change that abolishes Rh26 and weakens c expression.29 The lack of c antigen variants in humans compared to the other Rh antigens, and the preservation of expression of c on the red cells of nonhuman primates suggest that the two proline residues involved form a stable structure that is resistant to perturbations and changes in Rhce.30 In summary, point mutations and genetic exchange, mainly involving gene conversion events between RHD and RHCE, are primarily responsible for the large number of Rh antigens. Additional complexity results because many of the Rh epitopes are highly conformational and single amino acid changes in one part of the protein, including changes within the transmembrane

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regions, can affect the expression of cell-surface-exposed antigen epitopes.

RH Genotyping DNA-based molecular testing methods were introduced to the blood bank and transfusion medicine community approximately a decade ago, and much progress has been made in designing reproducible assays and validating gene targets. Assays for blood group antigens encoded by single nucleotide polymorphisms (SNPs) are highly reproducible and correlate with red cell phenotype. Genotyping for the two most important blood group systems, ABO and Rh, are more challenging because of the many different mutations that cause weak expression of A and B, and inactive O, and because of the numerous variant and hybrid RH alleles. Multiple regions of the genes must be sampled and hybrid genes are problematic for analysis. Genotyping for blood group antigens is performed principally in a reference laboratory setting, although testing platforms that used bead or microarray technology hold promise for implementation of high-throughput RH genotyping for donors and patients.31 RHD Zygosity Testing Serologic testing for red cell expression of D, C/c, and E/e can only predict the likelihood that a sample is homozygous (D/D) or heterozygous (D/⫺) for RHD. Genotyping enables zygosity to be determined by assaying for the presence of a recessive D-negative allele. In prenatal practice, paternal RHD zygosity testing is important to predict the fetal D status when the mother has antiD. Several different genetic events cause a D-negative phenotype, and multiple assays must be performed to accurately determine zygosity. These include detection of the region generated by deletion of RHD, and the 37-bp insert RHD pseudogene or the D-negative RHD-CE-D hybrid gene common in African Black ethnic groups. If the father is RHD homozygous, the fetus will be D-positive, and monitoring of the pregnancy will be required. If the father is heterozygous, the D type of the fetus should be determined to prevent invasive and unnecessary testing. Fetal Typing Genotyping is important in the prenatal setting to determine whether the fetus has inherited the paternal antigen to which the mother has a clinically significant antibody. Fetal DNA can be isolated from cells obtained by amniocentesis or chorionic villus sampling. Alternatively, the discovery that cell-free, fetal-derived DNA is present in maternal plasma or serum by approximately 5 weeks’ gestation allows maternal plasma to be used as a source of fetal DNA.32 Fetal DNA in maternal plasma is derived from apoptotic syncytiotrophoblasts, increases in concentration with gestational age, and is rapidly cleared following delivery.33 The small quantity of cell-free fetal DNA present relative to maternal DNA poses a challenge, and positive controls for isolation of sufficient fetal DNA are critical to validate negative results. When combined with sensitive real-time PCR methodology, isolation of fetal DNA from maternal plasma has been successfully applied

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to determine fetal D status.34 This has been more successful for D typing because the D-negative phenotype in the majority of samples is caused by the lack of RHD. Testing for the presence or absence of a gene is less demanding than testing for a single gene polymorphism or SNP. Theoretically, testing the maternal plasma for the presence of a fetal RHD could be used to eliminate the unnecessary administration of antepartum Rh Immune Globulin (RhIG) to the approximately 40% of D-negative women who are carrying a D-negative fetus. RhIG is not entirely risk free, and this approach would be cost-effective for some health-care systems.34 Distinction between Weak D and Partial D As indicated above, altered expression of D antigen is not uncommon. Weak D phenotypes have single amino acid changes that primarily affect the quantity of RhD in the membrane. Partial D phenotypes have amino acid changes that alter D epitopes, or often are hybrid proteins with portions of RhD joined to portions of RhCE. The distinction between weak D and partial D phenotypes is of clinical importance because the latter make anti-D. Routine serologic typing reagents cannot distinguish between these red cells; however, genotyping strategies that sample multiple regions of RHD can discriminate between weak D and partial D. Detecting Patients at Risk for Production of Antibodies to High-Incidence Antigens Alloimmunization is a serious complication of chronic transfusion, particularly in patients with SCD requiring long-term transfusion support. Many transfusion programs attempt to prevent or reduce the risk and incidence of alloantibody production in SCD patients by transfusing RBC units that are antigenmatched for D, C, E, and Kell. Although this approach reduces the incidence of alloimmunization, variant RHD and RHce genes are common in African Blacks and individuals of mixed ethnic backgrounds. The prevalence of RH alleles that encode altered D, C, and e antigens in this patient group explains why some SCD patients become immunized to Rh, despite Rh antigen-matching for D, C, and E.20,35 These antibodies often have complex, highincidence Rh specificities, and it can be difficult or impossible to find compatible units. Genotyping can identify those patients who are homozygous for variant RH alleles and at risk for production of alloantibodies to high-incidence Rh antigens and can identify compatible donors for transfusion.

Rhnull Phenotype Rhnull individuals lack expression of all Rh antigens. They suffer from a compensated hemolytic anemia, have variable degrees of spherocytosis, stomatocytosis, and increased red cell osmotic fragility.36 The phenotype is rare and occurs on two different genetic backgrounds: the “regulator” type, caused by a gene at an unlinked locus, and the “amorph” type, which maps to the RH locus.5 It is now clear that in the more common, regulator type of Rhnull, the suppression of Rh is caused by a lack of, or a

Chapter 7: Rh and LW Blood Group Antigens

mutant, Rh-associated glycoprotein (RhAG), also called Rh50 protein.37 RhAG is a 409-amino acid glycosylated protein that coprecipitates with RhD and RhCE.38 It shares 37% amino acid identity with the RhD/RhCE proteins and has the same predicted membrane topology. RhAG is not polymorphic and does not carry Rh antigens; however, it is important for targeting the Rh proteins to the membrane during erythroid development. RhAG has one N-glycan chain that carries ABO and Ii specificities.39 It is encoded by a single gene, RHAG, located at chromosome 6p11-21.1.38 The amorph type of Rhnull results from mutations in RHCE on a D-negative background.40 Amorph-type red cells express no Rh protein and have reduced amounts (⬃20%) of RhAG.

Rh-Membrane Complex Additional complication in Rh protein structures arises because they exist in the red cell membrane as complexes with several other proteins. Rh and RhAG are associated in the membrane as a core complex.41 The fact that several other proteins interact with the Rh-core complex is based on observations of Rhnull cells. These red cells have reduced expression of CD47, an integrinassociated protein (IAP) that has wide tissue distribution, binds β3 integrins, and is required for integrin-regulated Ca2⫹ entry into endothelial cells. Its function on the red cells is unknown; however, a role in red cell senescence has been suggested.42 Rhnull cells also have reduced glycophorin B (GPB), a sialoglycoprotein that carries S or s and U antigens. GPB appears to aid RhAG trafficking to the membrane, because the RhAG protein in GPB-deficient cells has increased glycosylation, reflecting longer dwell time in the endoplastic reticulum. Rhnull cells also lack LW, a glycoprotein of unknown function that belongs to the family of intercellular adhesion molecules (ICAM-4, see below). Band 3 (the anion exchanger) enhances the expression of the Rh antigens in transfected cells, suggesting that band 3 may also be associated with the Rh-core complex.43 Recent studies reveal that the Rh-core complex is linked to the membrane skeleton through interactions between CD47 and protein 4.244 and through a novel Rh/RhAG-ankyrin cytoskeleton connection.45 Red cell membrane protein-cytoskeleton and protein-protein interactions are an active area of investigation.

Rh Function Rh Glycoproteins (RhAG, RhBG, RhCG) The Rh blood group proteins are well known because of their importance in blood transfusion. However, the mammalian family of Rh proteins has been expanded with the discovery of RhAG in erythrocytes, and the related proteins, RhBG and RhCG, in other tissues. Protein sequences with similarities to the mammalian Rh proteins were first found in Caenorhabditis elegans, and these homologues, in turn, showed similarity to the ammonia transporters from bacteria, yeast (MEP), and plants (AMT).46 The relationship of the Rh glycoproteins to the AMT/MEP ammonia transporters from these other organisms has been substantiated

by functional transport data47-49 and structural modeling.50,51 The Rh proteins reveal the power of comparative genomics and proteomics, in which sequence analysis and homology modeling can give important insight into mammalian protein function. The nonerythroid Rh glycoproteins, RhBG and RhCG, are present in the kidney, liver, brain, and skin where ammonia production and elimination occur. In the kidney collecting segment and collecting duct, RhBG and RhCG are found on the basolateral and apical membranes, respectively, of the intercalated cells where they mediate transepithelial movement of ammonia from the interstitium to the lumen.52 In the liver, RhBG is found on the basolateral membrane of perivenous hepatocytes, where it may function in ammonia uptake. RhCG is also present in bile duct epithelial cells, where it is positioned to contribute to ammonia secretion into the bile fluid.53 The mechanism of ammonia transport by Rh glycoproteins is an active area of investigation. Expression of Rh glycoproteins in heterologous systems indicates that mammalian transport is an electroneutral process that is driven by the NH4⫹ concentration and the transmembrane H⫹ gradient.54,55 Functional studies of the kidney, liver, and brain Rh homologues, along with the erythrocyte RhAG/Rh proteins, promise to lead to development of a unifying hypothesis of ammonia transport in mammals by the Rh family of proteins.

RhCE and RhD The function of the more recently evolved erythrocyte blood group proteins, RhCE and RhD, has not yet been determined. When expressed in heterologous systems, they do not directly transport ammonia.56 RhCE and RhD lack the highly conserved histidine residues located in the membrane pore that are critical for ammonia transport. RhCE and RhD may be evolving a new function in the red cell membrane as phylogenetic analysis indicates the RhCE/D proteins are rapidly evolving, suggesting their function may be changing.

Immune Response to Rh Medical Aspects Human red cells can express more than 400 different blood group antigens. Typing patient and donor cells for every known antigen with the intention of providing perfectly matched blood would be a practical and fiscal impossibility. Fortunately, such extensive testing is not required for a number of reasons, the most important of which is that exposure to the majority of foreign red cell antigens through transfusion does not lead to the production of clinically significant alloantibodies. D is one notable exception. As many as 80% of D-negative patients exposed to D-positive red cells may develop high-titer, high-affinity, D IgG antibodies that may persist for the rest of their lives even if they are never exposed to the antigen again. The antibodies can cause HTRs and can cross the placenta causing HDFN when present in a D-negative female carrying a D-positive fetus. Therefore, because of its extraordinary immunogenicity and clinical significance, the D antigen is the only blood group antigen for which

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it is routine to prophylactically match blood before transfusion so as to avoid immunization. Although antibodies to C, c, E, and e can cause HTRs and HDFN, they are much less immunogenic than D (⬃1% rate of sensitization) and the use of RBCs lacking one or more of those antigens are indicated for patients only after sensitization has occurred (management of sickle cell patients may differ as described in preceding section). In practice, D-positive patients can be transfused with either D-positive or D-negative RBCs—the absence of D will cause no harm—but it is deemed prudent to reserve the rarer units of D-negative blood (⬃15% of donor units) for D-negative individuals who must receive them. In cases of trauma and/or massive transfusion in which the patient’s D status is unknown, efforts are made to provide D-negative blood, especially for females of childbearing potential, until the appropriate testing can be completed. When D-negative blood is in short or critical supply, it may be necessary to transfuse D-negative patients with D-positive units. In such scenarios, D-negative units are reserved for females of childbearing potential and for patients whose serum contains anti-D from a previous sensitization. Unlike ABO blood group antigens, which are expressed by all transfused blood cells including platelets, the D antigen is present only on red cells. Theoretically, the selection of platelet units for transfusion should be independent of the D status of the donor. However, a transfusion of pooled platelet concentrates may introduce as much as 5 mL of donor red cells, which may be sufficient to alloimmunize a D-negative patient. Therefore, the standard of care is to avoid transfusing D-negative patients, particularly females of childbearing potential, with platelet units derived from D-positive donors. If such units are unavailable and platelet transfusion must be undertaken, the administration of RhIG can be considered. A standard 300-µg dose of RhIG, which may inhibit the immunizing potential of up to 15 mL of D-positive red cells, would neutralize the effects of D-positive red cells from several mismatched platelet transfusions. With respect to the transfusion of plasma products, the D status of the donor is not an issue because plasma products do not contain cellular or soluble material capable of inducing anti-D immune responses.

Serologic Aspects The immune response to Rh, like that to other peptide antigens, is typically thymus-dependent, requiring T-cell help. Upon exposure to a foreign Rh antigen, an IgM response may develop, but this is quickly followed by the production of IgG antibodies. Consequently, nearly all examples of Rh antibodies are IgG molecules (mostly IgG1 and IgG3), which bind optimally to red cells at 37ºC and require the addition of an antiglobulin reagent to produce hemagglutination. Although IgG1 and IgG3 subclasses classically initiate complement activation, the vast majority of anti-Rh-containing sera do not do so. The usual explanation for this cites the relatively low copy number of Rh antigens per red cell, which results in Rh molecules situated too far apart on the cell surface to permit the simultaneous binding of C1q by multiple Rh IgG antibodies. Therefore, hemolysis from the transfusion

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of Rh-incompatible RBCs is generally extravascular because of the phagocytosis of IgG-coated erythrocytes by cells of the reticuloendothelial system. After anti-D, the Rh antibodies most commonly found in the sera of alloimmunized individuals are anti-E ⬎ anti-c ⬎ anti-e ⬎ anti-C. In approximately 50% of cases of warm-type autoimmune hemolytic anemia (WAIHA), autoantibodies are believed to be directed to Rh antigens by virtue of their “panreactivity” with all red cell phenotypes except Rhnull cells. However, direct binding of autoantibodies to putative epitopes common to D and C/E polypeptides or to other components of the Rh-membrane complex (RhAG, CD47, etc) has yet to be demonstrated in WAIHA. The difficulties in approaching this problem are largely technical in nature and relate to both the inability to produce workable quantities of pure patient autoantibody in vitro (ie, clone the autoantibody-producing B lymphocytes) and the inability to purify Rh proteins in a way that retains their native conformationally dependent epitopes.

Molecular Aspects The characterization of Rh antibodies on a molecular level, particularly that of anti-D, has been the focus of much study not only because of their clinical significance, but also because of the need to develop suitable in-vitro methods for their production.57 Ironically, because of better transfusion practice and the use of RhIG, alloimmunization of antigen-negative individuals is significantly less common (as are sera donors willing to be purposely hyperimmunized), so that supplies of Rh antibodies for use as typing reagents and for the preparation of RhIG are dwindling. To better understand the molecular make-up of Rh antibodies, investigations have focused on analyzing their variable regions in order to determine whether there are commonly shared genetic and/or structural features among Rh antibodies made by different individuals. Early work using rabbit antisera specific for different human heavy-chain variable region gene products suggested a restriction in the use of certain heavy-chain gene families by the anti-D contained in polyclonal sera from several dozen anti-D donors.58 Subsequent studies with rodent idiotypic antibodies demonstrated cross-reactive idiotypes among polyclonal anti-D preparations59,60 and among different examples of human monoclonal anti-C, -c, -D, -E, -e, and -G produced by transformed B cells.61 A more direct approach using nucleotide sequencing to examine immunoglobulin gene diversity examined a cohort of four IgM and 10 IgG monoclonal anti-D variable regions.62 A restricted use of the human heavy-chain variable region genes VH3-33 and VH434 was found with a shift in repertoire usage toward VH3-33 for anti-D that had isotype switched to the more clinically relevant IgG. The restriction of anti-D heavy chains to the use of these and other highly related VH genes has been confirmed and extended through the analysis of many additional examples of anti-D produced through both tissue culture and recombinant means.63-68 The use of molecular approaches such as site-directed mutagenesis,69 complementarity-determining region (CDR) sequence

Chapter 7: Rh and LW Blood Group Antigens

randomization,70 and heavy-chain/light-chain “shuffling”71 has demonstrated the genetic relatedness among anti-D molecules directed against different D epitopes as well as among antibodies with D and E specificity. These studies and others72,73 have supported the hypothesis that a restricted “Rh footprint” for D alloantibodies and a process termed “epitope migration”63 play a role in the molding of the anti-Rh immune repertoire.74 Although the precise significance of immunoglobulin germline gene restriction by Rh antibodies is not fully understood, it may have practical significance for the preparation of anti-D for both therapeutic and diagnostic use. For example, the VH genes used to encode anti-D are among the most cationic of the human germline VH genes75 and may account for the relatively high pI of polyclonal anti-D-containing antisera originally noted over 40 years ago.76 Although the cationic nature of the antibodies may be important for binding to D, it has also been suggested that a constitutive net-positive charge may be necessary to permeate the highly negative red cell zeta potential, thus permitting antibody to contact antigen.77 Second, while IgM D monoclonal antibodies are well-suited for antigen typing because they may serve as direct agglutinins, the fact that they are most often encoded by VH4-34 (the germline gene to which cold agglutinins are also restricted),78,79 may explain why many IgM monoclonal anti-D typing reagents falsely agglutinate D-negative cells when used at cooler-than-recommended temperatures. This phenomenon may also explain the body of literature claiming that the D antigen was present on numerous nonerythroid cells. Using IgM anti-D, these investigators may have been detecting antigens of the I/i blood group system.

the red cell membrane once, and the N-terminal extracellular region is organized into two immunoglobulin superfamily (IgSF) domains.80

Molecular Basis for Antigen Expression

LW Blood Group System

LWa is the common antigen, whereas LWb has an incidence of less than 1% in most Europeans.4(p407) The LWa/LWb polymorphism is caused by a single amino acid substitution, Gln70Arg, on the LW glycoprotein.81 An increased frequency of the uncommon LWb antigen in Latvians and Lithuanians (6%), Estonians (4%), Finns (3%), and Poles (2%) suggests that the LWb mutation originated in the people of the Baltic region. The LWab antigen was originally defined by an alloantibody made by the only known (genetically verified) LW(a⫺b⫺) person, who lacks expression of all LW antigens.5 The LW gene in this rare LW(a⫺b⫺) individual has a 10-bp deletion and a premature stop codon in the first exon.82 Rhnull red cells also lack LW antigens but do not have defective LW genes. Rh proteins appear to be required for LW to traffic to the membrane, and association with the D antigen is preferred. LW antigens require divalent cations (eg, Mg2⫹) for expression and have intramolecular disulfide bonds that are sensitive to dithiothreitol (DTT) treatment.5 This is helpful to differentiate anti-LW from anti-D, because the D antigen is resistant to DTT. Also helpful in identifying anti-LW is the fact that LW antigens are expressed equally well on D-positive and D-negative cord blood red cells.4(p407) Transient loss of LW antigens has been described in pregnancy and patients with diseases, particularly Hodgkin disease, lymphoma, leukemia, sarcoma, and other forms of malignancy. Transient loss of LW antigens is associated with the production of autoanti-LW that can appear to be alloantibody.4(p407)

History and Nomenclature

LW Function

The LW antigens are the “true” Rhesus antigens shared by humans and the rhesus monkey. As discussed earlier, the confusion occurred because LW antigens are more abundant on Dpositive than on D-negative red cells. When the situation was clarified, the term “Rh” remained associated with the human antigen, so the real Rhesus antigen was renamed LW in honor of Landsteiner and Wiener.5 The confusion can be understood today in the transfusion service when a weak example of anti-LWa often appears initially to be anti-D. The LW system has undergone additional terminology revisions. The historical terminology of LW1, LW2, LW3, LW4, and LW0 to describe phenotypes was based on both the LW and the D status of the red cells but is now obsolete.5 The phenotypes are LW(a⫹b⫺), LW(a⫺b⫹), LW(a⫹b⫹), and the rare LW(a⫺b⫺); the antigens are designated LWa, LWb, and LWab.

LW glycoprotein, ICAM-4, is a ligand with broad specificity for a number of β1, β2, β3, and β5 integrins including αLβ2 (LFA-1), αMβ2 (Mac-1), α4β1 (VLA-4), αVβ1, αVβ5, as well as platelet integrin α2bβ3.80,83-87 In addition, LW binds to the I domains of CD11a/CD18 and CD11b/CD18 on leukocytes.84,88 The function of LW glycoprotein on mature red cells is currently an area of active investigation. Evidence is emerging for both a normal homeostatic role in erythroblastic island formation through LW interaction with macrophage integrins89,90 as well as pathophysiologic roles in the development of vaso-occlusion in SCD mediated by endothelial cell integrins91,92 and of thrombosis mediated by activated integrins on platelets.87

Genes and Their Expressed Proteins

The molecular basis for the Rh antigens has now been elucidated. A gene deletion or silent RHD gene explains the absence of the D antigen in many Rh-negative individuals. The large number of amino acid differences between the RhD and RhCE proteins

LW is encoded by a single gene located on chromosome 19. The 42-kD LW glycoprotein is a member of the family of ICAMs and has been renamed ICAM-4 (CD242). LW passes through

Summary

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explains why exposure in an individual lacking D often results in a vigorous immune response characterized by a very heterogeneous population of antibodies. The proximity of RHD and RHCE, duplicated genes on the same chromosome, has resulted in numerous exchanges by gene conversion between them. This has generated new polymorphisms and explains the many antigens observed in this blood group system. Molecular analysis has also revealed that Rh antigen expression is affected not only by changes in extracellular amino acids, but also by intracellular changes, highlighting the conformational nature of these blood group antigens and complicating attempts to map the epitopes to specific amino acid residues. RH genotyping can be used to determine paternal RHD zygosity to predict HDFN and the fetus can be typed from amniocytes or from the maternal plasma. RH genotyping can also identify patients facing long-term transfusion therapy who are homozygous for variant RH alleles and at risk for production of alloantibodies to high-incidence Rh antigens. When partnered with RH genotyping of donors, this approach promises to have a positive impact on transfusion therapy outcomes by reducing alloimmunization, especially in patients with SCD. Rh antibodies show restriction in their use of particular variable region immunoglobulin germline genes. Antibodies to serologically distinct epitopes may be genetically related, and epitope migration may be an important process that helps shape the composition of the anti-Rh immune repertoire. Questions remain concerning the function of the Rh proteins. The discovery that Rh protein homologues also exist in the liver and kidney indicates that the Rh blood group antigens belong to a larger, conserved family of proteins and will contribute to the elucidation of their function. Similarly, the function of LW on the mature red cell is not entirely clear but its ability to interact as an adhesion molecule with a broad range of integrin-binding specificity suggests an important role in both normal red cell development as well as disease-associated processes such as vasoocclusion and unwanted thrombosis.

Disclaimer The authors have disclosed no conflicts of interest.

References 1. Bowman JM. RhD hemolytic disease of the newborn. N Engl J Med 1998;339:1775-7. 2. Diamond LK, Blackfan KD, Baty JM. Erythroblastosis fetalis and its association with universal edema of the fetus, icterus gravis neonatorum and anemia of the newborn. J Pediatr 1932;1:269-309. 3. Levine P, Burnham L, Katzin WM, Vogel P. The role of isoimmunization in the pathogenesis of erythroblastosis fetalis. Am J Obstet Gynecol 1941;42:925-37. 4. Daniels G. Human blood groups. Oxford: Blackwell, 2002.

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5. Issitt PD, Anstee DJ. Applied blood group serology. 4th ed. Durham, NC: Montgomery Scientific, 1998:425. 6. Tippett P. A speculative model for the Rh blood groups. Ann Hum Genet 1986;50:241-7. 7. Rosenfeld RE, Allen FH, Swisher SN, Kochwa S. A review of Rh serology and presentation of a new terminology. Transfusion 1962;2:287-312. 8. Colin Y, Cherif-Zahar B, Le Van Kim C, et al. Genetic basis of the RhD-positive and RhD-negative blood group polymorphism as determined by Southern analysis. Blood 1991;78:2747-52. 9. Cherif-Zahar B, Mattei MG, Le Van Kim C, et al. Localization of the human Rh blood group gene structure to chromosome 1p34.31p36.1 region by in situ hybridization. Hum Genet 1991;86:398-400. 10. Cherif-Zahar B, Bloy C, Le Van Kim C, et al. Molecular cloning and protein structure of a human blood group Rh polypeptide. Proc Natl Acad Sci U S A 1990;87:6243-7. 11. Arce MA, Thompson ES, Wagner S, et al. Molecular cloning of RhD cDNA derived from a gene present in RhD-positive, but not RhDnegative individuals. Blood 1993;82:651-5. 12. Mouro I, Colin Y, Cherif-Zahar B, et al. Molecular genetic basis of the human Rhesus blood group system. Nat Genet 1993;5:62-5. 13. Simsek S, de Jong CAM, Cuijpers HTM, et al. Sequence analysis of cDNA derived from reticulocyte mRNAs coding for Rh polypeptides and demonstration of E/e and C/c polymorphism. Vox Sang 1994;67:203-9. 14. Cartron J-P, Agre P. Rh blood group antigens: protein and gene structure. Semin Hematol 1993;30:193-208. 15. Wagner FF, Flegel WA. RHD gene deletion occurred in the Rhesus box. Blood 2000;95:3662-8. 16. Singleton BK, Green CA, Avent ND, et al. The presence of an RHD pseudogene containing a 37 base pair duplication and a nonsense mutation in Africans with the Rh D-negative blood group phenotype. Blood 2000;95:12-18. 17. Huang CH, Liu PZ, Chen JG. Molecular biology and genetics of the Rh blood group system. Semin Hematol 2000;37:150-65. 18. Wagner FF, Gassner C, Muller TH, et al. Molecular basis of weak D phenotypes. Blood 1999;93:385-93. 19. Flegel WA. Homing in on D antigen immunogenicity. Transfusion 2005;45:466-8. 20. Westhoff CM. The structure and function of the Rh antigen complex. Semin Hematol 2007;44:42-50. 21. Wagner T, Kormoczi GF, Buchta C, et al. Anti-D immunization by DEL red blood cells. Transfusion 2005;45:520-6. 22. Mouro I, Colin Y, Sistonen P, et al. Molecular basis of the RhCW (Rh8) and RhCX (Rh9) blood group specificities. Blood 1995;86:1196-201. 23. Faas BHW, Beckers EAM, Wildoer P, et al. Molecular background of VS and weak C expression in blacks. Transfusion 1997;37:38-44. 24. Daniels G, Faas BHW, Green CA, et al. The VS and V blood group polymorphisms in Africans: A serological and molecular analysis. Transfusion 1998;38:951-8. 25. Issitt PD. An invited review: The Rh antigen e, its variants, and some closely related serological observations. Immunohematology 1991;7:29-36. 26. Westhoff CM. The Rh blood group system in review: A new face for the next decade. Transfusion 2004;44:1663-73. 27. Westhoff CM, Silberstein LE, Wylie DE, Reid ME. 16Cys endoded by the RHce gene is associated with altered expression of the e antigen and is frequent in the Ro haplotype. Br J Haematol 2001;113:666-71.

Chapter 7: Rh and LW Blood Group Antigens

28. Noizat-Pirenne F, Mouro I, Gane P, et al. Heterogeneity of blood group RhE variants revealed by serological analysis and molecular alteration of the RHCE gene and transcript. Br J Haematol 1998;103:429-36. 29. Faas BHW, Ligthart PC, Lomas-Francis C, et al. Involvement of Gly96 in the formation of the Rh26 epitope. Transfusion 1997;37:1123-30. 30. Westhoff CM, Silberstein LE, Wylie DE. Evidence supporting the requirement for two proline residues for expression of c. Transfusion 2000;40:321-4. 31. Westhoff CM. Molecular testing for transfusion medicine. Curr Opin Hematol 2006;13:471-5. 32. Lo YM, Hjelm NM, Fidler C, et al. Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma. N Engl J Med 1998;339:1734-8. 33. Bianchi DW, Avent ND, Costa JM, van der Schoot CE. Noninvasive prenatal diagnosis of fetal Rhesus D: Ready for prime(r) time. Obstet Gynecol 2005;106:841-4. 34. Van der Schoot CE, Soussan AA, Koelewijn J, et al. Non-invasive antenatal RHD typing. Transfus Clin Biol 2006;13:53-7. 35. Vege S, Westhoff CM. Molecular characterization of GYPB and RH in donors in the American Rare Donor Program. Immunohematology 2006;22:143-7. 36. Ballas S, Clark MR, Mohandas N, et al. Red cell membranes and cation deficiency in Rhnull syndrome. Blood 1984;63:1046-55. 37. Cherif-Zahar B, Raynal V, Gane P, et al. Candidate gene acting as a suppressor of the RH locus in most cases of Rh-deficiency. Nat Genet 1996;12:168-73. 38. Ridgwell K, Spurr NK, Laguda B, et al. Isolation of cDNA clones for a 50 kDa glycoprotein of the human erythrocyte membrane associated with Rh (Rhesus) blood-group antigen expression. Biochem J 1992;287:223-8. 39. Moore S, Green C. The identification of specific Rhesus polypeptide blood group ABH-active glycoprotein complexes in the human red cell membrane. Biochem J 1987;244:735-41. 40. Huang CH, Chen Y, Reid ME, Seidl C. Rhnull disease: The amorph type results from a novel double mutation in RhCe gene on D-negative background. Blood 1998;92:664-71. 41. Ridgwell K, Eyers SAC, Mawby WJ, et al. Studies on the glycoprotein associated with Rh (Rhesus) blood group antigen expression in the human red blood cell membrane. J Biol Chem 1994;269:6410-6. 42. Oldenborg P-A, Zheleznyak A, Fang Y-F, et al. Role of CD47 as a marker of self on red blood cells. Science 2000;288:2051-3. 43. Beckmann R, Smythe JS, Anstee DJ, Tanner MJA. Functional cell surface expression of band 3, the human red blood cell anion exchange protein (AE1) in K562 erythroleukemia cells: Band 3 enhances the cell surface reactivity of Rh antigens. Blood 1998;92: 4428-38. 44. Dahl KN, Westhoff CM, Discher DE. Fractional attachment of CD47 (IAP) to the erythrocyte cytoskeleton and visual colocalization with Rh protein complexes. Blood 2003;101:1194-9. 45. Nicolas V, Le Van Kim C, Gane P, et al. Rh-RhAG/ankyrin-R, a new interaction site between the membrane bilayer and the red cell skeleton, is impaired by Rh(null)-associated mutation. J Biol Chem 2003;278:25526-33. 46. Marini AM, Urrestarazu A, Beauwens R, Andre B. The Rh (rhesus) blood group polypeptides are related to NH4⫹ transporters. Trends Biochem Sci 1997;22:460-1.

47. Marini AM, Matassi G, Raynal V, et al. The human Rhesus-associated RhAG protein and a kidney homologue promote ammonium transport in yeast. Nat Genet 2000;26:341-4. 48. Westhoff CM, Ferreri-Jacobia M, Mak DO, Foskett JK. Identification of the erythrocyte Rh blood group glycoprotein as a mammalian ammonium transporter. J Biol Chem 2002;277:12499-502. 49. Ripoche P, Bertrand O, Gane P, et al. Human Rhesus-associated glycoprotein mediates facilitated transport of NH(3) into red blood cells. Proc Natl Acad Sci U S A 2004;101:17222-7. 50. Khademi S, O’Connell J 3rd, Remis J, et al. Mechanism of ammonia transport by Amt/MEP/Rh: Structure of AmtB at 1.35 A. Science 2004;305:1587-94. 51. Conroy MJ, Bullough PA, Merrick M, Avent ND. Modelling the human rhesus proteins: Implications for structure and function. Br J Haematol 2005;131:543-51. 52. Weiner ID, Verlander JW. Renal and hepatic expression of the ammonium transporter proteins, Rh B glycoprotein and Rh C glycoprotein. Acta Physiol Scand 2003;179:331-8. 53. Weiner ID, Miller RT, Verlander JW. Localization of the ammonium transporters, Rh B glycoprotein and Rh C glycoprotein, in the mouse liver. Gastroenterology 2003;124:1432-40. 54. Westhoff CM, Siegel DL, Burd CG, Foskett JK. Mechanism of genetic complementation of ammonium transport in yeast by human erythrocyte Rh-associated glycoprotein. J Biol Chem 2004;279:17443-8. 55. Mak DO, Dang B, Weiner ID, et al. Characterization of ammonia transport by the kidney Rh glycoproteins RhBG and RhCG. Am J Physiol Renal Physiol 2006;290:F297-305. 56. Westhoff CM, Wylie DE. Transport characteristics of mammalian Rh and Rh glycoproteins expressed in heterologous systems. Transfus Clin Biol 2006;13:132-8. 57. Kumpel BM. Efficacy of RhD monoclonal antibodies in clinical trials as replacement therapy for prophylactic anti-D immunoglobulin: More questions than answers. Vox Sang 2007;93:99-111. 58. Natvig JB, Forre O, Michaelsen TE. Restriction of human immune antibodies to heavy-chain variable subgroups. Scand J Immunol 1976;5:667-75. 59. Natvig JB, Kunkel HG, Rosenfeld RE, et al. Idiotypic specificities of anti-Rh antibodies. J Immunol 1976;116:1536-8. 60. Forre O, Natvig JB, Michaelsen TE. Cross-idiotypic reactions among anti-Rh (D) antibodies. Scand J Immunol 1977;6:997-1003. 61. Thompson KM, Sutherland J, Barden G, et al. Human monoclonal antibodies against blood group antigens preferentially express a VH4-21 variable region gene-associated epitope. Scand J Immunol 1991;34:509-18. 62. Bye JM, Carter C, Cui Y, et al. Germline variable region gene segment derivation of human monoclonal anti-Rh(D) antibodies. J Clin Invest 1992;90:2481-90. 63. Chang TY, Siegel DL. Genetic and immunological properties of phage-displayed human anti-Rh(D) antibodies: Implications for Rh(D) epitope topology. Blood 1998;91:3066-78. 64. Perera WS, Moss MT, Urbaniak SJ. V(D)J germline gene repertoire analysis of monoclonal D antibodies and the implications for D epitope specificity. Transfusion 2000;40:846-55. 65. Siegel DL, Czerwinski M, Spitalnik SL. Structural analysis of monoclonal antibodies to blood group antigens. Transfus Clin Biol 2002;9s:83-97. 66. Dohmen SE, Mulder A, Verhagen OJ, et al. Production of recombinant Ig molecules from antigen-selected single B cells and restricted usage of Ig-gene segments by anti-D antibodies. J Immunol Methods 2005;298:9-20.

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67. Dohmen SE, Verhagen OJ, de Groot SM, et al. The analysis and quantification of a clonal B cell response in a hyperimmunized antiD donor. Clin Exp Immunol 2006;144:223-32. 68. Andersen PS, Haahr-Hansen M, Coljee VW, et al. Extensive restrictions in the VH sequence usage of the human antibody response against the Rhesus D antigen. Mol Immunol 2007;44:412-22. 69. Siegel DL, Chang TY. Epitope migration: Anti-Rh(D) antibodies as a model for human immunogenicity (abstract). Blood 1998;92(Suppl):671a. 70. Hughes-Jones NC, Bye JM, Gorick BD, et al. Synthesis of Rh Fv phage-antibodies using VH and VL germline genes. Br J Haematol 1999;105:811-6. 71. St-Amour I, Proulx C, Lemieux R, Bazin R. Modulations of antiD affinity following promiscuous binding of the heavy chain with naive light chains. Transfusion 2003;43:246-53. 72. Dohmen SE, Verhagen OJ, Muit J, et al. The restricted use of IGHV3 superspecies genes in anti-Rh is not limited to hyperimmunized anti-D donors. Transfusion 2006;46:2162-8. 73. Wagner FF, Ladewig B, Flegel WA. The RHCE allele ceRT: D epitope 6 expression does not require D-specific amino acids. Transfusion 2003;43:1248-54. 74. Chang T. Towards a quantitative model of immunogenicity: Counting pathways in sequence space. J Theor Biol 2000;206:255-78. 75. Boucher G, Broly H, Lemieux R. Restricted use of cationic germline VH gene segments in human Rh(D) red cell antibodies. Blood 1997;89:3277-86. 76. Abelson N, Rawson A. Studies of blood group antibodies. II. Fractionation of Rh antibodies by anion-cation cellulose exchange chromatography. J Immunol 1959;83:49-56. 77. Mollison PL, Engelfreit CP, Contreras M. Blood transfusion in clinical medicine. 10th ed. Oxford: Blackwell, 1997. 78. Silberstein LE, Jefferies LC, Goldman J, et al. Variable region gene analysis of pathologic human autoantibodies to the related i and I red blood cell antigens. Blood 1991;78:2372-86. 79. Pascuel V, Victor K, Leisz D, et al. Nucleotide sequence analysis of the V regions of two IgM cold agglutinins: Evidence that the VH421 gene segment is responsible for the major cross-reactive idiotype. J Immunol 1991;146:4385-91. 80. Bailly P, Tontti E, Hermand P, et al. The red cell LW blood group protein is an intercellular adhesion molecule which binds to CD11/ CD18 leukocyte integrins. J Immunol 1995;25:3316-20.

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81. Hermand P, Gane P, Mattei MG, et al. Molecular basis and expression of the LWa/LWb blood group polymorphism. Blood 1995;86:1590-4. 82. Hermand P, Le Pennec PY, Rouger P, et al. Characterization of the gene encoding the human LW blood group protein in LW⫹ and LW⫺ phenotypes. Blood 1996;87:2962-7. 83. Bailly P, Hermand P, Callebaut I, et al. The LW blood group glycoprotein is homologous to intercellular adhesion molecules. Proc Natl Acad Sci U S A 1994;91:5306-10. 84. Bailly P, Tontti E, Hermand P, et al. The red cell LW blood group protein is an intercellular adhesion molecule which binds to CD11/CD18 leukocyte integrins. Eur J Immunol 1995;25:3316-20. 85. Hermand P, Huet M, Callebaut I, et al. Binding sites of leukocyte beta 2 integrins (LFA-1, Mac-1) on the human ICAM-4/LW blood group protein. J Biol Chem 2000;275:26002-10. 86. Spring FA, Parsons SF, Ortlepp S, et al. Intercellular adhesion molecule-4 binds alpha(4)beta(1) and alpha(V)-family integrins through novel integrin-binding mechanisms. Blood 2001;98:458-66. 87. Hermand P, Gane P, Huet M, et al. Red cell ICAM-4 is a novel ligand for platelet-activated alpha IIbbeta 3 integrin. J Biol Chem 2003;278:4892-8. 88. Ihanus E, Uotila L, Toivanen A, et al. Characterization of ICAM-4 binding to the I domains of the CD11a/CD18 and CD11b/CD18 leukocyte integrins. Eur J Biochem 2003;270:1710-23. 89. Bony V, Gane P, Bailly P, Cartron JP. Time-course expression of polypeptides carrying blood group antigens during human erythroid differentiation. Br J Haematol 1999;107:263-74. 90. Lee G, Lo A, Short SA, et al. Targeted gene deletion demonstrates that the cell adhesion molecule ICAM-4 is critical for erythroblastic island formation. Blood 2006;108:2064-71. 91. Mankelow TJ, Spring FA, Parsons SF, et al. Identification of critical amino-acid residues on the erythroid intercellular adhesion molecule-4 (ICAM-4) mediating adhesion to alpha V integrins. Blood 2004;103:1503-8. 92. Zennadi R, Hines PC, De Castro LM, et al. Epinephrine acts through erythroid signaling pathways to activate sickle cell adhesion to endothelium via LW-alphavbeta3 interactions. Blood 2004;104:3774-81.

8

Other Protein Blood Groups Petr Jarolim Director, Clinical Chemistry, Brigham and Women’s Hospital and the Dana Farber Cancer Institute, and Associate Professor of Pathology, Harvard Medical School, Boston, Massachusetts, USA

Of the 29 currently known blood group systems, only seven have antigens formed by carbohydrates. Antigens of the remaining 22 blood group systems are carried by proteins. Two of these 22 systems—Rh (ISBT 004) and the closely associated LW (ISBT 016)—were discussed in detail in the previous chapter. The remaining blood group systems are summarized in Table 8-1. Many of the proteins carrying blood group antigens are functionally important; however, antibodies in only a few blood group systems represent a problem for the transfusion service. This chapter addresses only those antigens that elicit formation of clinically significant antibodies and briefly comments on the other blood group systems. Further details may be found in review articles2,3 and comprehensive textbooks.4-6

Kell and Kx Blood Group Systems (ISBT 006 and 019) Structure, Function, and Interaction of the Kell and XK Proteins Antigens of the Kell blood group system are carried by a 93-kD red cell membrane glycoprotein.7 The glycoprotein contains a short cytoplasmic N-terminal portion, a single membranespanning α-helical segment, and a large, 665 amino acid extracellular C-terminal portion held in a globular conformation by multiple disulfide bonds (Fig 8-1). Kell antigens are inactivated by reducing agents such as dithiothreitol, suggesting that disulfide bonds are important in maintaining its antigenic conformation.8 Proteolytic cleavage with chymotrypsin or trypsin also destroys Kell antigens. The Kell protein is a member of the neprilysin (M13) family of zinc metalloproteases. This family consists of Kell, neutral endopeptidase 24.11, two different endothelin-converting

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

enzymes, the product of the PEX gene, and XCE.9,10 Members of the M13 subfamily of membrane zinc endopeptidases have widely different roles, including processing of opioid peptides, Met- and Leu-enkephalin, oxytocin, bradykinin, angiotensin, endothelins, and parathyroid hormone. Kell protein has been shown to preferentially activate endothelin-311; however, the in-vivo physiologic role of Kell protein is probably complex, because K0 (null) persons are healthy. The Kell protein interacts in the membrane with the 37-kD protein XK, which plays an important role in the expression of Kell system antigens. In contrast to the Kell protein, XK spans the membrane 10 times and both of its N- and C-termini are located intracellularly (Fig 8-1). The function of XK is not known. Structurally, XK resembles the glutamate transporters but it has very little amino acid sequence homology with this group of transport proteins. The Kell and XK proteins are covalently associated in the membrane by a disulfide link between cysteine 72 of Kell and cysteine 347 of XK12-14 (Fig 8-1). The gene encoding the Kx antigen is located on the short arm of the X chromosome near the loci for X-linked chronic granulomatous disease (CGD) and Duchenne muscular dystrophy (DMD).

Kell in Transfusion Medicine The Kell blood group system is the second most important protein blood group system in transfusion medicine after Rh, because the antibodies can cause hemolytic transfusion reactions (HTRs) and hemolytic disease of the fetus and newborn (HDFN). The most important antigens in this system are KEL1, or K, and the antithetical KEL2, or k. K and k are codominant autosomal alleles; approximately 9% of Whites and 2% of Blacks are K positive (ie, KK or Kk); the remainder are K negative (ie, kk). Antigens of the Kell blood group system are highly immunogenic and, excluding ABO, K are second only to RhD in its potential to elicit production of alloantibodies. K antibodies are commonly found. Fortunately, because more than 90% of donor units are K negative, it is easy to obtain blood for transfusion to individuals with anti-K. In contrast, although anti-k is relatively rare, it is also of clinical significance, and only

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Table 8-1. Overview of the Protein Blood Group Systems Other than Rh and LW ISBT Number

System Name

Gene Name ISBT

Gene Name ISGN

Red Cell Membrane Component

Number of Antigens*

Examples of Antigens

002 005 006 008 009 010 011 012 013 014 015 017 019 020 021 022 023 024 025 026 029

MNS Lutheran Kell Duffy Kidd Diego Cartwright Xg Scianna Dombrock Colton Chido/Rodgers Kx Gerbich Cromer Knops Indian OK RAPH JMH GIL

MNS LU KEL FY JK DI YT XG SC DO CO CH/RG XK GE CROM KN IN OK RAPH JMH GIL

GYP, GYPB LU KEL DARC SLC14A1 SLC4A1 ACHE XG, MIC2 ERMAP ART4 AQP1 C4A, C4B XK GYPC CD55 CR1 CD44 BSG MER2 SEMA7A AQP3

Glycophorin A (CD235a) and glycophorin B (CD235b) Lutheran glycoprotein (CD239) Kell glycoprotein (CD258) Fy glycoprotein (chemokine receptor) (CD234) Kidd glycoprotein (urea transporter) Band 3, anion exchanger 1 (CD233) Acetylcholinesterase Xga glycoprotein (CD99) Sc glycoprotein (ERMAP) Do glycoprotein (ART4) Aquaporin-1 Complement component C4A/C4B Xk glycoprotein Glycophorin C (CD236) and glycophorin D Decay accelerating factor (CD55) Complement receptor 1 (CD35) Hermes antigen (CD44) Neurothelin, basoglin (CD147) Unknown Semaphorin 7A (CD108) Aquaporin 3

46 18 31 6 3 20 2 1 7 5 3 9 1 7 15 9 4 1 1 5 1

M, N, S, s, U Lua, Lub K, k, Jsa, Jsb, Kpa, Kpb Fya, Fyb Jka, Jkb Dia, Dib, Wra, Wrb Yta, Ytb Xga Sc1, Sc2, Sc3, Rd Doa, Dob Coa, Cob Ch1, Ch2, Ch3, Rg1, Rg2 Kx Ge2, Ge3, Ge4 Cra Kna Ina, Inb Oka MER2 JMH1 GIL

*Based on the ISBT Cape Town report.1 ISBT ⫽ International Society of Blood Transfusion; ISGN ⫽ International Society for Gene Nomenclature.

1 in 500 random-donor units is antigen negative. The other two sets of well-defined antithetical antigens are Kpa/Kpb and Jsa/Jsb (Fig 8-1). Mothers with anti-K are relatively rare, but since the introduction of Rh prophylaxis, anti-K accounts for nearly 10% of cases of severe HDFN. In contrast to RhD, anti-K titers are not good predictors of fetal anemia. In addition, affected Kellalloimmunized infants have lower reticulocyte counts and amniotic fluid bilirubin concentrations than RhD-sensitized infants. Anti-K has been hypothesized to suppress erythropoiesis at the progenitor cell level. It is important to determine if a fetus is at risk when the mother has anti-K. The prospective father should be typed and if he carries the K antigen, genotyping from amniocentesis can be performed using molecular techniques.

the Kx antigen exhibit depressed levels of the Kell protein.7,8 This phenotype, known as the McLeod syndrome, is associated with acanthocytic red cells and a mild chronic hemolytic anemia. The frequently reported neuromuscular symptoms in persons with McLeod phenotype may be caused by lack of Kx expression. Association of the McLeod phenotype with other rare syndromes, most frequently with CGD and DMD or Becker muscular dystrophy (BMD) is caused by large gene deletions encompassing XK together with adjacent genes CYBB that encodes a large subunit of cytochrome b558 and is associated with X-linked CGD, and DMD that encodes dystrophin.15,16

Kell Variants

Structure and Function of the Duffy Protein

There are two rare but clinically interesting variants in the Kell blood group system. Rare individuals have red cells that completely lack the common Kell antigens. Although these cells exhibit the null phenotype (K0), they are morphologically normal and survive normally in vivo. In contrast, individuals lacking

The Duffy gene encodes a glycoprotein of 336 amino acids with a molecular weight of 36 kD. The Duffy glycoprotein has seven transmembrane α-helical domains. The N-terminus is ectoplasmic and the C-terminus is intracellular (see Fig 8-2). Duffy protein functions as a chemokine receptor and is frequently

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Duffy Blood Group System (ISBT 008)

Chapter 8: Other Protein Blood Groups

COOH

Jsa/Jsb

Fya/Fyb

HELLH

Kpa/Kpb K/k C347

C72 Duffy

Jka/Jkb NH2 XK

Kell

Figure 8-1. Schematic representation of the Kell/XK complex in the red cell membrane. The XK protein is a membrane protein with 10 α-helical transmembrane segments, while Kell has only one transmembrane domain, most of which is exposed on the ectoplasmic side. Due to multiple disulfide bonds, the ectoplasmic portion of Kell is a globular structure; however, it is represented here schematically so that the positions of the main alleles can be shown. A disulfide bond between Cys72 of Kell and Cys347 of XK connects the two proteins. The position of the pentameric sequence HELLH is shown. Sequences HEXXH are involved in zinc binding and catalytic activity of zinc endopeptidases. The K/k polymorphism at amino acid 193 changes the consensus sequence for N-glycosylation at Asn191, which is not glycosylated in K. This difference in glycosylation may be important for the marked antigenicity of K. Positions of two additional sets of antithetical antigens Kpa/Kpb and Jsa/Jsb at amino acids 281 and 597 are indicated.

called the Duffy antigen receptor for chemokines (DARC).17,18 Duffy binds both CXC chemokines, such as interleukin-8 (IL-8) and melanocyte growth-stimulating activity (MGSA), as well as CC chemokines, such as regulated on activation, normal T-cell expressed and secreted (RANTES) and macrophage chemoattractant protein (MCP-1).17 It is currently not known why a red cell, with its limited metabolism and response to chemokine binding, would carry a significant number of chemokine receptors at its surface. One possible explanation is that the chemokine receptor acts as a scavenger for locally released chemokines. However, individuals who do not express the Duffy protein either on erythrocytes or in all tissues are phenotypically normal, suggesting that the Duffy protein is dispensable. Duffy also functions as a receptor for the human malarial parasite Plasmodium vivax, which infects the erythrocytes of Duffy-positive individuals.19 The binding site for P. vivax is located in the N-terminal ectoplasmic domain of Duffy. P. vivax

Kidd Figure 8-2. Schematic depiction of proteins carrying the Duffy and Kidd blood group antigens. The Duffy antigen receptor for chemokines (DARC) consists of seven transmembrane segments. The N-terminus is located extracellularly, functions as both the chemokine and P. vivax attachment site, and carries the Fya/Fyb polymorphism. Kidd protein has 10 transmembrane segments, both N- and C-termini are intracellular and the amino acid determining the Jka/Jkb polymorphism is located in the fourth ectoplasmic loop.

merozoites invade primarily reticulocytes, although reticulocytes and mature erythrocytes do not substantially differ in Duffy expression. This suggests that an additional receptor has to play a role in erythrocyte invasion.

Duffy in Transfusion Medicine The two main alleles of the Duffy blood group system are the antithetical codominant antigens Fya and Fyb whose genetic determinant is a Gly/Asp polymorphism in position 44 (Fig 8-2).20 These two alleles occur with similar frequencies in persons of European ancestry, Fya being somewhat more common. The Fy(a⫺b⫺) phenotype is extremely rare among Whites but approximately two-thirds of African Americans and more than 90% of native West Africans are Fy(a⫺b⫺). This is most likely caused by the genetic adaptation for resistance to P. vivax malaria. However, it is not clear why this genetic advantage would lead almost to fixation of the Fy(a⫺b⫺) phenotype

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Section I: Part I

in the indigenous population of West Africa. In contrast to the other malarial parasites, P. vivax causes a relatively mild form of malaria.17 A long-standing conundrum of immunohematology has been the well-known fact that transfused Fy(a⫺b⫺) people of African ancestry never develop anti-Fyb. This mystery was solved by the characterization of the mutation causing the Fy(a⫺b⫺) phenotype in native West Africans. The underlying mutation is a T→C substitution in the GATA site of the promoter region.21 This substitution prevents binding of the erythroid transcription factor GATA-1 and abolishes transcription of the gene in the erythroid cells, while leaving the transcription of the FY gene in other tissues unaffected. Because the mutation occurred in the FY*B allele, the Fyb antigen remains expressed in certain endothelial, epithelial, and brain cells, and, consequently, the transfusion recipient does not form antibodies against Fyb. Fya antibodies are found relatively frequently, constituting 6% to 10% of the clinically significant antibodies identified by immunohematology laboratories. For reasons not well understood, Fyb is a relatively poor immunogen and, consequently, anti-Fyb is considerably less common. Both immediate and delayed HTRs caused by Fya incompatibility have been described, ranging from mild to severe hemolysis. HDFN is usually mild; only a few cases of severe HDFN have been reported. In contrast, anti-Fyb is associated only rarely with cases of mild HDFN and is usually found in delayed HTRs, although on rare occasions it has caused severe acute hemolysis.

Kidd Blood Group System (ISBT 009) The two main antigens of the Kidd blood group system, Jka and Jkb, are found with almost identical frequencies in White populations. Jka is a better immunogen and anti-Jka is found more frequently than anti-Jkb. Anti-Jka may cause severe immediate or delayed HTRs and, occasionally, HDFN. It is one of the most dangerous immune antibodies because of its tendency to decrease to undetectable levels in between transfusions and its relatively low affinity for Jka-positive red cells. For these reasons, it accounts for a large proportion of delayed HTRs. Anti-Jkb may also cause immediate or delayed HTRs, albeit less severe than those caused by anti-Jka. Several cases of mild HDFN caused by anti-Jkb have been reported. The finding that cells of the Jk(a⫺b⫺) phenotype are resistant to lysis by 2M urea led to the discovery of the function of the protein carrying the Kidd antigens. Based on this finding and on in-vitro expression of the cloned Kidd cDNA,22-24 it is now known that the protein is a urea transporter, although the importance of urea transport for erythrocytes is not completely understood. Its presence or absence may not be critical for red cell structure and function, because carriers of the Jk(a⫺b⫺) phenotype have red cells indistinguishable from those of controls. Kidd is an integral protein with 10 transmembrane domains and both N- and C-termini located intracellularly (Fig 8-2). Of the five ectoplasmic loops, the longest, third loop is

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N-glycosylated, and the relatively short fourth loop carries an Asp280Asn polymorphism corresponding to the Jka/Jkb antigens.25 Kidd protein is expressed not only on the red cell surface but also on neutrophils and in the kidney.

MNS Blood Group System (ISBT 002) Structure and Function of Glycophorins A and B Antigens of the MNS blood group system are carried by glycophorin A (GPA) and glycophorin B (GPB). Both molecules are present in a very high copy number in the plasma membrane; 0.5-1.0 ⫻ 106 copies of glycophorin A and 1-3 ⫻ 105 molecules of glycophorin B. Glycophorins A and B are encoded by homologous genes at chromosome 4q28-q31 that undoubtedly arose by gene duplication. Both glycoproteins are integral membrane proteins with a single transmembrane α-helical segment and with the N-termini located extracellularly (Fig 8-3). Glycophorin A was the first protein whose primary structure was determined by amino acid sequencing.26 GPA carries the M and N antigens. The antigenic difference is caused by substitutions in position 1 and 5. Serine in position 1 and glycine in position 5 correspond to the M antigen, while the N allele has leucine and glutamic acid in these positions (Fig 8-3 and Table 8-2). An important requirement for recognition of these antigens by human antibodies is that serines in positions 2 and 4 and threonine in position 3 are O-glycosylated. GPB is homologous with GPA. Although the genes are greater than 95% identical, GYPB encodes a shorter protein because a point mutation at the 5⬘ splicing site of the third intron prevents incorporation of exon 3 into the translated mRNA. Because GYPB arose by duplication of the N allele of GYPA, GYPA*N, and the first 26 amino acids of GPB are therefore identical to those of GPA with N specificity, GPB expresses an N-like antigen designated as ‘N’ (Table 8-2 and Fig 8-3). The S and s alleles of GPB differ at amino acid position 29. The S allele contains methionine and the s allele, threonine. GPB also carries the U antigen whose epitope is adjacent to the point where GPB enters into the lipid bilayer (Fig 8-3). As is frequently the case with genes arising by duplication and located next to each other, unequal crossing over or gene conversion may easily occur. Consequently, numerous hybrid molecules containing portions of GPA and GPB have been described. This phenomenon is partly responsible for the 46 currently known antigens of the MNS blood group system, particularly for many of the Miltenberger (Mi) variants. GPA associates in the red cell membrane with the band 3 protein. The epitope of the Wrb antigen from the Diego blood group system (see below) is formed by both GPA and band 3. This clearly demonstrates the intimate association of GPA and band 3 in the plasma membrane.27

MNS in Transfusion Medicine The most commonly encountered antibodies are directed against the M, N, S, and s antigens. Anti-M is often found in the sera

Chapter 8: Other Protein Blood Groups

M/N ‘N’ Vga

Swa

Wrb

Wda

WARR Tra

S/s

Rba Fr

ELO

Ina Wu,NFLD Bpa

a

Hga,Moa

U

Dia

Out

In

N-terminus C-terminus Band 3

GPA

GPB

Figure 8-3. Antigens of the Diego and MNS blood group systems in a scheme of band 3 and of glycophorins A and B. The membrane domain of band 3 with 14 transmembrane segments is shown. Mutations underlying the Diego blood group antigens are located in the putative first, second, third, fourth, and seventh ectoplasmic loops. Positions of the M and N antigens in GPA and of the ‘N’, S, s, and U antigens in GPB are indicated. Arrows point to the sites in band 3 and GPA that are involved in formation of the Wrb epitope and, therefore, have to come into close contact in the membrane.

Table 8-2. N-Terminal Sequences of the M and N Alleles of Glycophorin A (GPA) and of the S and s Alleles of Glycophorin B (GPB)

Table 8-3. Comparison of the Most Frequently Encountered Antibodies in the MNS System

Antigen

Amino Acid Sequence

Anti-M and -N

Anti-S, -s, and -U

1 5 26

29

↓↓

↓↓

Naturally occurring Cold IgM Dosage Clinically insignificant

Exposure is required Warm IgG Minimal dosage Clinically significant

M (GPA)

SSTTGVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRA..

N (GPA)

LSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRA..

S (GPB)

LSTTEVAMHTSTSSSVTKSYISSQTNGEMGQLVHRFTVPA..

S (GPB)

LSTTEVAMHTSTSSSVTKSYISSQTNGETGQLVHRFTVPA.. ⬍---⬎

↑

M/N;’ N’

S/s

Differences between M and N reside in amino acids 1 and 5. First 26 amino acids of GPB are identical to GPA and GPB therefore expresses an N-like antigen ‘N’. The differences between the S and s antigens correspond to a M/T polymorphism in position 29 of GPB. S ⫽ serine; L ⫽ leucine; G ⫽ glycine; E ⫽ glutamic acid; M ⫽ methionine; T ⫽ threonine.

of persons who have not been exposed to human red cells. M antibodies are mostly IgM; however, they frequently contain an IgG component and, occasionally, are exclusively IgG. Anti-M is rarely clinically significant; hemolytic anti-M is usually IgG and reactive at 37ºC. Anti-N is rare, most likely because of the immune tolerance induced by the ‘N’ antigen on GPB. Strong and potentially clinically significant antibodies have been observed in persons of the rare phenotype M⫹N–S–s–U– who do not express GPB (hence ‘N’). In contrast to anti-M and anti-N, antibodies to S, s, and U usually occur after exposure to

allogeneic red cells. All are capable of causing HTRs and HDFN. The most important antibodies of the MNS system are compared in Table 8-3.

Diego Blood Group System (ISBT 010) Antigens of the Diego blood group system are carried by erythrocyte band 3 protein (anion exchanger 1), the most abundant integral protein of the red cell membrane together with GPA (see above). Band 3 is also one of the most important proteins for the structure and function of the membrane because it maintains red cell integrity by linking the red cell membrane to the underlying spectrin-based membrane skeleton. It also mediates exchange of chloride and bicarbonate anions across the plasma membrane, thereby significantly increasing the carrying capacity of blood for carbon dioxide. Band 3 consists of a cytoplasmic and a membrane domain. The membrane domain contains 14 transmembrane helices

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Section I: Part I

connected by ecto- and endoplasmic loops28,29 (Fig 8-3). The fourth loop of band 3 is N-glycosylated and the attached carbohydrate chain carries over half of the red cell ABO blood group epitopes.30 Several disorders of red cell structure and function have been associated with mutations in the band 3 gene, including Southeast Asian ovalocytosis,31 autosomal dominant spherocytosis,32-34 and distal renal tubular acidosis.35 Despite being the most abundant protein of the red cell membrane, band 3 until recently has not been known to carry any red cell antigens with the exception of the ABO antigens residing on the attached carbohydrate chains. It was only in 1992 that Spring et al36 reported that the Memphis II variant of erythroid band 3 protein carries the Dia blood group antigen. Dia was originally described in South American Indians by Layrisse et al in 1955.37 The antithetical antigen Dib was reported by Thompson et al in 1967.38 Dia and Dib represent codominantly expressed gene products. Dia is a low-incidence blood group antigen in persons of European ancestry who carry the antithetical high-incidence antigen Dib. Prevalence of Dia is as high as 8% in certain areas of Southeast Asia and reaches up to 40% in some groups of South American Indians.37 Dia was used as one of the markers for studying migration of people from Southeast Asia across the Bering Strait and southward through the American continents. Cloning and sequencing of band 3 gene identified the substitution 854 Pro→Leu in the last ectoplasmic loop of band 3 as the molecular basis of the Dia antigen. Dib corresponds to the wild type band 3 with proline in position 854.39 Subsequently, the low-incidence blood group antigen Wra was mapped to the fourth ectoplasmic loop.27 The antithetical Wrb antigen is seen only when both GPA and band 3 protein are expressed in the erythrocyte membrane, which helped to characterize the site of interaction between band 3 and GPA (Fig 8-3). During the last several years, numerous additional lowincidence antigens have been associated with single point mutations on band 3 and included in the Diego system.40,41 Positions of the mutated amino acids in the band 3 molecule are shown in Fig 8-3, which also indicates the regions of band 3 and glycophorin A that interact in the membrane and are involved in formation of the Wrb antigen. Some antigens of the Diego blood group system have been localized to the regions of band 3 protein that have been implicated in the adhesion of abnormal red cells, such as sickle cells or malaria-infected erythrocytes, to vascular endothelium.42,43 Erythrocytes from carriers of low-incidence blood group antigens in ectoplasmic loops of band 3 may serve as a model for evaluation of the sequence requirements for adhesion. The so-called “senescent” or “aging” red cell antigen may also be located in the ectoplasmic loops of band 3.

Gerbich Blood Group System (ISBT 020) As in the MNS system, antigens of the Gerbich system are located on glycophorins C and D. The glycophorin terminology is in fact

126

the only common feature of these two classes of glycophorins. There is otherwise no homology between the Gerbich and MNS genes. Unlike GPA and GPB, GPC and GPD are the products of a single gene and, unlike GYPA and GYPB, this gene is expressed in multiple tissues. The considerably less abundant and smaller GPD is produced from a shorter transcript than GPC. Although present in much smaller copy numbers than GPA and GPB, GPC plays an important role in the structural integrity of the red cell membrane. In the Leach phenotype, deletions of exons 3 and 4 or a frameshift mutation lead to complete absence of glycophorins C and D from the plasma membrane. The affected individuals have moderate elliptocytosis and decreased red cell deformability and mechanical stability. Antibodies in the Gerbich system are rare and, in the vast majority of cases, clinically insignificant.

Colton and GIL Blood Group Systems (ISBT 015 and 029) Antigens of these two blood group systems are carried by members of the large aquaporin family. Antibodies against the two antigens of the Colton blood group system, the high-frequency Coa and the less common, antithetical Cob, are rare and have only rarely been associated with mild HTRs and mild HDFN. These two antigens, and the Co3 antigen that is present on all red cells except those of the very rare null phenotype Co(a⫺b⫺), are carried by aquaporin-1,44 a member of a large family of water channels.45 It is present in the membrane as a tetramer. Expression of aquaporin-1 in Xenopus oocytes is associated with dramatic swelling and lysis of the cell.46 At the same time, the Co(a⫺b⫺) phenotype is associated with only slightly abnormal red cells and with normal kidney function despite the fact that aquaporin-1 is the major water channel of human kidney.47,48 The GIL antigen is carried by aquaporin-3, which differs from aquaporin-1 in that it transports glycerol, water, and urea.49

Lutheran Blood Group System (ISBT 005) The Lutheran blood group system contains multiple antigens; however, clinically significant antibodies are rarely encountered. The most important antigens are Lua and Lub. The frequency of Lua is less than 10% in most populations, while Lub is a highfrequency antigen with an average prevalence of 99.8% in all populations. Lutheran antigens are poorly developed at birth and, not surprisingly, anti-Lua has been associated only rarely with mild cases of HDFN. It does not cause transfusion reactions. Lub is somewhat more immunogenic and anti-Lub has caused mild or moderate HTRs and mild HDFN. Of historical note, Lu and Se (Chapter 6) were the first two loci for which an autosomal linkage in humans was demonstrated. Lutheran antigens reside on B-CAM/LU (Fig 8-4), a pair of spliceosomes (protein products arising from the same gene because of alternative splicing of hnRNA) that belong to the

Chapter 8: Other Protein Blood Groups

nonadherent cell lines with CD44 cDNA confers an adherent phenotype.55 As with the Lutheran antigens, expression of Ina and Inb is suppressed by In(Lu).

B-CAM/LU (Lutheran)

Xg Blood Group System (ISBT 012) CD147 (Oka)

LW

The only antigen of the Xg system, Xga, is carried by a glycoprotein of 180 amino acids.56 The function of Xg glycoprotein in erythrocytes is not known. Designation of this blood group system reflects the fact that the PBDX gene,57 which encodes Xg, is located on the X-chromosome. Xg is 48% homologous to CD99, an adhesion molecule. Xga antibodies are clinically insignificant.

Scianna Blood Group System (ISBT 013)

Figure 8-4. Members of the immunoglobulin superfamily carrying blood group antigens. B-CAM/LU contains five immunoglobulin domains and carries antigens of the Lutheran blood group system. CD147, or neurothelin, contains two Ig domains and carries the Oka antigen. CD147 is similar to the LW protein, which was discussed in Chapter 7.

The seven antigens of the Scianna blood group system are carried by the erythrocyte membrane-associated protein (ERMAP), potentially a receptor/signal transduction molecule specific for erythroid cells.58 Mild HDFN and mild postransfusion hemolysis caused by anti-Sc-2 and anti-Sc-3 antibodies have been reported.

Chido/Rodgers Blood Group System (ISBT 017) immunoglobulin superfamily.50,51 Basal cell adhesion molecule (B-CAM) is involved in adhesion of the basal surface of epithelial cells to the basement membrane. B-CAM/LU is a receptor for laminin.52 Expression of B-CAM/LU is increased on red cells from patients with sickle cell disease52 and on a number of malignant epithelial tumors, which also lose the polarity of B-CAM/LU expression found in normal tissues.51 The null phenotype, Lu(a⫺b⫺), is rare but quite interesting. It may result from three different patterns of inheritance. A recessive pattern of inheritance is associated with an exceedingly rare null allele. In the most common dominant type of inheritance, the expression of Lu can be suppressed by a single copy of the suppressor gene In(Lu). In addition to reducing the expression of Lutheran antigens to levels undetectable by standard serologic techniques, In(Lu) also decreases the expression of antigens P1, i, Ina and Inb (see below), and AnWj, a receptor for Haemophilus influenzae. The third cause of the Lu(a⫺b⫺) phenotype is the presence of an X-linked recessive suppressor gene XS2.53

Indian Blood Group System (ISBT 023) The four antigens of this system, Ina, Inb, INFI, and INJA reside on CD44, an adhesion molecule expressed in leukocytes, fibroblasts, epithelial cells, and other tissues. CD44 is an important lymphocyte marker that functions as a hyaluronan receptor,54 and a lymphocyte homing receptor (Fig 8-4). Transfection of

Antigens of the Chido/Rodgers blood group system are the only protein antigens that are not produced by red cells but instead adhere to the red cell surface (Lewis antigens are glycolipids). They are carried by the complement component C4. Although antibodies against the nine known antigens of the system are generally benign, a severe anaphylactic reaction following a transfusion of platelets to a patient with anti-Ch3 has been described.59

Knops Blood Group System (ISBT 022) Antigens are located on the C3b/C4b complement receptor 1 (CR1, CD35). CR1 protects erythrocytes from autohemolysis by inhibiting the classical and alternative complement pathways through cleavage of C4b and C3b. CR1 is a large 190- to 280-kD molecule. It contains 30 complement control protein domains (CCPD) of about 60 amino acids. Seven CCPDs form a long homologous repeat (LHR) of about 450 amino acids. Various forms of CR1 contain up to 6 LHRs. Erythrocyte CR1 binds immune complexes and carries them to the liver and spleen for removal. Expression of CR1 on erythrocytes varies widely from 20 to 1500 molecules and is decreased in hemolytic anemias, AIDS, systemic lupus erythematosus, and other autoimmune disorders. Plasmodium falciparum-infected erythrocytes deficient in CR1 have greatly reduced rosetting capacity, indicating an essential role for CR1 in rosette formation and raising the possibility that CR1 polymorphisms in Africans that influence the interaction between erythrocytes and parasite-encoded protein

127

Section I: Part I

PfEMP1 may protect against severe malaria.60 CR1 could therefore be a potential target for future therapeutic interventions to treat severe malaria.61,62

Blood Group Antigens on PhosphatidylinositolLinked Proteins—Cartwright (ISBT 011), Dombrock (ISBT 014), Cromer (ISBT 021), and JMH (ISBT 026) The common denominator of these antigens is the linkage of the carrier protein to the glycosylphosphatidylinositol (GPI) anchor (Fig 8-5). The Cartwright (Yt) blood group system consists of two antigens, Yta and Ytb, which are located on red cell acetylcholinesterase. Most examples of anti-Yta are benign. The five antigens of the Dombrock system are located on a recently characterized glycoprotein.63 Homology studies suggest that the Dombrock molecule is a member of the adenosine 5⬘-diphosphate (ADP)-ribosyltransferase ectoenzyme gene family. Dombrock expression is developmentally regulated during erythroid differentiation and occurs at highest levels in the fetal liver. Doa and Dob antigens differ in a single amino acid substitution within the RGD motif of the molecule.63,64 The Cromer blood group system contains 15 antigens located on decay accelerating factor (DAF, CD55), a complement regulatory protein. Although DAF is the first complement regulatory protein identified, it plays only a minor role in complementmediated lysis, the more important being the MIRL (CD59) molecule. This was clearly demonstrated in the case of the null

phenotype, Inab, which is associated with lack of DAF expression on all circulating cells but not with increased hemolysis.65 The only antigen of the JMH blood group system resides on a GPI-linked protein semaphorin 7A (CD108) that is part of a plasma membrane complex associated with intracellular protein kinases.66 CD108 is expressed in multiple tissues and may play a role in signal transduction. Semaphorin 7A is a surprisingly diverse molecule67 and four new antigens have been recently added to the JMH blood group system.1 Antibodies in these four blood group systems have been associated only occasionally with mild HTRs or HDFN. Expression of all GPI-linked antigens is, not surprisingly, decreased in paroxysmal nocturnal hemoglobinuria, a disorder caused by defects in the X-linked phosphatidylinositol glycan class A (PIG-A) gene, which participates in an early step of GPI anchor synthesis.

OK and RAPH Blood Group System (ISBT 024 and 025) Each of these two blood group systems contains only one antigen. Oka is a high-incidence antigen; rare Ok(a⫺) individuals have so far been reported only in Japan. Oka is carried on an N-glycosylated protein with an apparent molecular weight of 35 to 69 kD, which is expressed in multiple tissues. The Ok glycoprotein is a member of the immunoglobulin superfamily. As with LW, its extracellular domain contains two immunoglobulin domains. The function of the Ok glycoprotein in erythrocytes is not known. MER2 is the only antigen of the RAPH system. All MER2 antibodies so far reported have been found in patients on hemodialysis. The molecular basis of the antigen is not known.

NH2

Summary Protein

O⫽C Ethanolamine

Glycan core

N-glucosamine

Phosphatidylinositol

Figure 8-5. Schematic representation of a GPI-anchored protein. The membrane anchor is provided by phosphatidylinositol. The inositol moiety binds to a glycan core via a molecule of N-glucosamine. The glycan core is attached via ethanolamine to the C-terminus of the protein.

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The century-long history of modern transfusion medicine and immunohematology practice led to the characterization of an enormous number of blood group antigens with often confusing terminology. These antigens have been arranged into a complex framework of blood group systems, collections, and low- and high-frequency antigens. Fortunately, advances in biochemical and molecular biology techniques in the past two decades led to a detailed structural characterization of most proteins carrying blood group antigens and to a better understanding of the relation between gene variations, amino acid polymorphisms, protein structure, and immunogenicity of individual antigens. Better understanding of the molecular biology, biochemistry, and immunogenicity of proteins carrying blood group antigens will undoubtedly contribute to accurate compatibility testing and to safe transfusion of red cells.

Disclaimer The author has disclosed no conflicts of interest.

Chapter 8: Other Protein Blood Groups

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22. Olives B, Neau P, Bailly P, et al. Cloning and functional expression of a urea transporter from human bone marrow cells. J Biol Chem 1994;269:31649-52. 23. Olives B, Martial S, Mattei MG, et al. Molecular characterization of a new urea transporter in the human kidney. FEBS Lett 1996;386:156-60. 24. Olives B, Mattei MG, Huet M, et al. Kidd blood group and urea transport function of human erythrocytes are carried by the same protein. J Biol Chem 1995;270:15607-10. 25. Olives B, Merriman M, Bailly P, et al. The molecular basis of the Kidd blood group polymorphism and its lack of association with type 1 diabetes susceptibility. Hum Mol Genet 1997;6:1017-20. 26. Tomita M, Marchesi VT. Amino-acid sequence and oligosaccharide attachment sites of human erythrocyte glycophorin. Proc Natl Acad Sci U S A 1975;72:2964-8. 27. Bruce LJ, Ring SM, Anstee DJ, et al. Changes in the blood group Wright antigens are associated with a mutation at amino acid 658 in human erythrocyte band 3: A site of interaction between band 3 and glycophorin A under certain conditions. Blood 1995;85:541-7. 28. Lux SE, John KM, Kopito RR, Lodish HF. Cloning and characterization of band 3, the human erythrocyte anion-exchange protein (AE1). Proc Natl Acad Sci U S A 1989;86:9089-93. 29. Tanner MJA, Martin PG, High S. The complete amino acid sequence of the human erythrocyte membrane anion-transport protein deduced from the cDNA. Biochem J 1988;256:703-12. 30. Fukuda M, Fukuda MN. Changes in red cell surface glycoproteins and carbohydrate structures during the development and differentiation of human erythroid cells. J Supramol Structure 1981;17: 313-24. 31. Jarolim P, Palek J, Amato D, et al. Deletion in erythrocyte band 3 gene in malaria-resistant Southeast Asian ovalocytosis. Proc Natl Acad Sci U S A 1991;88:11022-6. 32. Jarolim P, Rubin HL, Liu S-C, et al. Duplication of 10 nucleotides in the erythroid band 3 (AE1) gene in a kindred with hereditary spherocytosis and band 3 protein deficiency (band 3PRAGUE). J Clin Invest 1994;93:121-30. 33. Jarolim P, Rubin HL, Barabec V, et al. Mutations of conserved arginines in the membrane domain of erythroid band 3 protein lead to a decrease in membrane-associated band 3 and to the phenotype of hereditary spherocytosis. Blood 1995;85:634-40. 34. Jarolim P, Murray JL, Rubin HL, et al. Characterization of 13 novel band 3 gene defects in hereditary spherocytosis with band 3 deficiency. Blood 1996;88:4366-74. 35. Jarolim P, Shayakul C, Prabakaran D, et al. Autosomal dominant distal renal tubular acidosis is associated in three families with heterozygosity for the R589H mutation in the AE1 (band 3) Cl-/HCO3exchanger. J Biol Chem 1998;273:6380-8. 36. Spring FA, Bruce LJ, Anstee DJ, Tanner MJA. A red cell band 3 variant with altered stilbene disulphonate binding is associated with the Diego (Dia) blood group antigen. Biochem J 1992;288:713-16. 37. Layrisse M, Arends T, Dominguez-Sisco R. Nuevo grupo sanguineo encontrado en descencientes de Indios. Acta Med Venez 1955; 3:132. 38. Thompson PR, Childers DM, Hatcher DE. Anti-Dib. First and second examples. Vox Sang 1967;13:314-18. 39. Bruce LJ, Anstee DJ, Spring FA, Tanner MJA. Band 3 Memphis variant II. Altered stilbene disulfonate binding and the Diego (Dia) blood group antigen are associated with the human erythrocyte band 3 mutation Pro854→Leu. J Biol Chem 1994;269:16155-8.

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Section I: Part I 40. Jarolim P, Murray J, Rubin H, et al. Blood group antigens Wda, Rba, and WARR are located in the third ectoplasmic loop of erythrocyte band 3 protein. Transfusion 1997;37:607-15. 41. Jarolim P, Murray JL, Rubin HL, et al. A Thr552→Ile substitution in erythroid band 3 gives rise to the Warrior blood group antigen. Transfusion 1997;37:398-405. 42. Crandall I, Collins WE, Gysin J, Sherman IW. Synthetic peptides based on motifs present in human band 3 protein inhibit cytoadherence/sequestration of the malaria parasite Plasmodium falciparum. Proc Natl Acad Sci U S A 1993;90:4703-7. 43. Thevenin BJM, Crandall I, Ballas SK, et al. Band 3 peptides block the adherence of sickle cells to endothelial cells in vitro. Blood 1997;90:4172-9. 44. Smith BL, Preston GM, Spring FA, et al. Human red cell aquaporin CHIP. I. Molecular characterization of ABH and Colton blood group antigens. J Clin Invest 1994;94:1043-9. 45. Preston GM, Agre P. Isolation of the cDNA for erythrocyte integral Membrane protein of 28 kilodaltons: member of an ancient channel family. Proc Natl Acad Sci U S A 1991;88:11110-14. 46. Preston GM, Carroll TP, Guggino WB, Agre P. Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science 1992;256:385-7. 47. Preston GM, Smith BL, Zeidel ML, et al. Mutations in aquaporin-1 in phenotypically normal humans without function CHIP water channels. Nature 1994;265:1585-7. 48. Mathai JC, Mori S, Smith BL, et al. Functional analysis of aquaporin-1 deficient red cells—The Colton-null phenotype. J Biol Chem 1996;271:1309-13. 49. Roudier N, Ripoche P, Gane P, et al. AQP3 deficiency in humans and the molecular basis of a novel blood group system, GIL. J Biol Chem 2002;277:45854-9. 50. Eyler CE, Telen MJ. The Lutheran glycoprotein: A multifunctional adhesion receptor. Transfusion 2006;46:668-77. 51. Campbell IG, Foulkes WD, Senger G, et al. Molecular cloning of the B-CAM cell surface glycoprotein of epithelial cancers: A novel member of the immunoglobulin superfamily. Cancer Res 1994;54:5761-5. 52. Udani M, Zen Q, Cottman M, et al. Basal cell adhesion molecule Lutheran protein—The receptor critical for sickle cell adhesion to laminin. J Clin Invest 1998;101:2550-8.

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53. Norman PC, Tippett P, Beal RW. An Lu(a⫺b⫺) phenotype caused by an X-linked recessive gene. Vox Sang 1986;51:49-52. 54. Aruffo A, Stamenkovic I, Melnick M, et al. CD44 is the principal cell surface receptor for hyaluronate. Cell 1990;61:1303-13. 55. St. John T, Meyer J, Idzerda R, Gallatin WM. Expression of CD44 confers a new adhesive phenotype on transfected cells. Cell 1990;60:45-52. 56. Ellis NA, Tippett P, Petty A, et al. PBDX is the XG blood group gene. Nat Genet 1994;8:285-90. 57. Ellis NA, Ye TZ, Patton S, et al. Cloning of PBDX, an MIC2-related gene that spans the pseudoautosomal boundary on chromosome Xp. Nat Genet 1994;6:394-400. 58. Velliquette RW. Review: The Scianna blood group system. Immunohematol 2005; 21:70-6. 59. Westhoff CM, Sipherd BD, Wylie DE, Toalson LD. Severe anaphylactic reactions following transfusions of platelets to a patient with anti-Ch. Transfusion 1992;32:576-9. 60. Moulds JM. A review of the Knops blood group: Separating fact from fallacy. Immunohematol 2002;18:1-8. 61. Rowe JA, Moulds JM, Newbold CI, Miller LH. P. falciparum rosetting mediated by a parasite-variant erythrocyte membrane protein and complement-receptor 1. Nature 1997;388:292-5. 62. Rowe JA, Rogerson SJ, Raza A, et al. Mapping of the region of complement receptor (CR) 1 required for Plasmodium falciparum rosetting and demonstration of the importance of CR1 in rosetting in field isolates. J Immunol 2000;165:6341-6. 63. Gubin AN, Njoroge JM, Wojda U, et al. Identification of the Dombrock blood group glycoprotein as a polymorphic member of the ADPribosyltransferase gene family. Blood 2000;96:2621-7. 64. Reid ME. Complexities of the Dombrock blood group system revealed. Transfusion 2005;45(Suppl):92S-99S. 65. Lublin DM. Cromer and DAF: Role in health and disease. Immunohematol 2005;21:39-47. 66. Mudad R, Rao N, Angelisova P, et al. Evidence that CDwl08 membrane protein bears the JMH blood group antigen. Transfusion 1995;35:566-70. 67. Seltsam A, Strigens S, Levene C, et al. The molecular diversity of Sema7A, the semaphorin that carries the JMH blood group antigens. Transfusion 2007;47:133-46.

9

Anemia and Red Blood Cell Transfusion Jeffrey L. Carson1 & Paul Hébert2 1

Richard C. Reynolds Professor of Medicine, Chief, Division of General Internal Medicine, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, New Brunswick, New Jersey, USA 2 Professor of Medicine, Surgery, Anesthesiology, and Epidemiology; University of Ottawa; Critical Care Physician, The Ottawa Hospital; Senior Scientist, Ottawa Health Research Institute Ottawa, Ontario Canada

Red cell transfusion is an extremely common medical intervention. About 75 million units of Red Blood Cells (RBCs) are collected worldwide,1 and in the United States, 15.8 million allogeneic units were donated in 2006.2 In Northern England, about 50% of units are given to medical patients, and 40% to surgical patients; hip replacement and coronary artery bypass graft (CABG) surgery were the most common surgical indications.3 RBC units are also frequently administered to critically ill patients, as a supportive therapy to patients receiving chemotherapy and marrow transplants, and to patients with blood loss from medical conditions such as gastrointestinal bleeding.4 This chapter reviews the current knowledge about red cell transfusion and the risk posed by anemia. The adaptive physiologic mechanisms in response to acute blood loss are described, as well as more chronic decreases in red cell mass. These discussions include a review of oxygen transport, adaptive mechanisms in anemia, and microcirculatory effects of anemia. Some of these fundamental principles are followed by a review of clinical outcomes in patients with anemia. Similarly, the clinical consequences (both risks and benefits) of red cell transfusion are outlined. This overview summarizes preclinical, observational, and controlled trials. Guidelines on transfusion practices are examined. The first three sections are based on systematic reviews of the literature. The fourth section describes an approach to decision making in red cell transfusion that is occasionally evidencebased, but frequently based on the authors’ views.

Physiologic Adaptations to Blood Loss and Anemia

oxygen is bound according to its partial pressure (PO2). The oxygen-binding affinity of hemoglobin is graphically represented by a sinusoidal relationship between hemoglobin oxygen saturation and PO2. This relationship, referred to as the oxyhemoglobin dissociation curve, enables both efficient loading in the lungs at high PO2 and efficient unloading in the tissues at low PO2 (Fig 9-1). However, the affinity of hemoglobin for oxygen (the degree to which oxygen molecules saturate the hemoglobin binding sites at a given PO2) is altered by various disease states and may play a significant adaptive role in the response to anemia. The amount of oxygen delivered, either to the whole body or to specific organs, is the product of blood flow and arterial oxygen content. For the whole body, oxygen delivery (DO2) is the product of total blood flow or cardiac output (CO) and arterial oxygen content (CaO2), represented by the following equation (#1): DO2
CO  CaO2 When ambient air is breathed under normal conditions, the oxygen present in arterial blood is bound to hemoglobin. When fully saturated, 1 g of hemoglobin binds 1.39 mL of oxygen. In addition, a small amount of hemoglobin also is dissolved in plasma water. The negligible amount of dissolved oxygen is directly proportional to the partial pressure and is calculated by multiplying PO2 by a constant (k
0.00301 mL/mm Hg), called the solubility coefficient. Thus, under most circumstances, arterial oxygen content can be approximated from the portion bound to hemoglobin with the following equation (#2): CaO2 (mL/L)
%Sat  1.39 (mL/g)  Hb (g/dL)

Overview of Oxygen Transport Hemoglobin is a complex molecule that consists of four globin moieties, each incorporating an iron-containing heme ring where

When CaO2 from equation 2 is substituted into equation 1, the results are as shown in the following equation (#3): DO2
CO  (%Sat  1.39  Hb)

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

Where CO is cardiac output in liters per minute, %Sat is hemoglobin oxygen saturation in percent, and Hb is hemoglobin concentration in g/dL.

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500

Left shifted Higher pH Lower temperature Decreased 2,3-DPG

80

O2 Consumption (mL/min)

% Saturation of Hemoglobin

100

Right shifted Lower pH Higher temperature Increased 2,3-DPG

60 40 20

0

25

50 75 PO2 (mm Hg)

100

125

Figure 9-1. Oxyhemoglobin dissociation curve. The P50 is a measurement of the partial pressure of oxygen when hemoglobin is 50% saturated. The solid line represents oxygen-binding affinity to the hemoglobin molecule at standard temperature (37ºC) and pH (7.4). The P50 of this curve is usually between 26 and 28 mm Hg. The dashed lines represents hypothetical shifts in the curve. A rightward shift represents a decreased affinity of hemoglobin for oxygen or an increase in P50. It may be caused by increased levels of 2,3-diphosphoglycerate (2,3-DPG), temperature, or decreased pH. A shift to the left represents an increased affinity of oxygen for hemoglobin and is measured by a decrease in P50. A leftward shift may result from decreased temperature, levels of 2,3-DPG or increased pH.

Tissue hypoxia and anoxia eventually occur if oxygen delivery decreases to a level at which it is no longer adequate to meet the metabolic demands of the tissues. From Equations 1 and 3, it is apparent that tissue hypoxia can be caused by decreased oxygen delivery resulting from decreases in either hemoglobin concentration (anemic hypoxia), cardiac output (stagnant hypoxia), or hemoglobin saturation (hypoxic hypoxia). Each of the determinants of DO2 has substantial physiologic reserves, thereby enabling the human body to adapt to significant increases in oxygen requirements or decreases in one of the determinants of DO2 as a result of various diseases. In health, the amount of oxygen delivered to the whole body exceeds resting oxygen requirements twofold to fourfold. For example, with a hemoglobin level of 15 g/dL, a hemoglobin oxygen saturation of 99%, and a cardiac output of 5 L/minute, oxygen delivery is 1032 mL/minute. At rest, the amount of oxygen required or consumed by the whole body ranges from 200 to 300 mL/minute. A decrease in hemoglobin concentration to 10 g/dL would result in an oxygen delivery of 688 mL/minute. Despite this 33% decrease in oxygen delivery, there remains a twofold excess of oxygen delivered compared with oxygen consumed. However, a further decrease in hemoglobin concentration to 5 g/dL with all other parameters (including cardiac output) remaining constant, decreases oxygen delivery to a critical level of 342 mL/minute. Under stable experimental conditions, this dramatic decrease in oxygen delivery still exceeds oxygen consumption. However, at the critical level or threshold of oxygen— DO2(crit)—oxygen delivery equals oxygen consumption. Further

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400 300 200 100 0

0

Anaerobic threshold

0

200

400 600 800 O2 Delivery (mL/min)

1000

1200

Figure 9-2. Relationship between oxygen consumption and oxygen delivery. The solid line shows the byphasic consumption and delivery. The dashed line illustrates the postulated changes in the relation with diseases such as sepsis and acute respiratory distress syndrome. The anaerobic threshold is shifted to the right, suggesting that patients need increased levels of delivery to avoid ongoing ischemic damage to vital organs.

decreases in hemoglobin concentration result in inadequate oxygen delivery to tissues. There is, therefore, a biphasic relationship between oxygen delivery and consumption (Fig 9-2). One phase is an oxygen delivery-independent portion, in which oxygen consumption is independent of oxygen delivery above the DO2(crit) delivery threshold. The other is a delivery or supplydependent portion, in which oxygen delivery is directly related to oxygen consumption. The latter portion of this relationship below the DO2(crit) indicates the presence of tissue hypoxia. Both laboratory and clinical studies have attempted to determine the DO2(crit). In the most rigorous clinical study,5 the investigators found a threshold value of 4 mL/minute/kg. In other clinical and laboratory studies, investigators found values in the range of 6 to 10 mL/minute/kg.5,6 The DO2(crit), or the anaerobic threshold, is not a single, fixed value but varies substantially depending on factors such as basal metabolic rate, the specific organ or tissue, some disease states, and perhaps such complex factors as patient age or genetic composition. In the previous example, cardiac output did not increase as would otherwise be expected in anemia. Once it is oxygenated, blood is distributed to all organs and tissues through the arterial tree into the microcirculation. Organ blood flow is controlled by arterial tone in medium-sized vessels and is primarily responsive to changes in autonomic stimulation and the release of locally generated vasodilating substances. Within organ systems, red cells are carried into the microcirculation, where oxygen is released to the tissues through a thinwalled capillary network. Once released, oxygen diffuses through the interstitial space, finally finding its way into the cell and its mitochondria to be used in cellular respiration. Each of these physiologic mechanisms can be altered in disease, as described later. Additional adaptive changes in the microcirculation enhance oxygen delivery in anemia.7

Chapter 9: Anemia and Red Blood Cell Transfusion

Adaptive Mechanisms in Anemia In anemia, oxygen-carrying capacity decreases, but tissue oxygenation is preserved at hemoglobin levels well below 10 g/dL (Table 9-1). After the development of anemia, adaptive changes include a shift in the oxyhemoglobin dissociation curve, hemodynamic alterations, and microcirculatory alterations. The shift to the right of the oxyhemoglobin dissociation curve in anemia is primarily the result of increased synthesis of 2,3-diphosphoglycerate (2,3-DPG) in red cells.8-10 This rightward shift enables more oxygen to be released to the tissues at a given PO2, offsetting the effect of reduced oxygen-carrying capacity of the blood. In-vitro studies have shown rightward shifts in the oxyhemoglobin dissociation curve with increases in temperature and decreases in pH.11 Although clinically important shifts have been documented in a number of studies, hemoglobin oxygen saturation generally is measured in arterial specimens processed at standard temperature and pH. Therefore, current measurement techniques do not reflect oxygen-binding affinity or unloading conditions in the patient’s microcirculatory environment, which may be affected by temperature, pH, and a number of disease processes. The shift in the oxyhemoglobin dissociation curve caused by decreases in pH (increase in hydrogen ion concentration) is the Bohr effect.11,12 Because changes in pH rapidly affect the ability of hemoglobin to bind oxygen, this mechanism has been postulated to be an important early adaptive response to anemia.13 However, the equations describing the physical process indicate that a very large change in pH is needed to modify the partial pressure of oxygen at which hemoglobin is 50% saturated

with oxygen (P50) by a clinically important amount (⬃10 mm Hg). As a result, the Bohr effect is unlikely to have significant clinical consequences.11,12 Several hemodynamic alterations occur after the development of anemia. The most important determinant of cardiovascular response is the patient’s volume status, or more specifically left ventricular preload. The combined effect of hypovolemia and anemia often occurs as a result of acute blood loss. Acute anemia can cause tissue hypoxia or anoxia through both diminished cardiac output (stagnant hypoxia) and decreased oxygen-carrying capacity (anemic hypoxia).14 The body primarily attempts to preserve oxygen delivery to vital organs by compensatory increases in myocardial contractility and heart rate as well as increased arterial and venous vascular tone mediated through increased sympathetic discharge. In addition, a variety of mechanisms redistribute organ blood flow. The adrenergic system plays an important role in altering blood flow to and within specific organs. The renin-angiotensin-aldosterone system is stimulated to retain both water and sodium. Losses ranging from 5% to 15% in blood volume result in variable increases in resting heart rate and diastolic blood pressure. Orthostatic hypotension often is a sensitive indicator of relatively small losses in blood volume not sufficient to cause a marked decrease in blood pressure. Larger losses result in progressive increases in heart rate and decreases in arterial blood pressure accompanied by evidence of organ hypoperfusion. The increased sympathetic tone diverts an ever decreasing global blood flow (cardiac output) away from the splanchnic, skeletal, and cutaneous circulation

Table 9-1. Physiologic Changes Associated with Anemia Oxyhemoglobin dissociation curve ● Anemia shifts the oxyhemoglobin curve to the right because of increased 2, 3-biphosphoglycerate levels. ● Anemia causes clinically significant rightward shifts in the oxyhemoglobin curve because of the Bohr effect. ● The shift in the oxyhemoglobin curve has been clearly established in many forms of anemia (excluding hemoglobinopathies). ● The shift in the oxyhemoglobin curve has been clearly established in a number of human diseases. Cardiac output ● Cardiac output increases with increasing degrees of normovolemic anemia. ● Increased cardiac output in normovolemic anemia is a result of increased stroke volume. ● The contribution of increased heart rate to the increase in cardiac output following normovolemic anemia is variable. Other hemodynamic alteration ● Changes in blood viscosity result in many of the hemodynamic changes in normovolemic anemia. ● Normovolemic anemia is accompanied by increased sympathetic activity. ● Normovolemic anemia causes increased myocardial contractility. ● Normovolemic anemia causes a decrease in systemic vascular resistance. ● Normovolemic anemia results in a redistribution of cardiac output toward the heart and brain and away from the splanchnic circulation. ● Maximal global oxygen delivery occurs at hemoglobin values of 10 g/dL to 11 g/dL. ● Global oxygen delivery declines above and below hemoglobin values of 10 g/dL to 16 g/dL. Coronary and cerebral blood flow ● Coronary blood flow is increased during anemia. ● Cerebral blood flow is increased during anemia. ● Coronary artery disease in the presence of moderate degrees of anemia (hemoglobin values below 9 g/dL) results in impaired left ventrical contractility or ischemia. ● Moderate anemia does not aggravate cerebral ischemia in patients with cerebrovascular disease.

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toward the coronary and cerebral circulation. Once vital organ systems such as the kidneys, the central nervous system, and the heart are affected, the patient is considered in hypovolemic shock. Although the American College of Surgeons Committee on Trauma15 has categorized the cardiovascular and systemic response to acute blood loss according to degree of blood loss, many of these responses are modified by the rapidity of blood loss and patient characteristics, such as age, coexisting illnesses, preexisting volume status, hemoglobin value, and the use of medications having cardiac effects (β-blockers) or peripheral vascular effects (antihypertensives). The compensatory changes in cardiac output most thoroughly studied are the cardiovascular consequence of normovolemic anemia. When intravascular volume is stable or high after the development of anemia (as opposed to hypovolemic anemia and shock), increases in cardiac output have been consistently reported. Indeed, an inverse relationship between hemoglobin level (or hematocrit) and cardiac output has been clearly established in well-controlled laboratory studies (Fig 9-3).13,16-19 Similar clinical observations have been made in the perioperative setting20 and for chronic anemia.16 Unfortunately, the strength of inferences from clinical studies is limited by confounding factors arising from major coexisting illnesses such as cardiac disease, a lack of appropriate control patients, and significant weaknesses in study design. Researchers have attempted to determine the level of anemia at which cardiac output begins to rise. Reported thresholds for this phenomenon identified in primary clinical and laboratory studies have ranged from 7 to 12 g/dL of hemoglobin.16 Two major mechanisms are thought to be responsible for the physiologic processes underlying increased cardiac output during normovolemic anemia: 1) reduced blood viscosity and 2) increased sympathetic stimulation of the cardiovascular effectors.21 Blood viscosity affects both preload and afterload,

Cardiac Output (L/min)

15

10

5

0

0

2.5

5.0 7.5 10.0 Hemoglobin (g/dL)

12.5

15.0

Figure 9-3. The theoretic effect of hemoglobin concentration on cardiac output. The curves illustrate how cardiac output increases as hemoglobin concentration decreases. The solid curve is meant to describe the increase in a healthy adult. The dashed line on the top shows how the cardiac output response can be accentuated in a young athlete and the lower dashed line might correspond to someone with poor cardiovascular function.

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two major determinants of cardiac output,21-23 whereas sympathetic stimulation primarily increases the two other determinants, heart rate and contractility. Unlike the situation for hypovolemic anemia, in this setting the effects of blood viscosity appear to predominate.22,23 Complex interactions exist among blood flow, blood viscosity, and cardiac output. In vessels, blood flow alters whole-blood viscosity, and blood viscosity modulates cardiac output. Under experimental conditions in a rigid hollow cylinder, blood flow is directly related to the fourth power of the diameter and to driving pressure. It is inversely related to the length of the vessel and to blood viscosity (Poiseuille-Hagen law).7,21 Also, blood viscosity increases as flow decreases because of increasing aggregation of red cells. Thus, viscosity is highest in postcapillary venules, where flow is the lowest, and viscosity is lowest in the aorta, where flow is the highest. In postcapillary venules, there is a disproportionate decrease in blood viscosity as anemia worsens, consequently augmenting venous return at a given venous pressure. If cardiac function is normal, the increase in venous return or left ventricular preload is the most important determinant of increased cardiac output during normovolemic anemia. The conclusion is based on experiments in which viscosity was maintained during anemia by means of high-viscosity colloidal solutions. In such studies, the cardiovascular effects of hemodilution were attenuated22 compared with similar levels of hemodilution accompanied by reduced whole-blood viscosity. Decreased left ventricular afterload, another cardiac consequence of decreased blood viscosity, also may be an important mechanism for the increase in cardiac output as anemia worsens.22 Sympathetic stimulation can result in increased cardiac output through enhanced myocardial contractility24 and increased venomotor tone.18,25 The effects of anemia on left ventricular contractility in isolation have not been clearly determined, given the complex changes in preload, afterload, and heart rate. Only one before-and-after hemodilution study was performed with loadindependent measures to document increased left ventricular contractility.24 Chapler and Cain18 summarized several well-controlled animal studies indicating that venomotor tone increases as a result of stimulation of the aortic chemoreceptors. If sympathetic stimulation is significant in the specific clinical setting, contractility is increased from stimulation of the β-adrenergic receptors.21,26 The inverse relationship between cardiac output and hemoglobin level has led investigators to attempt to determine the hemoglobin level that maximizes oxygen transport. Richardson and Guyton27 evaluated the effects of hematocrit on cardiac performance in a canine model. They established that optimal oxygen transport occurred between hematocrits of 40% and 60%. Others determined maximum oxygen delivery to be in the lower end of the range, at a hematocrit of 40% to 45% (hemoglobin 13 to 15 g/dL).28,29 However, in one of the most widely quoted studies addressing this topic,30 investigators found peak oxygen transport to occur at a hematocrit of 30% (hemoglobin concentration, 10 g/dL). Unfortunately, global indices of optimal oxygen delivery mask any differences in blood flow between

Chapter 9: Anemia and Red Blood Cell Transfusion

specific organs.31,32 In addition, attempting to identify a single optimal hemoglobin concentration that maximizes oxygen delivery neglects the large number of factors interfering with adaptive mechanisms during management of patients other than healthy, young patients with anemia. Will the transfusion of allogeneic RBCs reverse any adaptive response to acute or chronic normovolemic anemia? Assuming that oxygen-carrying capacity is not impaired during storage and that hematocrit is restored after a transfusion, the cardiovascular consequences will be reversed. However, the storage process alters the properties of red cells. These alterations may impair flow and oxygen release from hemoglobin9 in the microcirculation.

Microcirculatory Effects of Anemia and Red Cell Transfusion At the level of the microcirculation, three putative adaptive mechanisms increase the amount of oxygen supplied to tissues by capillary networks. In a model of the microcirculation proposed by Krogh,33 oxygen supply to the tissues is enhanced through recruitment of previously closed capillaries, increased capillary flow, and increased oxygen extraction from existing capillaries. The degree of anemia, the specific tissue bed, and a variety of disease processes affect microcirculatory blood flow and oxygen supply.7,34 As the degree of hemodilution becomes more pronounced and hematocrit decreases, blood viscosity decreases disproportionately in capillary networks. This occurs because the hematocrit is highest in the capillary network, as a consequence, there is a larger decrease in capillary viscosity. Stored red cells have properties that differ from those of their in-vivo counterparts; many are related to the duration of storage. Characteristically, older RBC units have lower levels of 2,3-DPG. The result is a leftward shift in the oxyhemoglobin dissociation curve, which can impede delivery of oxygen to the tissues.13 In addition, storage of red cells decreases the deformability of their membrane.35 As a consequence, stored red cells may impede flow in the microcirculation36 and may have limited ability to release oxygen to tissues. However, these storage lesions are reversible within 24 to 48 hours.37 There are reports38 suggesting that disease processes such as sepsis impair red cell deformability. In conjunction with significant systemic microcirculatory dysfunction, the decrease in red cell deformability may dramatically affect tissue oxygen delivery in sepsis and septic shock.38 This body of evidence suggests that RBC transfusions increase systemic oxygen delivery but may have adverse effects on microcirculatory flow.

Interaction between Pathophysiologic Processes and Anemia Several disease processes affecting either the entire body or specific organs potentially limit adaptive responses and make patients more vulnerable to the effects of anemia. Specifically, heart, lung, and cerebrovascular diseases have been proposed to increase the risk of adverse consequences of anemia. Age, severity

of illness, and therapeutic interventions also may affect adaptive mechanisms. The heart, specifically the left ventricle, is particularly prone to adverse consequences of anemia. This is because the myocardium consumes 60% to 75% (extraction ratio) of all oxygen delivered by the coronary circulation.28,31,39 Such a high extraction ratio is unique to the coronary circulation. As a result, oxygen delivery to the myocardium can increase substantially only with an increase in blood flow.40 In addition, most left ventricular perfusion is restricted to the diastolic period, because pressures inside the left ventricle are too high to allow adequate coronary blood flow during systole. Thus, any shortening of its duration (eg, tachycardia) decreases blood flow. Laboratory studies have been performed to investigate the effects of normovolemic anemia on the coronary circulation.29,39,41 There appear to be minimal consequences of anemia with hemoglobin levels in the range of 7 g/dL if the coronary circulation is normal.19,24 However, myocardial dysfunction and ischemia either occur earlier or are greater in anemic animal models with moderate to high-grade coronary stenosis compared with controls with normal hemoglobin values.42 Data from studies with human subjects are inconsistent. Several clinical studies involving patients with coronary artery disease undergoing normovolemic hemodilution have not shown any increase in cardiac complications or silent ischemia during electrocardiographic monitoring.43 In addition, a retrospective analysis involving 224 patients undergoing CABG surgery did not show a significant association between hemoglobin level and coronary sinus lactate level (an indicator of myocardial ischemia).44 However, in two recent cohort studies, moderate anemia was poorly tolerated by perioperative45 and critically ill patients46 with cardiovascular disease. Thus, retrospective studies seem to support preclinical reports. It is also plausible that anemia results in considerable increases in morbidity and mortality among patients with other cardiac diseases, including heart failure and valvular heart disease, presumably because of the greater burden of the adaptive increase in cardiac output. During normovolemic anemia, cerebral blood flow increases as hemoglobin values decrease. Investigators have observed increases ranging from 50% to 500% of baseline value in laboratory studies47 and in one study with human subjects.48 Cerebral blood flow increases because of overall increases in cardiac output, which is preferentially diverted to the cerebral circulation. As oxygen delivery begins to decrease, cerebral tissues are able to increase the amount of oxygen extracted from blood. A number of factors, including degree of hemodilution, type of fluid used for volume expansion, volume status (preload), and extent of cerebrovascular disease, are capable of potentially modifying global or regional cerebral blood flow during anemia.49 The increase in global cerebral blood flow combined with the potential for improved flow characteristics across areas of vascular stenosis (improved rheologic properties of blood because of decreased viscosity) prompted a number of laboratory and clinical49-52 studies to investigate hemodilution as therapy for acute ischemic stroke.50-52

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Section I: Part I

The results of laboratory studies suggest that moderate degrees of anemia alone should rarely result in or worsen cerebral ischemia. None of the randomized clinical trials demonstrated that hemodilution in acute ischemic stroke improved clinical outcomes in patients. Because of the variety of variables that affect clinical outcomes, the negative findings may not fully rule out the possibility that hemodilution offers therapeutic benefit. Thus, the currently available evidence indicates that cerebrovascular disease does not appear to predispose patients to serious morbidity from anemia. Changes in oxygen delivery to the brain during normovolemic anemia (either increases or decreases in blood flow) do not uniformly affect various cerebral pathologic conditions. For example, patients with high intracranial pressure from traumatic brain injury may be adversely affected by increased cerebral blood flow. However, after subarachnoid hemorrhage, mild degrees of normovolemic or hypervolemic anemia may improve overall oxygen delivery, possibly by overcoming the effects of cerebral vasospasm and thereby improving cerebral blood flow through decreased viscosity.53 However, the effects of moderate to severe anemia in subarachnoid hemorrhage have not been assessed in laboratory or clinical studies. One of the major consequences of redistributing some of the available cardiac output toward the coronary and cerebral circulation during normovolemic anemia is the shunting of flow away from other organs, including the kidneys and intestines. Critically ill patients may be adversely affected by this redistribution,54 which could result in increased intestinal ischemia, bacterial translocation, and multiple-system organ failure.55 Critical illness also can tax many of the body’s adaptive responses. Specifically, cardiac performance may be impaired56 or may already be at maximal capacity in response to increased metabolic demands. Pathologic processes affecting the microcirculation, particularly prevalent among critically ill patients, also may affect the patient’s response to anemia and transfusions.

lactate production, depressed ventricular function, and death have occurred at hemoglobin levels of 3 g/dL or less. Some animals survive with hemoglobin levels as low as 1 to 2 g/dL.57 However, results of studies with animals suggest a decreased ability to tolerate anemia in the presence of cardiac disease. In dogs with experimentally induced coronary stenosis varying from 50% to 80%, ST-segment changes or locally depressed cardiac function occurred at hemoglobin levels in the range of 7 to 10 g/dL.58,59

Human Studies Studies involving patients who refuse blood transfusion for religious reasons provide critical insight into the effect of anemia on humans. The largest study was performed with 1958 adult surgical patients who refused transfusion for religious reasons.45 The mortality was greatest among patients with the lowest preoperative hemoglobin concentrations. Among patients with underlying cardiovascular disease, the risk of death was markedly greater than for patients without cardiovascular disease, especially in those patients with a hemoglobin concentration less than 10 g/dL. Among patients without underlying cardiovascular disease, the difference in mortality at hemoglobin levels greater than or less than 10 g/dL was not as great (Fig 9-4). None of the 99 patients with hemoglobin concentrations between 7 and 8 g/ dL died.60 However, the risk of death rose sharply below a hemoglobin concentration of 5 g/dL. These results, as well as data on animals and physiologic data, suggest that anemia is not tolerated as well in the presence of cardiovascular disease. In a series of studies, the effect of anemia was evaluated among healthy volunteers who underwent isovolemic reduction of hemoglobin level to 5 g/dL. Transient and asymptomatic electrocardiographic changes were found in five of the 87 volunteers

16

Cardiovascular Disease-No Cardiovascular Disease-Yes

Clinical Outcomes of Anemia and Red Cell Transfusion Every medical decision must weigh risk vs benefit. The decision to administer RBC units must consider the risks of blood transfusion (see Chapters 44 through 56), the risk of anemia, and the level of anemia at which blood transfusion prevents the associated adverse outcomes.

Odds Ratio

13 10 7 4 1 6

Risk of Anemia Preclinical Laboratory Studies The critical hemoglobin threshold is similar in different animals.1 Results of studies suggest that healthy animals can tolerate hemoglobin levels between 3 and 5 g/dL after normovolemic hemodilution. Electrocardiographic changes consistent with ischemia occur at hemoglobin levels less than 5 g/dL, whereas

136

7

8

9

10

11

12⫹

Preoperative Hemoglobin g/dL Figure 9-4. Association between preoperative hemoglobin level and mortality among patients with and without cardiovascular disease.45 In a population of patients who refused blood transfusion, the risk of death was higher among patients with cardiovascular disease (top line) than among patients without cardiovascular disease (bottom line) for each preoperative hemoglobin level. (Used with permission from Carson et al.45)

Chapter 9: Anemia and Red Blood Cell Transfusion

included in two studies.61,62 These changes occurred when the hemoglobin level was between 5 and 7 g/dL and in patients with faster heart rates.62 Changes in critical oxygen delivery were not measured. Subtle but reversible changes in cognition were identified in nine volunteers younger than 35 years at a hemoglobin level between 5 and 7 g/dL.63 Self-rated fatigue was found in eight volunteers when the hemoglobin level decreased to 7 g/dL. Fatigue increased as hemoglobin levels decreased to 5 g/dL.64 The results of these studies suggest that important clinical effects can be detected in young, healthy humans with hemoglobin levels between 5 and 7 g/dL. It is uncertain how these results apply to older patients with comorbid factors who are also under stress from surgery or acute illness. An analysis of 31,000 patients 65 years of age or older undergoing major noncardiac surgery examined the association between preoperative hematocrit and mortality or cardiac morbidity (cardiac arrest or Q-wave myocardial infarction).65 Mortality rose monotonically when the hematocrit was less than 36%. Cardiac events were more frequent when the hematocrit was less than 39%. These results, in contrast to experimental data, suggest that mild anemia is associated with increased mortality and morbidity. However, anemia may be only a marker of underlying disease and studies are needed to demonstrate improved outcome if anemia is corrected by transfusion.

Efficacy of Transfusion Observational Studies Selected observational studies of the effect of RBC transfusion on clinical outcomes are described in Table 9-2. The results vary, although only one study has demonstrated a beneficial effect of RBC transfusion. The validity of these studies is uncertain because the decision to transfuse often correlates with the illness burden of the patient. It is likely that comorbidity was not adequately adjusted for in these studies. For example, there are many observational studies demonstrating that blood transfusion increases risk of bacterial infection,76 yet none of the randomized clinical trials have confirmed this finding. Only randomized clinical trials can overcome this limitation. Clinical Trials in Adults Ten randomized clinical trials contrasted the effects of different transfusion thresholds (Table 9-3).77-86 The clinical settings varied, although each trial randomly assigned patients to transfusion on the basis of a restrictive or a liberal strategy. Restrictive triggers (specified hemoglobin concentrations that had to be attained) ranged from 7 to 9 g/dL. Two trials specified a hematocrit of 25% or 30% for RBC transfusion. The liberal transfusion strategies specified the following triggers: 100% of normal red cell volume, two RBC units (immediately in one trial, postoperatively in another) irrespective of clinical state, and transfusion sufficient to maintain hemoglobin level at or above 10 g/dL in three trials and above 9 g/dL in another. Two trials specified the liberal triggers as transfusion to maintain hematocrit at 32% and 40% or greater. There is

overlap between the liberal and restrictive transfusion groups in these trials. One trial involving patients in the intensive care unit (ICU) contributed 47% of the patients and 82% of the recorded deaths. There were a total of 1780 trial participants. Of the 10 trials, only three included more than 100 patients, and only one of these evaluated a transfusion strategy that included assessment for symptoms. In one of these trials, 428 patients undergoing first-time, elective CABG surgery were randomly assigned to study arms with transfusion triggers of 9 g/dL vs 8 g/dL.85 The differences between perioperative hemoglobin levels were small, and the mean reduction in hemoglobin level during the admission was equal between the groups. The event rates were very low, and there were no differences in any outcome. The second trial included 127 patients undergoing knee arthroplasty. Patients were randomly assigned to receive autologous blood transfusion immediately after the operation (first unit in recovery room and second unit on return to the ward) vs autologous blood only if the hemoglobin level decreased to less than 9 g/dL.86 The mean postoperative hemoglobin level was approximately 0.7 g/dL different, although only 25% of the restrictive group received a transfusion. There were no differences in outcome. In a third trial (pilot study), 84 patients with hip fracture undergoing surgical repair were randomly assigned to groups receiving either transfusion at a 10 g/dL threshold or transfusion for symptoms (transfusion was also allowed if hemoglobin level was less than 8 g/dL).83 The mean hemoglobin level before randomization was 9.1 g/dL. The lowest mean hemoglobin level after randomization in the group with symptoms was 8.8 g/dL, and the highest mean hemoglobin level in the threshold group was 11.1 g/dL. There were no differences in outcomes (including functional recovery, mortality, and morbidity), although 60 days after the operation, five patients in the symptomatic group had died, as had two patients in the 10 g/dL group. In all of these trials (and the other trials in Table 9-3), the numbers of patients were much too small to evaluate the effect of lower transfusion triggers on clinically important outcomes such as mortality, morbidity, and functional recovery. The Transfusion Requirements in Critical Care (TRICC) trial81,84 is the only adequately powered study to evaluate clinically important outcome. In the main study, the investigators randomly assigned 838 volume-resuscitated ICU patients to a restrictive strategy in which patients received allogeneic RBC transfusions at hemoglobin levels of 7 g/dL (and were maintained between 7 to 9 g/dL) or to a liberal strategy of receiving RBCs at 10 g/dL (and were maintained between 10 and 12 g/dL). Average hemoglobin levels (8.5 vs 10.7 g/dL) and RBC units transfused (2.6 vs 5.6 units) were significantly lower in the restrictive as opposed to the liberal group. The 30-day mortality was slightly lower in the restrictive transfusion group (18.7% vs 23.3%), although the finding was not statistically significant (p
0.11). Mortality was lower in the restrictive transfusion group in patients less than 55 years of age (p 0.02) and less ill patients defined by APACHE II scores less than 20 (p 0.02). Furthermore, the restrictive transfusion group had fewer patients

137

Section I: Part I

Table 9-2. Summary of Nonrandomized Studies Study

Clinical Setting

Subjects

Outcomes

Nelson et al (1993)66

Vascular surgery (n
27)

High-risk patients who had undergone elective infra-inguinal bypass vascular procedures Anemic group: n
13 Nonanemic group: n
14

MI occurred in 14% of pts with Hct 28% compared to 77% with Hct 28% No deaths in either group

Hébert et al (1997)67

Critical care (n
4,470)

Critically ill patients admitted to ICU Survivors: n
3,469 Nonsurvivors: n
1,001

Survivors had higher hemoglobin levels than nonsurvivors

Paone et al (1997)68

Cardiac surgery (n
100)

Patients undergoing isolated CABG surgery Transfusion group: n
13 No transfusion group: n
87

No difference in clinical outcomes Length of ICU stay longer in transfused patients (2.60.3 days) vs nontransfused group (1.60.1 days)

Patients received allogeneic RBCs on bypass for low SvO2 (55%) and transfused postoperatively for a Hct 20% or at any Hct level if deemed clinically warranted Hogue et al (1998)69

Urologic surgery (n
190)

Patients undergoing radical prostatectomy Hct 28% group: n
81 Hct 28% group: n
100

Patients with Hct levels 28% immediately after surgery were significantly (p
0.05) more likely to have ischemic episodes

Carson et al (1998)70

Orthopedic surgery (n
8,787)

Hip fracture patients, aged 60 years or older, who underwent surgical repair Transfused group: n
3,699 Nontransfused group: n
5,088

No difference in 30- or 90- day mortality

Spiess et al (1998)71

Cardiac surgery (n
2,202)

CABG surgery patients, mean age SD
64.3 10.2 years High ICU Hct 34% group: n
410 Medium ICU Hct group 25% to 33%: n
1,544 Low ICU Hct 24% group: n
248

MI highest in ICU Hct (8.3%) then medium ICU Hct (5.5%) and low ICU Hct (3.6%)

Wu et al (2001)72

Myocardial infarction (n
78,794)

MI patients 65 years old or older 5.0 to 24.0% n
380 24.1 to 27.0% n
838 27.1 to 30.0% n
2,106 30.1 to 33.0% n
4,848 33.1 to 36.0% n
9,885 36.1 to 39.0% n
16,218 39.1 to 48.0% n
44,699

Transfusion was associated with reduction in 30-day mortality in patients with Hct 5.0% to 33%

Vincent et al (2002)73

Critical care (n
1,136)

41% of patients transfused during 28-day period

Transfusion was associated with increased mortality; odds ratio 1.37; 95% confidence interval 0.02-1.84

Rao et al (2004)74

Acute coronary syndrome (n
24,112)

Subjects included in three clinical trials Received transfusion: n
2,400 No transfusion: n
21,711

Transfusion was associated with an increased 30-day mortality (hazard ratio, 3.94; 95% confidence interval, 3.26-4.75) and 30-day mortality or MI (hazard ratio, 2.92; 95% confidence interval, 2.55-3.35)

Sabatine et al (2005)75

Acute coronary syndrome (n
39,922)

Subjects from 16 clinical trials 4.6% of patients with ST-segment elevated MI received transfusion

In ST-segment elevated MI, transfusion was associated with a decreased risk of cardiovascular death when hemoglobin 12 g/dL

2.7% of patients with non-ST-segment elevated MI received transfusion

In non-ST-segment elevated MI, transfusion was associated with increased risk of cardiovascular death regardless of hemoglobin concentration

Hct
hematocrit; ICU Hct
intensive care unit hematocrit; MI
myocardial infarction; CABG
coronary artery bypass graft.

138

Table 9-3. Results of Randomized Controlled Trials in Adults Study

Setting

Subjects: Eligibility and Comparability

Transfusion Strategy

Mean Blood Usage, Units/Pt (sd)

Proportion Transfused (n)

Mean Hb / Hct Levels (sd)

30-Day Mortality (n)

Length of Hospital Stay (mean  sd)

Topley et al (1956)77

Trauma (n
22)

1 L blood loss; considered to be at no clinical risk in raising the blood volume  100% of normal, or allowing it to reach 30% below normal

Liberal: to achieve red cell volume 100% of normal Restrictive: maintain red cell volume 70%–80% of normal

11.3 (6.9)

100% (10)

–

–

4.8 (6.7)

67% (8)

Lowest Hb: 15.6  2.0 g/dL Lowest Hb: 11.3  0.7 g/dL

–

–

Acute severe upper gastrointestinal hemorrhage

Liberal: patients received at least 2 RBC units immediately on admission to hospital Restrictive: patients did not receive RBCs during the first 24 hours unless Hb 0.02) HPA-1a (PlA1, Zwa) HPA-1b (PlA2, Zwb) HPA-3a (Baka, Leka) HPA-3b (Bakb) HPA-4a (Pena, Yukb)

IIIa IIIa IIb IIb IIIa

0.85 0.15 0.61 0.39 0.99

   ? 

    

 — — — —

 — — — —

() () () () ()

GPIIb/IIIa: private/low-frequency (gene frequency < 0.02) HPA-4b (Penb, Yuka) HPA-6b (Tua, Caa) HPA-7b (Moa) HPA-8b (Sra) HPA-9b (Maxa) HPA-10b (Laa) HPA-11b (Groa) HPA-14b (Oea) HPA-16b (Duva) Vaa

IIIa IIIa IIIa IIIa IIb IIIa IIIa IIIa IIIa IIb/IIIa

0.01 0.003 0.01 0.003 0.002 0.01 0.001 0.005 0.01 0.002

         

— — — — — — — — — —

— — — — — — — — — —

— — — — — — — — — —

— — — — — — — — — —

GP Ia/IIa: public HPA-5a (Brb, Zavb) HPA-5b (Bra, Zava, Hca)

Ia Ia

0.89 0.11

 

? 

— 

— 

() ()

GP Ia/IIa: private HPA-13b (Sita) Swia

Ia Ia

0.0025 0.002

 

— —

— —

— —

— —

GP Ib/IX: public HPA-2a (Kob) HPA 2b (Koa, Siba)

Ibα Ibα

0.89 0.11

— 

— ?

— —

— —

? ()

GP Ib/IX: private HPA-12b (Iya)

Ibβ

0.01



—

—

—

—

CD109: public HPA-15a (Govb) HPA-15b (Gova)

CD109 CD109

0.53 0.47

 

? —

— —

— —

— ()

GP38 Dya

38 kD

0.01



—

—

—

—

— —

— —

— —

— —

 

Platelet nonspecific alloantigens ABO HLA *Modified with permission from Warkentin & Smith.197

GP
glycoprotein; NAIT
neonatal alloimmune thrombocytopenia; PTP
posttransfusion purpura; PAT
passive alloimmune thrombocytopenia; TAT
transplantationassociated alloimmune thrombocytopenia; PTR
platelet transfusion refractoriness. 
relatively common; 
established but rare; —
not reported; ()
probable association, but definitive link inconclusive; ?
possible association but not established.

0.007) is almost 1000 times less than predicted with the theoretical ratio (6.4). Immunogenetics is a major factor determining alloimmunization against HPA-1a. There is a strong association

364

between formation of anti-HPA-1a and HLA-DRB3*0101 and HLA-DQB1*0201 (odds ratio, 25 and 40, respectively).207 In contrast, no HLA association exists for immunization against HPA1b.208 Thus, it appears that persons with certain HLA genotypes

Chapter 23: Management of Immune Thrombocytopenia

Table 23-5. Observed Frequencies of Neonatal Alloimmune Thrombocytopenia and Posttransfusion Purpura in Relation to Expected (Theoretic) Frequency of the HPA-1ab (PlA1/A2), HPA-5ab (Bra/b), and HPA-3ab (Baka/b) Alloantigen Systems* Target Alloantigen

HPA-3b HPA-1b HPA-3a HPA-5b HPA-1a HPA-5a

Observed Percentage of Cases of NAIT‡ Pregnancies at Theoretic Risk of NAIT† (Descending Order)

Observed Cases of PTP§

14.5 10.8 9.3 8.7 1.9 1.1

0 29 9 11 105 7

0 0 1 6 44 0

*Modified with permission from Warkentin & Smith.197 †

Percentage of pregnancies at theoretic risk of NAIT for a given target alloantigen is determined as follows: x(1  x)2  100, where x is the gene frequency of the target alloantigen. Note the lack of correlation between the theoretical and observed risk for NAIT. ‡

Data are from Mueller-Eckhardt et al204 and represent serologic investigations using a defined protocol over an 18-month period ending June 30, 1988.

§

For comparison, the serologic findings for cases of PTP are shown for which only one platelet alloantigen specificity was identified (from January 1990 to August 2006 at the BloodCenter of Wisconsin).206

Table 23-6. Severity of Thrombocytopenia by Platelet Count Nadirs ( 1000/µL) in Relation to Target Glycoprotein for Various Alloimmune Thrombocytopenic Syndromes* Glycoprotein GPIIb/IIIa HPA-1a (IIIa) HPA-1b (IIIa) HPA-3ab (IIb) HPA-4ab (IIIa) Mean GPIa/Iia HPA-5b HPA-5a Mean

NAIT

PTP

PAT

TAT

17 (n
81) 9 (n
2) 10 (n
5) 13 (n
7) 16

6 (n
43) 5 (n
4) 3 (n
4) 6 (n
1) 6

8 (n
9) — — — 8

8 (n
4) — — — 8

44 (n
48) 35 (n
5) 43

26 (n
1) — 26

35 (n
1) — 35

43 (n
1) — 43

*Data from Warkentin & Smith197and Brunner-Bolliger et al.210 The data show that alloimmune thrombocytopenic syndromes that involve GPIIb/ IIIa are more likely to cause severe thrombocytopenia than are those involving GPIa/IIa. The data are combined for alloimmune thrombocytopenic syndromes involving either allele of the HPA-3ab and HPA-4ab alloantigen systems, whereas the data are shown separately for the alleles of the HPA-1ab and HPA-5ab systems. NAIT
neonatal alloimmune thrombocytopenia; PTP
posttransfusion purpura; PAT
passive alloimmune thrombocytopenia; TAT
transplantation-associated alloimmune thrombocytopenia.

NAIT
neonatal alloimmune thrombocytopenia; PTP
posttransfusion purpura.

are much more likely to generate an alloimmune response when GPIIIa bears the leucine33 substitution that determines the HPA1a phenotype. Overall, on the basis of the observed allelic frequencies, the expected theoretical ratio of NAIT for anti-HPA5b, compared with anti-HPA-5a, should be approximately 8 (Table 23-5). A similar ratio (47:3, or 15.7) has been observed. However, although the expected and observed ratios are similar (contrast the HPA-1a/1b system), a role for immunogenetics and alloimmunization exists also for the HPA-5a/5b system.209

Severity of Alloimmune Thrombocytopenia In general, the severity of thrombocytopenia is greater for alloimmune thrombocytopenia that involves the GPIIb/ IIIa complex, compared with the GPIa/IIa complex (Table 23-6197,204,206,210). Because there are approximately 20 times more GPIIb/IIIa molecules compared with GPIa/IIa complexes (40,000 vs 2000), this suggests that greater numbers of alloantibodies binding to the more numerous GPIIb/IIIa receptors results in greater platelet destruction.

Neonatal Alloimmune Thrombocytopenia Neonatal alloimmune thrombocytopenia is a transient but potentially life-threatening thrombocytopenic disorder limited to fetal and neonatal life. It is caused by maternal IgG alloantibodies that cross the placenta and cause premature destruction of platelets bearing paternally derived platelet alloantigens (analogous to

hemolytic disease of the fetus and newborn). Neonatal alloimmune thrombocytopenia occurs in approximately 1 to 1.5 per 1000 live births.211 Approximately 75% of cases in a population of European ancestry are caused by fetomaternal incompatibility for the platelet-specific alloantigen HPA-1a, and 20% by HPA-5b.204 Other alloantigens implicated in NAIT, including private alloantigens identified in only one or a few families [eg, HPA6b (Tua/Ca), HPA-7b (Moa)], are shown in Table 23-4. In east Asian populations, anti-HPA-4b (Penb) is more common than anti-HPA-1a. Although HLA or ABO alloantibodies have been claimed to cause NAIT, in most cases, undetected platelet-specific alloantibodies or another diagnosis caused the thrombocytopenia.212 The typical clinical presentation of NAIT is isolated severe thrombocytopenia in an otherwise healthy neonate, especially if fetomaternal incompatibility involves an alloantigen on the GPIIb/IIIa complex (Table 23-6). Petechiae are found in 90%; gastrointestinal tract hemorrhage in 30%; and hemoptysis, hematuria, and retinal bleeding in fewer than 10% of patients. Approximately 15% have intracranial hemorrhage.204 The thrombocytopenia usually resolves within 1 to 3 weeks. Serious sequelae of fetal and neonatal intracranial bleeding include hydrocephalus, porencephalic cysts, and epilepsy. Firstborn offspring constitute approximately one-half of patients. This suggests that unlike the situation for hemolytic disease of the

365

Section II: Part I

fetus and newborn, sensitization can occur early during the first pregnancy.204 Subsequently affected siblings usually have thrombocytopenia to a similar or greater extent, an observation used to emphasize preventive treatment in subsequent pregnancies. Laboratory investigation of possible NAIT involves three steps. First, there must be a high index of suspicion: isolated thrombocytopenia in an otherwise well infant should be assumed to indicate NAIT until proved otherwise. The second step is to type maternal and paternal platelets to determine whether they are incompatible for a major platelet alloantigen. Commonly, the mother lacks certain platelet alloantigens that often are associated with alloimmune thrombocytopenia; for example, maternal homozygous HPA-1bb (PlA1-negative) status confers risk of NAIT caused by anti-HPA-1a. The third step is to determine whether the mother has platelet alloantibodies in her serum. Sometimes, no alloantibodies can be detected in maternal serum despite severe neonatal thrombocytopenia. Indeed, for approximately one-fourth of HPA-1bb mothers with infants believed to have had NAIT, anti-HPA-1a cannot be detected.204 The potential for low-incidence platelet-specific alloantigens to explain fetomaternal incompatibility means that maternal serum should be tested against paternal platelets whenever possible. There remains debate as to whether the titer of alloantibodies predicts severity of fetal thrombocytopenia.

Neonatal Treatment The optimal treatment of a neonate in whom NAIT is suspected because of severe thrombocytopenia is to increase the platelet count urgently to safe levels, even before serologic confirmation of the diagnosis. In some centers (eg, National Blood Service in the United Kingdom), HPA-1bb and HPA-5bb platelets can be obtained upon request. These should be effective for more than 95% of patients.213 When matched platelets are not available, washed and irradiated maternal platelets should be given to the neonate. These platelets are obtained by apheresis, and are washed to remove the maternal alloantibodies. Irradiation is performed to prevent graft-vs-host disease caused by maternal lymphocytes. In an emergency, immediate administration of whole-blood-derived platelets obtained from random donors may be of benefit to a bleeding infant.214 Giving high-dose IVIG to the neonate increases the platelet count of approximately 65% of patients.215 This treatment should be combined with maternally derived platelets. Corticosteroids are not recommended. Prenatal Management About one-half of the time, NAIT is suspected during the prenatal period, usually because the mother previously bore an affected infant, although the diagnosis sometimes is suggested in utero when fetal ultrasonography shows cerebral hemorrhage, hydrocephalus, or hydrops fetalis. One tenet of management is that thrombocytopenia in a subsequently affected offspring is generally as severe as, or more severe than, a previously affected sibling. Neonatal alloimmune thrombocytopenia caused by antiHPA-1a is more likely to cause fetal morbidity and mortality than

366

that caused by anti-HPA-5b and usually requires more aggressive treatment. When the father is known to be heterozygous for the implicated alloantigen (a situation that occurs approximately 25% and 20% of the time for NAIT involving the HPA-1a/1b and HPA-5a/5b systems, respectively), prenatal fetal typing (usually performed with genetic methods) is important, because it identifies the infant who is homozygous for the maternal antigen and is not at risk, obviating further treatment. For pregnancies at risk, general advice to the mother includes avoiding aspirin and nonsteroidal antiinflammatory medications. Two general approaches have been taken to manage pregnancies at high risk of severe NAIT213,216: regular administration of high-dose IVIG, repeated in-utero platelet transfusions, or both. The initial step is to obtain a fetal platelet count by means of percutaneous umbilical blood sampling, generally starting at 20 to 24 weeks of gestation. Because of the risk of fetal exsanguination, maternal platelets should be on hand for transfusion if the fetal platelet count is shown to be less than 50,000/µL.216 Intravenous immune globulin is given at a dosage of 1 g/kg/week starting within 1 week of documentation of fetal thrombocytopenia. Fetal blood sampling is repeated 4 to 6 weeks later; if no response is seen, glucocorticoid salvage treatment (prednisone, 60 mg/day) is started.216 However, not all fetuses respond to this approach. Another approach, which has been used in certain European centers, involves regular intrauterine platelet transfusions by means of percutaneous umbilical blood sampling, including a short time before delivery. This approach has led to good outcomes in situations in which previous siblings were severely affected.217 However, each fetal platelet transfusion carries risk of hemorrhage and death218 that likely depends on the experience of the fetomaternal unit. There is no consensus on which approach is preferred. Regardless of the antenatal management, there is consensus that delivery should be by means of elective cesarean section, performed as soon as fetal maturity is documented. The major reason for this mode of delivery is that it allows an organized, multidisciplinary approach to the peripartum care of the newborn. This approach includes urgent determination of the cord platelet count, provision of washed, irradiated maternal platelets (or antigen-negative platelets) and, usually, the use of high-dose IVIG (1 g/kg/day for 2 consecutive days) to treat severe neonatal thrombocytopenia.

Posttransfusion Purpura Posttransfusion purpura is a very rare disorder that typically manifests as severe thrombocytopenia and bleeding that begin 5 to 10 days after blood transfusion—usually Red Blood Cells (RBCs)—in a patient previously sensitized by pregnancy or transfusion.206 In 85% to 95% of cases, women are affected; the median age is 52 years. The observation that previous blood transfusions can be sensitizing explains why, on occasion, males develop PTP. Sometimes the presumably sensitizing transfusion occurs only a few weeks earlier; consequently, PTP can present after just a few weeks of intermittent transfusions.206 Although thrombocytopenia usually lasts 1 to 4 weeks, the duration can be as short as 3 days219 to as long as 4 months

Chapter 23: Management of Immune Thrombocytopenia

or more. The platelet count usually is less than 10,000/µL (Table 23-6). Mucocutaneous bleeding (wet purpura, petechiae, epistaxis, gastrointestinal, urinary tract) is common, and approximately 5% to 10% of patients die, usually because of intracranial hemorrhage. Because effective treatments are available (see below), it is important to diagnose PTP promptly to minimize morbidity and mortality. Diagnostic confusion with HIT can result because both syndromes can present 5 to 10 days following surgery, and sometimes PF4/heparin antibodies are present because of concomitant exposure to heparin.220,221

Pathogenesis Almost invariably, high-titer, platelet-specific alloantibodies are found in the patient’s serum or plasma. Anti-HPA-1a is detected in 60% of cases, although several other platelet alloantigens have been implicated [HPA-1b, -2b, -3a, -3b, -4b, -5a, 5b, -15b, and the isoantigen CD-36 (Naka)].206 More than one specificity is observed in approximately 15% of cases. As in NAIT, the HLA-DRB3*0101 antigen is found in most HPA-1anegative patients with PTP. Although platelet-specific alloantibodies are causative, the pathogenesis of PTP remains obscure, and the conundrum is why autologous platelets are destroyed. The currently favored hypothesis is that PTP represents a situation in which alloantibodies resulting from reexposure to an incompatible platelet alloantigen evince autospecificity (“pseudospecificity”). Although the platelet-specific alloantibodies are detectable for years following an episode of PTP, the autoreactive (or panreactive) antibodies are detectable only during the acute (thrombocytopenic) phase of PTP. In keeping with this view, Taaning and Tønnesen222 reported that panreactive GPIIb/IIIa antibodies are readily detected during, but not after, an episode of PTP. Kiefel et al223 reported that antibody with allospecificity for HPA-1a, but not for HPA-1b, could be eluted from both autologous and donor HPA-1bb platelets that had been sensitized with acute phase serum from a PTP patient, suggesting that use of adsorption/elution methods may help distinguish a reactivity profile of PTP sera from that seen with NAIT. One report224 suggests that such alloantibodies with autoreactivity could arise spontaneously, because a woman with HPA-1bb platelets and no history of blood transfusion developed “ITP” with antibodies showing specificity for HPA-1a. Cure of the thrombocytopenia by splenectomy was accompanied by disappearance of the HPA-1a-like antibodies. Studies by another group of investigators225 indicate that two distinct types of antibodies—some with alloreactivity and others with autoreactivity—develop during the acute phase of PTP. Treatment High-dose IVIG is the treatment of choice for PTP. More than 90% of patients respond, attaining a platelet count greater than 100,000/µL in an average of 4 days.226 Although some physicians also give corticosteroids, this agent probably does not influence the course of disease and should be considered adjunctive rather than primary therapy. In rare instances, splenectomy may be

considered for a patient refractory to IVIG, corticosteroids, and compatible platelet transfusion.227 Whole-blood-derived (unselected) platelets—which are likely to bear the HPA-1a antigen—are usually destroyed quickly, and can cause febrile or even anaphylactoid reactions. Antigen-negative platelets are the preferred component; however, the efficacy of HPA-1a-negative platelet transfusions (for patients with PTP caused by anti-HPA-1a) is also uncertain. Some reports indicate lack of benefit.228 RBC units should be washed229 or filtered228 before administration to remove platelet antigens. Only four patients are known to have developed recurrent PTP with subsequent transfusions.206 Accordingly, for a patient who has recovered from PTP, future precautions usually include avoidance of incompatible blood components (only autologous, washed, or platelet alloantigen-compatible RBCs are given, or platelet alloantigen-compatible plasma or platelet products are given). However, PTP recurrence is uncommon even if incompatible blood is given, possibly because residual high-titer platelet alloantibodies immediately clear the alloantigens. Patients with a history of PTP must not donate blood because their plasma can trigger passive alloimmune thrombocytopenia.

Passive Alloimmune Thrombocytopenia Passive alloimmune thrombocytopenia is characterized by abrupt onset of thrombocytopenia within a few hours after transfusion of a blood component.197 It is caused by the passive transfer of platelet-reactive alloantibodies in the component that rapidly clear the incompatible recipient platelets. Protein-specific platelet-antibody studies confirm that the alloantibodies bind to the recipient’s platelets. However, although the alloantibody can be detected in the donor’s plasma, it may not be detectable in the recipient’s plasma. This finding suggests that almost 100% of the transfused alloantibody binds soon after transfusion.210,230 Only two alloantigens (HPA-1a and -5b) have been implicated in this syndrome.197,210,230 In general, the severity of bleeding parallels the degree of thrombocytopenia; thus, spontaneous mucocutaneous bleeding usually occurs only in patients with severe thrombocytopenia caused by anti-HPA-1a. The duration of thrombocytopenia is generally less than 1 week. It is important to investigate suspected passive alloimmune thrombocytopenia, because the risk that numerous recipients can develop this syndrome means that the implicated blood donor must not donate blood in the future.

Transplantation-Associated Alloimmune Thrombocytopenia In rare instances, alloimmune mechanisms explain thrombocytopenia that occurs in the setting of HSCT or transplantation of solid organs.

Hematopoietic Transplantation Panzer et al231 reported a 32-year-old man with chronic myeloid leukemia who had severe thrombocytopenia (platelet count, 17,000/µL) beginning 18 months after allogeneic marrow transplantation from his HLA-matched sister. High-dose IVIG gave

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transient increases in platelet count, and persisting remission followed splenectomy. Antibodies with HPA-1a specificity were eluted from the patient’s platelets. This led to further investigations, which showed that a small number of residual, nonneoplastic lymphoid cells of host origin produced anti-HPA-1a against the HPA-1a-positive platelets formed by donor-derived megakaryocytes. Thus, host-vs-donor alloimmune thrombocytopenia resulted from mixed chimerism, in which residual host lymphoid cells derived from the HPA-1a-negative individual developed an alloimmune response against platelets derived from the engrafted HPA-1a-positive marrow. A similar situation attributable to anti-HPA-5b after allogeneic marrow transplantation for chronic myeloid leukemia has been reported.232 However, in this patient, HPA-5b alloantibodies were detectable both before and after transplantation, and the early posttransplantation thrombocytopenia gradually improved as elutable anti-HPA-5b became more difficult to detect. Alloimmune thrombocytopenia may have played a role in two cases with transfusion-refractory thrombocytopenia associated with a rise in titer of anti-HPA-1a (compared with the pretransplantation state) that developed following autologous HSCT performed for metastatic carcinoma of the breast. However, these reports are not conclusive, because it is difficult to distinguish a PTP-like illness (which implies destruction of engrafted autologous donor marrow-derived platelets) from typical posttransplantation platelet transfusion refractoriness.233

Solid-Organ Transplantation In rare instances, immunocompetent lymphoid cells within a transplanted solid organ cause alloimmune thrombocytopenia in the recipient of the organ. A dramatic scenario was reported by West et al.234 All three organ recipients (two of a kidney, one of a liver) had severe thrombocytopenia and bleeding within 5 to 8 days after transplantation from a multiparous female organ donor with normal platelet counts. The two recipients of renal transplants had thrombocytopenia refractory to high-dose IVIG and platelet transfusions. One of these patients died, but the other recovered after splenectomy performed 50 days after transplantation. The liver transplant recipient had organ rejection, which was accompanied by correction of the platelet count when he received a new liver allograft. HPA-1a alloantibodies were detected in the organ donor and posttransplant (but not pretransplant) recipient serum. These cases illustrate that passenger immunocompetent lymphoid cells occasionally induce severe alloimmune thrombocytopenia when introduced into an alloincompatible environment.

Platelet Transfusion Refractoriness Platelet transfusion refractoriness, which is failure to achieve the expected platelet increment after two consecutive platelet transfusion episodes, has several explanations (Table 23-7). Nonimmune, patient-dependent factors are probably the most important, which means that poor platelet count recoveries can persist even when HLA alloimmunization is prevented with

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Table 23-7. General Causes of Platelet Transfusion Refractoriness, Listed in Probable Descending Order of Frequency* ● ●

●

● ● ●

Nonimmune mechanisms Septicemia, fever, disseminated intravascular coagulation, amphotericin B therapy, hypersplenism, fixed platelet count requirements in severe thrombocytopenia Platelet-nonspecific alloantibodies HLA alloantibodies ABO alloantibodies Platelet-specific alloantibodies Drug-dependent antibodies (eg, vancomycin) Platelet-reactive antibodies

*Modified with permission from Warkentin & Smith.197

leukocyte-reduced blood components235 and when HLA- or ABO-compatible platelets are given. There is anecdotal evidence that platelet-specific alloantibodies sometimes cause refractoriness. However, prospective studies have shown that this is a relatively infrequent occurrence. For example, Novotny et al236 found that even when HLA alloantibody formation was largely prevented with blood components filtered before storage, platelet-specific alloantibodies at most explained 4 of 79 (5%) of cases of refractoriness. There are occasions, however, on which the transfusion service needs to provide HLA- and platelet-specific antigen-compatible platelet products to manage certain of these patients.237

Summary A variety of platelet-antibody and other assays have improved the ability of the clinician to make an accurate diagnosis of immune thrombocytopenia in many diverse clinical settings that can involve pathogenic autoantibodies, alloantibodies, and drugdependent antibodies. The treatment decisions that arise depend upon several relevant factors, including the nature of the specific diagnosis, the expected prognosis, and the presence of clinically evident bleeding or thrombosis.

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Chapter 23: Management of Immune Thrombocytopenia

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224. Taaning E, Kilmann S. A case of autoimmune thrombocytopenic purpura serologically similar to post-transfusion purpura. Vox Sang 1992;62:108-12. 225. Watkins NA, Smethurst PA, Allen D, et al. Platelet αIIbβ3 recombinant autoantibodies from the B-cell repertoire of a posttransfusion purpura patient. Br J Haematol 2002;116:677-85. 226. Mueller-Eckhardt C, Kiefel V. High-dose IgG for post-transfusion purpura—revisited. Blut 1988;57:163-7. 227. Cunningham CC, Lind S. Apparent response of refractory posttransfusion purpura to splenectomy. Am J Hematol 1989;30:112-3. 228. Win N, Matthey F, Slater GP. Blood components—transfusion support in post-transfusion purpura due to HPA-1a immunization. Vox Sang 1996;71:191-3. 229. Gabriel A, LaBnigg A, Kurz M, Panzer S. Post-transfusion purpura due to HPA-1a immunization in a male patient: Response to subsequent multiple HPA-1a-incompatible red-cell transfusions. Transfus Med 1995;5:131-4. 230. Warkentin TE, Smith JW, Hayward CPM, et al. Thrombocytopenia caused by passive transfusion of anti-glycoprotein Ia/IIa alloantibody (anti-HPA-5b). Blood 1992;79:2480-4. 231. Panzer S, Kiefel V, Bartram CR, et al. Immune thrombocytopenia more than a year after allogeneic marrow transplantation against donor platelets with anti-PlA1 specificity: Evidence for a hostderived immune reaction. Br J Haematol 1989;71:259-64.

232. Bierling P, Pignon JM, Kuentz M, et al. Thrombocytopenia after bone marrow transplantation caused by a recipient origin Br a allo-antibody: Presence of mixed chimerism 3 years after the graft without hematologic relapse. Blood 1994;83:274-9. 233. Roy V, Verfaillie CM. Refractory thrombocytopenia due to antiPLA1 antibodies following autologous peripheral stem cell transplantation: Case report and review of literature. Bone Marrow Transplant 1996;17:115-7. 234. West KA, Anderson DR, McAlister VC, et al. Alloimmune thrombocytopenia after organ transplantation. N Engl J Med 1999;341:1504-7. 235. The Trial to Reduce Alloimmunization to Platelets Study Group. Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions. N Engl J Med 1997;337:1861-9. 236. Novotny VMJ, van Doorn R, Witvliet MD, et al. Occurrence of allogeneic HLA and non-HLA antibodies after transfusion of prestorage filtered platelets and red cells: A prospective study. Blood 1995;85:1736-41. 237. Kekomäki S, Volin L, Koistinen P, et al. Successful treatment of platelet transfusion refractoriness: The use of platelet transfusions matched for both human leucocyte antigens (HLA) and human platelet alloantigens (HPA) in alloimmunized patients with leukaemia. Eur J Haematol 1998;60:112-8.

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Bleeding from Acquired Coagulation Defects and Antithrombotic Therapy Thomas J. Raife,1 Jeffrey S. Rose,2 & Steven R. Lentz3 1

Associate Professor of Pathology, and Director, Division of Transfusion Medicine and Blood Banking, Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA 2 Hematology/Oncology Fellow, Department of Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA 3 Hamilton Professor of Hematology, Director, Division of Hematology, Oncology, and Blood and Marrow Transplantation, and Professor of Medicine, Department of Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA

Acquired disorders of hemostasis may occur in association with specific clinical conditions or may arise spontaneously in otherwise healthy patients. Unlike inherited coagulation disorders, acquired coagulation disorders are often diagnosed in adult patients, although they may present at any age. Bleeding symptoms can vary from mild bruising and mucosal bleeding to prolonged postoperative bleeding and severe hemorrhage. If the patient does not have a history of significant hemostatic challenge such as surgery or trauma, it may be difficult to distinguish an acquired coagulation defect from an undiagnosed congenital disorder such as mild hemophilia or von Willebrand disease. Management of acute bleeding in patients with acquired coagulation disorders requires prompt recognition of the specific hemostatic defect to guide therapy. The initial laboratory evaluation should usually include the prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time, fibrinogen level, and platelet count. In most patients, the bleeding time test is not recommended, because it is not informative about the risk of bleeding or the proper treatment. Automated hemostatic assays, including use of the platelet function analyzer (PFA-100, Dade Behring, Deerfield, IL) and thromboelastography, are receiving increasing attention as potentially more informative alternatives to the bleeding time test.1,2 The acquired coagulation defects discussed in this chapter, and their characteristic effects on laboratory tests of hemostasis, are summarized in Table 24-1. Congenital disorders of coagulation are considered in Chapter 28, and hemostatic support in the perioperative setting and solid organ transplantation are reviewed in Chapters 38 and 40, respectively.

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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Liver Disease Pathophysiology Liver disease and liver transplantation are associated with multiple hemostatic defects, including quantitative and qualitative abnormalities of coagulation factors, thrombocytopenia, and platelet dysfunction. The liver produces most of the soluble components of the coagulation pathways and many of the regulatory proteins, including Factors II, V, VII, IX, X, XI, XII, and XIII, as well as prekallikrein, high-molecular-weight kininogen, plasminogen, α2-macroglobulin, antiplasmins, antithrombin, thrombin activatable fibrinolysis inhibitor, tissue factor pathway inhibitor, ADAMTS13 metalloprotease, protein C, and protein S.3,4 Factor VIII, the factor that is deficient in patients with classical hemophilia, is also produced by the liver. However, plasma levels of Factor VIII are determined by release of von Willebrand factor (vWF) from endothelium; therefore, levels may be elevated, rather than decreased, in patients with liver disease. The liver also plays a key role in clearance of activated coagulation factors and fibrinolytic fragments that interfere with hemostatic mechanisms. Significant liver dysfunction can, therefore, lead to bleeding from impaired biosynthesis of multiple hemostatic factors combined with failed clearance of inhibitory factors. In both acute and chronic liver disease, the extent of hepatocellular damage generally correlates with the magnitude of the hemostatic defect, as assessed by laboratory tests and clinical bleeding. In acute hepatitis, plasma levels of the vitamin-Kdependent Factors II, VII, IX, and X are quantitatively decreased. Deficiency of Factor VII usually precedes that of other factors because of its short (6-hour) biological half-life. Levels of Factor IX tend to be less depressed than other vitamin-K-dependent factors. Levels of Factor V are variably decreased in acute hepatitis and are often decreased in chronic liver disease. Levels of fibrinogen are usually elevated in acute hepatitis as part of the acute phase response. In chronic hepatitis, levels of fibrinogen are typically

Chapter 24: Bleeding from Acquired Coagulation Defects and Antithrombotic Therapy

Table 24-1. Laboratory Tests of Hemostasis in Patients with Acquired Coagulation Defects Disorder or Therapy

PT

aPTT

TT

Fibrinogen

Platelet Count

Liver disease

↑

(↑)

↑

(↓)

(↓)

Vitamin K deficiency or warfarin therapy

↑

↑

N

N

N

Disseminated intravascular coagulation

↑

↑

↑

↓

↓

Inhibitor to Factor VIII (acquired hemophilia)

N

↑

N

N

N

Acquired von Willebrand syndrome

N

(↑)

N

N

N

Inhibitor to Factor V

↑

↑

N

N

N

Acquired Factor X deficiency

↑

↑

N

N

N

Lupus anticoagulant

(↑)

(↑)

N

N

N

Heparin therapy

N

↑

↑

N

N

Direct thrombin inhibitor therapy

↑

↑

N

N

N

Fibrinolytic therapy

↑

↑

↑

↓

N

PT
prothrombin time; aPTT
activated partial thromboplastin time; TT
thrombin time. Test results are indicated as normal (N), increased (↑), or decreased (↓). Parentheses indicate a mild or variable effect.

normal or mildly decreased, with levels less than 100 mg/dL indicative of end-stage liver disease and poor prognosis. Biosynthesis of dysfunctional hemostatic proteins also contributes to the bleeding diathesis of liver disease. Impaired vitamin K utilization results in the production of dysfunctional, noncarboxylated vitamin-K-dependent factors. Production of abnormal fibrinogens (dysfibrinogenemia) and accumulation of fibrin degradation products (FDPs) can impair coagulation. Dysfibrinogenemia caused by abnormal posttranslational processing of fibrinogen is common in chronic active hepatitis and cirrhosis. The abnormal fibrinogens have been shown to have antithrombin activity and to impair fibrin polymerization, which may result in the formation of fibrin clots with impaired stability. Elevated FDP levels are caused by disseminated intravascular coagulation (DIC) and impaired hepatic clearance of FDPs. Thrombocytopenia and platelet dysfunction are important components of the hemostatic deficit in liver disease. In chronic liver disease, portal hypertension with resultant splenomegaly and sequestration of platelets is the primary cause of thrombocytopenia. In acute viral hepatitis thrombocytopenia is usually modest, although platelet-dependent hemostatic mechanisms may be defective even when the platelet count is normal.

Laboratory Features The PT is the most sensitive laboratory screening test for coagulation defects in liver disease. The PT is particularly sensitive to decreased levels of Factor VII because of its short half-life. Factor VII is a reliable indicator of hepatic synthesis of vitamin-K-dependent hemostatic factors. The aPTT and thrombin time are also prolonged because of deficiencies of multiple coagulation factors and the inhibitory effects of dysfunctional fibrinogens and FDPs. When the thrombin time is prolonged, despite normal levels of fibrinogen and minor elevations of FDPs, dysfibrinogenemia should be suspected. In acute hepatitis, the fibrinogen level is usually normal, but may be

elevated when there is an acute phase response. A significant decline in plasma fibrinogen occurs only when acute liver disease is severe. Prolonged clotting times usually correct to normal when the patient’s plasma is mixed 1:1 with normal plasma. Levels of individual vitamin-K-dependent coagulation factors, as well as the non-vitamin-K-dependent Factor V, are often decreased in liver disease. In contrast, levels of Factor VIII are typically normal or elevated. It is usually not necessary to measure coagulation factors in patients with liver disease. However, measurement of Factors V, VII, and VIII can be useful in some patients to help distinguish between vitamin K deficiency (in which Factor VII is decreased and Factors V and VIII are normal), synthetic liver dysfunction (in which Factors V and VII are decreased and Factor VIII is normal or elevated), and consumption of coagulation factors (in which all three factors are generally decreased).

Management of Bleeding Bleeding associated with severe liver disease is a major clinical challenge requiring surgical, pharmacologic, and transfusion support. Because the hemostatic defect invariably includes deficiency of coagulation factors, often with coexisting thrombocytopenia, transfusion of plasma and platelets are the mainstay of therapy. Plasma or platelets may be transfused to treat active bleeding when the international normalized ratio (INR) is greater than 1.5 or the platelet count is below 50,000/µL, respectively. The utility of transfusion of plasma when the INR is more modestly elevated (ie, between 1.1 and 1.4) is uncertain.5 The INR is a very poor predictor of bleeding in conjunction with surgical procedures, and the prophylactic transfusion of plasma to correct modest elevations of the INR before procedures has not been demonstrated to have a significant impact on clinical outcomes.5 The prophylactic transfusion of plasma to correct modest elevations of the INR or in advance of minor procedures should be discouraged.

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In bleeding patients, a typical initial dose of plasma is 15 mL/ kg, with doses ranging up to 30 mL/kg in severe liver disease. The hemostatic benefit of plasma transfusion is often quite transient, necessitating frequent dosing, which can cause volume overload. The PT is used to monitor efficacy and to determine requirements for repeated administration. The PT may remain prolonged by up to 3 seconds despite hemostatically adequate levels of vitamin-K-dependent factors. It is often difficult to correct such mild elevations of the PT, and attempts to do so may consume large quantities of plasma without success. This difficulty may be related to the short half-life of Factor VII, the poor recovery of Factor IX from transfused plasma, and the loss of transfused clotting factors into third space fluid. Prolongation of the PT by more than 6 seconds often indicates that multiple factors are deficient. Fibrinogen levels are seldom decreased enough from liver dysfunction to cause major bleeding. However, severe hypofibrinogenemia (fibrinogen level less than 100 mg/mL) in a patient with active bleeding should be treated with cryoprecipitate. Each unit of cryoprecipitate contains 150 mg to 250 mg of fibrinogen. A typical initial dose of 10 to 15 units should increase the plasma concentration of fibrinogen by 50 to 75 mg/dL. In Europe, fibrinogen concentrates fractionated from plasma and treated to inactivate or remove virus are available for this purpose. An actively bleeding patient with a platelet count below 50,000/µL generally should be treated with transfusion of platelets. As with transfused plasma, multiple physiologic factors often diminish the recovery of transfused platelets. Although liver disease can cause qualitative platelet dysfunction, the therapeutic role of platelet transfusion in the absence of thrombocytopenia is unproven.3 Vitamin K should be administered to most patients with coagulopathy and liver disease, but its effectiveness is often poor; even if the PT is shortened, clinical bleeding may persist. Desmopressin acetate (DDAVP), which stimulates release of vWF (and possibly Factor VIII) from endothelial cells, is sometimes used. However, Factor VIII and vWF are often elevated in liver disease, so the value of DDAVP is uncertain and has not been rigorously tested.6 An evolving approach to the rapid treatment of the coagulopathy of liver disease is the use of recombinant activated Factor VII (rFVIIa).7 Doses of rFVIIa ranging from 5 to 120 µg/kg have been used before liver biopsy, with prompt normalization of the PT occurring in most patients. However, in this and several other settings of hemostatic challenge in liver disease, normalization of the PT associated with the use of rFVIIa has not been demonstrated to prevent bleeding or improve clinical outcomes. Thus, the utility of rFVIIa in treating the coagulopathy of liver disease remains unproven. Prothrombin complex concentrates, which contain vitaminK-dependent coagulation factors, are gaining renewed interest for treating the coagulopathy of liver disease.8 Older prothrombin complex concentrates were occasionally associated with serious thromboembolic complications, but newer formulations appear to be safer and have been used successfully to rapidly reverse the coagulopathy of liver disease.

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Vitamin K Deficiency Pathophysiology Vitamin K comprises a group of fat-soluble vitamins that are available from many dietary sources and from gastrointestinal flora. Because there are two independent sources of vitamin K, deficiency solely from inadequate dietary absorption or inadequate production by gastrointestinal flora is rare. Deficiency usually arises from disruption of both sources. Vitamin K is a coenzyme in the hepatocellular pathway that synthesizes coagulation Factors II, VII, IX, and X, as well as the regulatory factors protein C, protein S, and protein Z. Vitamin K is required for the γ-carboxylation of several amino terminal glutamyl residues during posttranslational processing. The carboxylated glutamyl residues provide negative charge densities that allow formation of ionic bridges between the factors and phospholipid surfaces and also facilitate protein-protein interactions between coagulation factors. The ability of the vitamin-Kdependent clotting factors to assemble on anionic phospholipid surfaces, such as platelet membranes, is essential for normal hemostasis. In conditions of vitamin K deficiency, plasma levels of vitamin-K-dependent coagulation factors are quantitatively decreased, and production of dysfunctional noncarboxylated factors further contributes to impaired hemostasis.

Hemorrhagic Disease of the Newborn Hemorrhagic disease of the newborn is a postnatal bleeding disorder caused by inadequate production of vitamin-K-dependent coagulation factors. A transient physiologic decrease in coagulation factors normally occurs during the newborn period because of synthetic limitations of the immature liver. With maternal or newborn vitamin K deficiency, plasma concentrations of vitamin-K-dependent factors can decline to levels that are inadequate to maintain hemostasis. Bleeding in hemorrhagic disease of the newborn can be severe and can include melena, intracranial hemorrhage, bleeding from circumcision, generalized ecchymosis, and intramuscular hemorrhage. Factors that predispose to hemorrhagic disease of the newborn include prematurity, delayed bacterial colonization of the gut, liver disease, inadequate maternal or infant vitamin K intake, and prenatal exposure to warfarin or anticonvulsant drugs. Because breast milk is a poor source of vitamin K, postnatal deficiency of vitamin K tends to be more severe in breast-fed infants who do not receive vitamin K supplementation. The PT is prolonged in hemorrhagic disease of the newborn, and levels of all of the vitamin-K-dependent factors are decreased. Levels of fibrinogen and Factors V and VIII are usually normal, as is the platelet count. Treatment relies on administration of vitamin K1 (phytonadione). Intramuscular injection of 0.5 to 1 mg of vitamin K1 usually produces normal neonatal levels of vitamin-K-dependent factors within 24 hours. In cases of

Chapter 24: Bleeding from Acquired Coagulation Defects and Antithrombotic Therapy

severe hemorrhage, transfusion of plasma should be given along with vitamin K1. Treatment with vitamin K1 may be less effective in premature infants because of liver immaturity. Caution should be used with administration of vitamin K1 in very large doses, which can produce hemolysis, hyperbilirubinemia, and kernicterus. Prophylactic administration of vitamin K1 to newborns (1 mg parenterally or 2 mg orally) diminishes the transient decrease in vitamin-K-dependent factors and prevents hemorrhagic disease of the newborn. This practice is mandated by law in many countries, which accounts for the rarity of the disorder in the developed world.

Other Causes of Vitamin K Deficiency Malabsorption syndromes, including celiac disease, sprue, inflammatory bowel disease, and parasitic infestations, can impair absorption of dietary vitamin K. Absorption of vitamin K also can be impaired in severe biliary stasis or after ingestion of bile acid sequestering resins. Ingestion of high-dose aspirin, highdose vitamin E, warfarin, or other anticoagulants is a common cause of vitamin K deficiency, either from decreased absorption, or vitamin K antagonism. Antibiotic therapy contributes to vitamin K deficiency by inhibiting the synthetic capacity of vitaminK-producing bacteria. Certain antibiotics, such as cephalosporins that contain an N-methyl thiotetrazol ring, interfere directly with vitamin K activity. The combination of inadequate dietary intake of vitamin K and use of broad spectrum antibiotics is an insidious cause of vitamin K deficiency in hospitalized patients.

Treatment A single oral dose of 5 to 10 mg of vitamin K1 usually restores adequate levels of vitamin-K-dependent coagulation factors within 24 hours. Larger doses may be required in patients with severe deficiency, particularly those associated with warfarin or other anticoagulants. In cases of ingestion of warfarin-like rodent poisons, doses of vitamin K1 up to 100 mg daily (orally or intravenously) may be required because of the potency and extremely long biological half-lives of these poisons. If emergent reversal of the bleeding diathesis is required, transfusion of plasma (15 mL/ kg) should raise levels of vitamin-K-dependent factors to 30% to 50% of normal. Both rFVIIa and prothrombin complex concentrates have been used successfully to urgently reverse the effects of vitamin K antagonists in small numbers of patients.9–11 General guidelines for the reversal of warfarin anticoagulation are summarized below in the section on Antithrombotic Therapy.

Disseminated Intravascular Coagulation Pathophysiology DIC is a syndrome of diverse etiology that is characterized by pathologic activation of procoagulant and fibrinolytic pathways. Systemic activation of these pathways creates two seemingly

paradoxical clinical problems: tissue injury caused by disseminated microvascular thrombosis, and hemorrhage caused by consumption of coagulation factors and accelerated fibrinolysis. Some patients, such as those with DIC caused by meningococcemia, experience severe thrombosis of the skin and other organs but have little clinical evidence of bleeding. Other patients with DIC present with bleeding from surgical sites, severe ecchymosis, and diffuse oozing from phlebotomy sites and mucosal surfaces. Still other patients have clinical manifestations of both thrombosis and hemorrhage. DIC is usually a secondary phenomenon, and the clinical entities that underlie its development are impressively diverse. They include obstetric accidents, intravascular hemolysis, sepsis, viral illnesses, crush injuries, burns, head injuries, autoimmune and inflammatory disorders, malignancies, toxins, and medications. The most common causes of DIC in the developed world are obstetric accidents and infection. Worldwide, venomous snake bite is estimated to be among the most common causes of DIC. DIC often occurs in patients who have features of the systemic inflammatory response syndrome (SIRS). In SIRS, pathologic processes evoke the production of inflammatory mediators that stimulate expression of tissue factor and release of plasminogen activators from endothelial cells.12 Tissue factor initiates coagulation pathways, leading to the production of thrombin, which in turn stimulates platelet aggregation, activation of coagulation Factors V and VIII, and cleavage of fibrinogen to form fibrin matrices. Generalized production of thrombin can lead to diffuse microvascular deposition of fibrin and subsequent organ failure. Thrombin is normally regulated by the thrombomodulin/protein C anticoagulant system, but this regulatory system is impaired in DIC because of downregulation of thrombomodulin and consumption of protein C. Plasminogen activators activate plasmin, which proteolytically cleaves both fibrin clots (fibrinolysis) and soluble fibrinogen (fibrinogenolysis). Generation of plasmin in the systemic circulation causes proteolytic consumption of fibrinogen and accumulation of FDPs, both of which contribute to bleeding in DIC. Platelet activation and aggregation lead to thrombocytopenia and bleeding in DIC. Consumption of platelets in DIC can be extremely rapid, overwhelming the capacity of the marrow to replenish the circulating pool of platelets. In addition, partial activation of platelets and accumulation of FDPs may produce a functional defect in the platelets that remain in the circulation. Severe deficiency of the vWF-cleaving metalloprotease ADAMTS13 has been reported in DIC and may contribute to platelet microvascular thrombosis.13

Clinical Features DIC is a spectrum of disorders with acute and chronic manifestations. In fulminant acute DIC, thrombosis and hemorrhage often produce multi-organ failure manifested by renal, pulmonary, hepatic, cutaneous, and central nervous system dysfunction. Metabolic instability, hypotension, fever, proteinuria,

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Section II: Part I

and hypoxia are common. Hematologic signs include generalized ecchymosis, petechiae, or skin necrosis, and bleeding from mucosal surfaces, venipuncture sites, and surgical sites. In DIC associated with meningococcemia, the appearance of rapidly progressing retiform skin lesions is a poor prognostic sign. The skin lesions in these patients are caused by widespread thrombosis of dermal and subdermal vessels. The thrombotic diathesis of DIC also can include significant large vessel thrombosis. Chronic DIC reflects low-grade systemic activation of hemostatic pathways and is associated with underlying vascular disease, autoimmune disorders, chronic inflammatory disorders, malignancies, and chronic liver disease. Because hemostatic compensatory mechanisms usually keep pace with consumption, bleeding may be less prominent than thrombotic complications, except with severe thrombocytopenia. Clinical manifestations are variable and may include deep venous thrombosis or migratory thrombophlebitis as well as epistaxis and ecchymosis.

Laboratory Features Many laboratory tests have been developed to aid in the diagnosis of DIC. These tests include assays for activated coagulation factors, specific activation fragments of coagulation factors, complexes of hemostatic factors with inhibitors, fibrin monomer, and various products of fibrin degradation. Although these assays offer the potential to refine the diagnostic criteria and pathophysiologic understanding of DIC syndromes, a more commonly available set of laboratory tests suffices in most clinical circumstances. The initial laboratory evaluation should include the PT, aPTT, platelet count, fibrinogen concentration, and a test for FDPs or D-dimer. Collectively, these assays reflect the degree of depletion of hemostatic factors and the extent of fibrinolytic activity. The PT and aPTT assess the collective function of coagulation factors and provide an indication of available hemostatic resources. A low or falling fibrinogen level in combination with other coagulation abnormalities is strong evidence for DIC. FDPs and D-dimer are almost always elevated in DIC because of accelerated fibrinolysis. FDP levels reflect both fibrinolysis and fibrinogenolysis, while D-dimer detects only fibrinolysis and is a more specific indicator of fibrin formation. FDPs and D-dimer do not distinguish between the systemic fibrinolysis of DIC and the localized fibrinolysis that occurs following trauma or surgical procedures. Thus these tests must be interpreted together with fibrinogen levels and other hemostatic assays. In chronic DIC, consumption of coagulation factors is partially compensated by their increased hepatic synthesis. Levels of fibrinogen and Factor VIII are often elevated as acute phase reactants. The PT and aPTT are normal or shortened. Increased FDP and D-dimer levels are of particular value in the diagnosis of chronic DIC.

Treatment Treatment of DIC requires control of the triggering pathologic process. Management of bleeding and thrombosis is an

380

important adjunctive measure, and can provide time for definitive therapies, such as antimicrobial therapy, surgery, or cancer treatment, to become effective. Prevention of ischemic organ injury is of paramount concern. Other supportive measures may include volume expansion and correction of hypotension to improve microcirculation and optimize tissue oxygenation. The use of heparin in DIC highlights the dilemma of a disorder that simultaneously produces bleeding and thrombosis. While it may be counterintuitive to consider using an anticoagulant in the face of pathologic bleeding, the devastating consequences of systemic thrombosis make heparin a rational treatment for DIC. Moreover, by slowing consumption of coagulation factors, heparin may paradoxically improve the bleeding diathesis. Heparin inhibits thrombin activity and slows the consumptive process while reducing microvascular fibrin deposition. Heparin has been found to prevent complications of DIC in certain syndromes such as DIC associated with acute promyelocytic leukemia or solid tumors, but its value in treating DIC in other clinical settings is less certain. The initial dose of heparin in DIC is usually relatively low (for example, 8 U/kg/hour by continuous intravenous infusion), and titrated according to the hemostatic response. Declining levels of FDPs and D-dimer, rising levels of fibrinogen, and shortening of the aPTT demonstrate the efficacy of heparin in slowing consumption of coagulation factors and decreasing fibrin formation. Despite theoretical concerns about “feeding the fire” of thrombosis, transfusion of platelets, fibrinogen, and other coagulation factors can be lifesaving in some patients with severe bleeding caused by DIC. In patients with active bleeding or impending biopsy and laboratory evidence of severe consumption of fibrinogen (fibrinogen level less than 100 mg/dL) or other coagulation factors (PT greater than 1.5  control value), transfusion of cryoprecipitate (0.2 unit/kg) or plasma (15 mL/kg) is indicated. It is important to remember that many patients who have definitive laboratory evidence of DIC may not have clinically significant thrombosis or hemorrhage. Such patients typically do not require any specific therapy other than treatment of the underlying process that is triggering DIC. When bleeding associated with DIC is refractory to heparin and transfusion therapy, antifibrinolytic agents such as εaminocaproic acid are occasionally used. In addition to stabilizing clots, antifibrinolytic agents also reduce FDP production. However, inhibition of fibrinolysis has the potential to exacerbate fibrin deposition, resulting in severe thrombotic complications. Therefore, antifibrinolytic agents should be reserved for life-threatening hemorrhage, and should be considered for use only in conjunction with heparin in the setting of DIC. Potential newer therapeutic agents for DIC include antithrombin and activated protein C, as well as small molecules that directly inhibit thrombin or Factor Xa.14 Antithrombin is a serine protease inhibitor that inactivates thrombin, Factor Xa, and other coagulation factors. Heparin is a cofactor for antithrombin, and if antithrombin becomes depleted in DIC, the effectiveness of heparin anticoagulation may be limited. Antithrombin has been reported to be beneficial as an adjunctive to heparin therapy in

Chapter 24: Bleeding from Acquired Coagulation Defects and Antithrombotic Therapy

DIC when its levels are depleted to less than 70% of normal.14,15 Protein C is an anticoagulant agent that inhibits thrombin production by inactivating Factors Va and VIIIa. Protein C and activated protein C have shown efficacy in clinical trials of DIC associated with bacterial sepsis.16,17 These new agents and others in development offer the promise of improved treatment of DIC in the future.14

Coagulation Factor Inhibitors Normal blood plasma contains several proteins that inhibit activated coagulation factors. These natural coagulation factor inhibitors include antithrombin, heparin cofactor II, α1-protease inhibitor, α2-macroglobulin, C1 inhibitor, plasminogen activator inhibitors, and tissue factor pathway inhibitor. They function to limit the extent of hemostatic and fibrinolytic reactions and thereby localize thrombi to sites of vascular injury. In contrast to natural coagulation factor inhibitors, pathologic inhibitors are usually immunoglobulins that bind directly to coagulation factors and either inhibit their activity or increase their clearance. Pathologic inhibitors, also known as circulating anticoagulants, may occur as an immunologic response to coagulation factor therapy in patients with hereditary hemophilia, or may arise spontaneously as autoantibodies in patients without a history of abnormal hemostasis.18 Patients who spontaneously develop inhibitors of coagulation Factor VIII often present with a severe bleeding diathesis, and are said to have “acquired hemophilia.” Spontaneous inhibitors of other coagulation factors are encountered less frequently, but also can cause abnormal bleeding. When a coagulation factor inhibitor is suspected, it is essential to distinguish between an inhibitor of a specific coagulation factor, which often causes bleeding, and a nonspecific inhibitor such as a lupus anticoagulant, which may predispose to thrombosis rather than bleeding.

Factor VIII Inhibitors (Acquired Hemophilia) Acquired hemophilia is a rare disorder that is caused by the spontaneous development of an autoantibody inhibitor of coagulation Factor VIII. The annual incidence has been estimated to be about one case per million.18 Autoantibodies to Factor VIII occur mainly in adults; they may arise in the postpartum period or in association with immunologic disorders such as systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, or lymphoproliferative disorders. Approximately 50% of cases occur in elderly patients without any underlying medical condition. The characteristic clinical presentation of acquired hemophilia is the appearance of pathologic hemorrhage in a patient with no history of abnormal bleeding. Patients may present with rapidly enlarging ecchymosis, soft tissue hematoma, gross hematuria, hemarthrosis, or gastrointestinal bleeding. Bleeding is sometimes severe and life threatening. With no known history of a bleeding disorder, the presence of an inhibitor is often not

recognized before surgical procedures, and some patients are diagnosed only after experiencing excessive postoperative bleeding.18 The presence of a circulating inhibitor to Factor VIII can be readily detected in the hemostasis laboratory. Typically, the aPTT is prolonged, and it does not correct to the normal reference range when repeated on a 1:1 mixture of the patient’s plasma with normal plasma. Occasionally, the 1:1 mixture must be incubated for up to 2 hours to allow the inhibitor to completely inactivate Factor VIII before the aPTT is performed. The PT is usually normal. The Factor VIII activity level is low (often less than 10% of normal), but levels of other coagulation factors (such as Factors IX, XI, and XII) are normal. If multiple coagulation factors are affected, a nonspecific inhibitor (lupus anticoagulant) should be suspected. The diagnosis of a specific Factor VIII inhibitor is confirmed by a quantitative assay of the inhibitor level (often expressed in “Bethesda units” or BU). One Bethesda unit is defined as the amount of inhibitor that neutralizes half the Factor VIII activity in a 1:1 mixture with normal plasma. The natural history of acquired hemophilia is variable. In some cases, such as those that present in the postpartum period, the inhibitor disappears spontaneously within weeks to months.19 In other cases, the inhibitor persists for many years. In patients with active bleeding, the immediate goal of treatment is to control acute hemorrhage (Table 24-2). Invasive procedures, intramuscular injections, and the use of antiplatelet agents should be avoided if possible. If the bleeding is mucosal, an antifibrinolytic agent such as ε-aminocaproic acid should be given. If the inhibitor level is low (less than 10 BU), large doses of human Factor VIII (starting dose of 100 to 150 U/kg, followed by continuous infusion of 10 U/kg/hour) can overwhelm the inhibitor. Many clinicians prefer to use recombinant human Factor VIII, rather than plasma-derived Factor VIII concentrates, to minimize exposure to multiple blood donors.18 Factor VIII levels should be measured frequently to assess the response to Table 24-2. Management of Acute Bleeding Episodes in Patients with Acquired Factor VIII Inhibitors Low inhibitor level (10 BU) ● Recombinant human Factor VIIa ● Porcine Factor VIII concentrate ● Prothrombin complex concentrate ● Activated prothrombin complex concentrate ● Plasmapheresis or immunoadsorption, followed by human Factor VIII concentrate or recombinant human Factor VIII Adjunctive measures ● Immobilization ● Compression ● Avoid aspirin and other antiplatelet agents ● ε-aminocaproic acid (if bleeding is mucosal)

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treatment; the usual goal of Factor VIII replacement in a patient with active bleeding is to maintain a Factor VIII activity level that is greater than 50% of normal. Patients with very low inhibitor levels (less than 3 BU) may respond to DDAVP, which acts by stimulating release of endogenous vWF and Factor VIII from endothelial storage sites. DDAVP is administered either intravenously or nasally, and can be repeated every 24 hours for up to 3 days. Side effects of DDAVP include fluid retention and hyponatremia; therefore, this drug should not be used in elderly patients with a history of cardiovascular disease. Repeated use of DDAVP for more than three doses may result in loss of its hemostatic effectiveness because of depletion of vWF from storage sites.6 If the inhibitor level is high (greater than 10 BU), the likelihood of achieving a therapeutic response to human Factor VIII is low, and alternative treatments should be considered. Plasmapheresis or immunoadsorption columns have been used to acutely decrease the plasma level of a circulating inhibitor to allow successful treatment with human Factor VIII.20 Some patients respond well to porcine Factor VIII concentrate (starting dose of 50 to 100 U/kg), because most acquired inhibitors have low cross-reactivity with porcine Factor VIII. However, continuous or repeated use of porcine Factor VIII often results in the development of inhibitors of porcine Factor VIII, which limits therapeutic options for management of future episodes of bleeding. It is recommended, therefore, that use of this product be restricted to life- and limb-threatening emergencies. Other therapeutic options for the management of acute bleeding in patients with acquired hemophilia include rFVIIa, prothrombin complex concentrates, and activated prothrombin complex concentrates (also called Factor VIII inhibitor bypassing concentrates). These products contain activated coagulation factors that appear to bypass Factor-VIII-dependent clotting reactions. Because the goal of therapy is to bypass (rather than replace) Factor VIII, it is not necessary to measure Factor VIII levels when using these products. It is important to distinguish between prothrombin complex concentrates, which contain Factor VIII bypassing activity, and highly purified plasma-derived Factor IX concentrates or recombinant Factor IX (eg, BeneFIX, Wyeth BioPharma, Madison, NJ), which cannot be used to bypass Factor VIII inhibitors. The long-term management of patients with acquired Factor VIII inhibitors should be directed toward eradication of the inhibitor. Although some inhibitors disappear spontaneously, even after many years, a trial of immunosuppressive therapy should be considered in most patients. Some patients respond to corticosteroids alone.18 Other immunosuppressive treatments include cyclophosphamide, azathioprine, cyclosporin A, intravenous immunoglobulin, and rituximab.19

Lupus Anticoagulants Lupus anticoagulants are autoantibodies that nonspecifically inhibit phospholipid-dependent coagulation reactions in vitro. Although first recognized in patients with systemic lupus

382

erythematosus, nonspecific inhibitors are actually encountered more frequently in patients without lupus. Lupus anticoagulants are induced by certain medications, including procainamide, hydralazine, quinidine, and chlorpromazine, or may occur in association with human immunodeficiency virus infection. Lupus anticoagulants also arise spontaneously in patients who are otherwise healthy. Lupus anticoagulants represent a subset of a larger group of “antiphospholipid” autoantibodies that recognize phospholipidprotein complexes. Antiphospholipid antibodies are usually either IgG or IgM; other immunoglobulin classes are rare. Lupus anticoagulants often prolong the aPTT, but rarely prolong the PT. The aPTT usually does not correct completely to normal when the patient’s plasma is mixed 1:1 with normal plasma, but correction in mixing studies can be observed with lupus anticoagulants of low titer or low avidity. Some lupus anticoagulants do not prolong either the aPTT or PT. Several alternative clotting assays, including the dilute Russell viper venom time and kaolin clotting time, have been developed as high-sensitivity screening tests for lupus anticoagulants. Regardless of whether the aPTT or one of the alternative assays is used to detect the lupus anticoagulant, a confirmatory test should be performed to establish that the inhibitor is phospholipid dependent.21 Paradoxically, although lupus anticoagulants prolong clotting times in vitro, they are often associated clinically with thrombosis rather than with bleeding. Thrombotic events in patients with lupus anticoagulants can be either venous (such as deep venous thrombosis and pulmonary embolism) or arterial (such as myocardial infarction, stroke, and peripheral arterial occlusion). A syndrome of severe microvascular thrombosis resulting in acute multi-organ failure (the “catastrophic antiphospholipid antibody syndrome”) has been described in some patients.22 In addition to thrombosis, patients with lupus anticoagulants may experience pregnancy loss, thrombocytopenia, neurologic symptoms, and livedo reticularis. The mechanisms by which lupus anticoagulants predispose to these clinical conditions are incompletely understood, but may involve autoantibody-mediated activation of procoagulant cell surface receptors23 or disruption of the anticoagulant properties of annexin A5.24 Individual patients with antiphospholipid antibodies usually have positive laboratory test results for lupus anticoagulants, anticardiolipin antibodies, anti-β2-glycoprotein I antibodies, or a combination. However, none of the currently available laboratory tests for antiphospholipid antibodies predict with certainty which patients have an increased risk for clinical complications, and it is likely that only a subset of individuals with abnormal test results are actually predisposed to thrombosis. Patients with lupus anticoagulants, either with or without anticardiolipin antibodies, have a higher risk for thrombotic complications than those with isolated anticardiolipin antibodies.25 Women with persistence of antiphospholipid antibodies also have an increased risk for miscarriage and other complications of pregnancy. Identification of a lupus anticoagulant in a bleeding patient is important for several reasons. First, because most lupus

Chapter 24: Bleeding from Acquired Coagulation Defects and Antithrombotic Therapy

anticoagulants do not cause a bleeding diathesis (with the rare exception of those associated with severe thrombocytopenia or hypoprothrombinemia; see below), a search for another abnormality of hemostasis should be undertaken. Second, recognition that prolongation of the aPTT is caused by a lupus anticoagulant may prevent inappropriate transfusion of plasma and other blood components. Third, all patients with lupus anticoagulants should be considered to be at high risk for thrombosis, particularly in the settings of surgery, trauma, or pregnancy. Patients with lupus anticoagulants may have coexistent autoimmune thrombocytopenia. The thrombocytopenia is often mild, but can be associated with abnormal bleeding if the platelet count decreases to less than 50,000/µL. Another circumstance in which lupus anticoagulants are directly associated with increased risk of bleeding, rather than thrombosis, is the hypoprothrombinemia-lupus anticoagulant syndrome. Patients with this syndrome appear to have an autoantibody inhibitor that causes accelerated clearance of prothrombin from plasma.26 The PT is typically prolonged out of proportion to the aPTT. The diagnosis can be confirmed by performing a specific assay for prothrombin (Factor II) activity or antigen. A prothrombin level below 20% of normal can produce severe hemorrhage. Treatment of bleeding in such patients is challenging. Transfusion of plasma may be ineffective. Treatment options for acute bleeding include intravenous IgG and rFVIIa.27,28 Immunosuppressive therapy with corticosteroids, danazol, or rituximab may be effective for long-term management.29,30 In patients with venous or arterial thrombosis, anticoagulant therapy with heparin followed by long-term anticoagulation with warfarin is usually indicated. The presence of a lupus anticoagulant may interfere with monitoring of heparin using the aPTT, and alternative monitoring methods such as Factor Xa inhibition assays may be needed to measure the heparin level. Alternatively, fixed-dose low-molecular-weight (LMW) heparin can often be used without laboratory monitoring. Lupus anticoagulants also can influence the INR in patients who are treated with warfarin. In such patients, it may be necessary to monitor warfarin therapy by measuring the level of a specific vitamin-K-dependent factor (such as Factor II or X).31

Acquired von Willebrand Syndrome Acquired deficiency of vWF arises spontaneously in previously healthy individuals or occur in association with neoplastic, rheumatologic, or hematologic disorders. Up to 50% of patients have monoclonal gammopathies or lymphoproliferative disorders.32 The clinical presentation of acquired von Willebrand syndrome is similar to that of congenital von Willebrand disease, except that it occurs in individuals with no personal or family history of abnormal bleeding. Symptoms vary from mild cutaneous and mucosal bleeding (ecchymosis, epistaxis, gingival hemorrhage, or menorrhagia) to severe, life-threatening hemorrhage. The diagnosis should be suspected in patients who have a prolonged bleeding time or a prolonged platelet function analyzer (PFA-100) closure time. The aPTT is either prolonged or normal.

Levels of vWF activity (ristocetin cofactor activity), vWF antigen, and Factor VIII are often depressed. Most cases are caused by autoantibody inhibitors of vWF. The inhibitors cause rapid clearance of vWF from the circulation, and inhibitory activity often cannot be demonstrated in the patient’s plasma by mixing studies. Variant forms of acquired von Willebrand syndrome occur in some patients with solid tumors, particularly Wilms’ tumor. In these patients, the deficiency of vWF is not mediated by autoantibodies, but is caused instead by absorption of vWF to malignant cells or cell products.33 Selective loss of highmolecular-weight multimers of vWF can be seen in patients with aortic valve stenosis.34 Management of bleeding in patients with vWF inhibitors can be difficult. As in patients with other bleeding disorders, invasive procedures, intramuscular injections, and the use of antiplatelet agents should be avoided if possible. In patients with an underlying hematoproliferative disorder, treatment with chemotherapeutic or cytoreductive agents may lead to resolution of the long-term bleeding diathesis.32 In patients with acute bleeding, therapeutic options include DDAVP or infusion of Factor VIII concentrates that contain large quantities of vWF [eg, Humate P (ZLB Behring, King of Prussia, PA); Koate-DVI (Bayer Corporation, West Haven, CT); Alphanate (Grifols, Los Angeles, CA)]. Although these partially purified Factor VIII concentrates are derived from pooled plasma, they are subjected to virus attenuation procedures during processing, so the risk of viral transmission is considered to be low. Highly purified Factor VIII concentrates or recombinant human Factor VIII cannot be used, because these products generally contain little or no vWF. Purified vWF concentrates are available in Europe. Cryoprecipitate can be given as a source of vWF, but its use is discouraged because of a greater risk of exposure to blood-borne pathogens. Replacement therapy may be only transiently effective, because of the short half-life of vWF caused by excessive clearance from the circulation. Antifibrinolytic agents such as ε-aminocaproic acid may be beneficial, particularly in patients with mucosal bleeding. Treatment with rFVIIa may be efficacious in some patients with vWF inhibitors.35 Administration of high-dose intravenous IgG (2 g/kg over 2 to 5 days) may result in a rapid increase in vWF levels. Repeated administration of intravenous IgG every 3 weeks may be effective in controlling chronic bleeding.36

Inhibitors of Factor V Autoantibody inhibitors of Factor V can arise in patients following surgery, transfusions, or antibiotic therapy. The majority of cases have been reported in patients who were exposed to topical hemostatic agents, such as bovine thrombin or fibrin sealant, that contain trace amounts of bovine Factor V. The inhibitor presumably arises as an alloantibody to bovine Factor V that cross-reacts with human Factor V.37 Inhibitors to Factor V may occur after a single exposure to fibrin sealant, but reexposure appears to increase the likelihood of inhibitor development.38 Typically, both the PT and aPTT are prolonged, and do

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not correct to normal when performed on a 1:1 mixture of the patient’s plasma with normal plasma. The thrombin time also is prolonged because of the presence of a coexisting inhibitor to bovine thrombin. The diagnosis can be confirmed by performing a quantitative assay of the Factor V inhibitor level. The clinical presentation is quite variable, ranging from apparently normal hemostasis to severe hemorrhage, possibly because of differential effects of the inhibitor on plasma and platelet pools of Factor V. Treatment options for acute bleeding are limited; some patients respond to transfusion of platelets, immunoadsorption, or plasmapheresis.39 In a majority of patients, the inhibitor disappears within weeks to months, and immunosuppressive therapy does not appear to influence the time course of the disease.

Acquired Factor X Deficiency Acquired deficiency of Factor X can occur in patients with primary (AL) amyloidosis. The deficiency arises from accelerated clearance of Factor X from the circulation resulting from adsorption of Factor X to amyloid fibrils.40 Both the PT and the aPTT are prolonged, but unlike the situation in patients with circulating inhibitors, the PT and aPTT usually correct to normal when performed on a 1:1 mixture of the patient’s plasma with normal plasma. The hemostatic defect in these patients is multifactorial; in addition to Factor X deficiency, patients with amyloidosis may have excessive fibrinolysis and amyloid infiltration of blood vessels. Management of bleeding in such patients is problematic. Replacement therapy in the form of plasma or prothrombin complex concentrate is often ineffective, and antifibrinolytic agents such as ε-aminocaproic acid are of limited benefit.

Other Coagulation Factor Inhibitors Autoantibody inhibitors of other coagulation factors are rarely encountered. As with inhibitors of Factor V, inhibitors of prothrombin prolong both the PT and the aPTT. Inhibitors of Factors IX or XI prolong only the aPTT. Specific factor and inhibitor assays can be performed to distinguish the type of inhibitor present. Depending on the inhibitor, treatment options for bleeding episodes include plasma, prothrombin complex concentrates, Factor IX concentrates, or rFVIIa. Adjunctive treatment with antifibrinolytic agents such as ε-aminocaproic acid also may be beneficial. Inhibitors of Factor XIII do not affect either the PT or aPTT; specific assays of fibrin stabilization (such as the urea clot lysis assay) are necessary to identify these inhibitors.41 In patients with acute bleeding, large doses of cryoprecipitate or Factor XIII concentrate can be given to try to overwhelm the inhibitor, but this approach may have limited effectiveness.

Acquired Platelet Function Disorders Acquired disorders of platelet function occur much more frequently than congenital platelet abnormalities. Acquired platelet dysfunction is caused by drugs such as aspirin, medical

384

conditions such as chronic renal insufficiency, or procedures such as cardiopulmonary bypass. The risk of bleeding in patients with acquired platelet dysfunction is variable and unpredictable, and abnormal bleeding generally occurs only in the presence of additional hemostatic defects. Signs of platelet dysfunction manifest typically as mucocutaneous bleeding with excessive bruising, gingival bleeding, or epistaxis. Diagnostic tests of platelet function include the bleeding time and the platelet function analyzer (PFA100). Although useful as screening tests, these tests lack diagnostic specificity, and they cannot be used to predict bleeding risk. The bleeding time may be prolonged because of abnormalities of cutaneous connective tissue, and both the bleeding time and the PFA-100 test results may be abnormal in the presence of anemia (hematocrit less than 30%) or thrombocytopenia (platelet count less than 100,000/µL). Many medications can alter platelet function and lead to a delayed PFA-100 closure time or prolonged bleeding time, so a thorough medication history must be obtained before these tests are performed. Formal platelet aggregation testing may be useful in the evaluation of selected patients.

Drug-Induced Platelet Dysfunction A large number of drugs and medications have been reported to impair platelet function (Table 24-3). Among the most frequently encountered are aspirin, nonsteroidal anti-inflammatory drugs, and antiplatelet agents such as ticlopidine, clopidogrel, and abciximab. The antiplatelet effects of aspirin are irreversible, so recovery of normal hemostasis relies on the release of new platelets into the circulation rather than the disappearance of the drug from the plasma. The ability of platelets to aggregate is partially restored within 4 to 5 days after aspirin ingestion is stopped, but platelet dysfunction may persist for up to 7 days after aspirin is discontinued.42 If possible, aspirin should be discontinued 7 to 10 days before a surgical or endoscopic procedure, unless it has been prescribed for the secondary prevention of stroke or myocardial infarction.43 Most other nonsteroidal anti-inflammatory drugs inhibit platelet reactivity in a reversible manner, and platelet function usually returns to normal within 24 hours after the

Table 24-3. Some Drugs that Cause Impairment of Platelet Function Aspirin

Nitroglycerin

Nonsteroidal anti-inflammatory drugs

Isosorbide dinitrate

Ticlopidine

Nitroprusside

Clopidogrel

Nitroglycerin

Abciximab

Isosorbide dinitrate

Eptifibatide

Nitroprusside

Tirofiban

Nitroglycerin

β-Lactam antibiotics

Isosorbide dinitrate

Prostacyclin

Nitroprusside

Dipyridamole

Nitroglycerin

Dextrans

Chapter 24: Bleeding from Acquired Coagulation Defects and Antithrombotic Therapy

drug is discontinued. Nonsteroidal anti-inflammatory drugs that selectively inhibit cyclooxygenase-2, such as celecoxib, appear to have very little effect on platelets and can be used to treat pain in patients with preexisting bleeding disorders such as hemophilia. The risk of clinical bleeding caused by aspirin or other nonsteroidal anti-inflammatory agents is generally low, and platelet transfusions should not be given prophylactically. However, ingestion of these drugs can increase the risk for serious bleeding in patients who also have additional hemorrhagic risk factors. If severe hemorrhage caused by defective platelet function is suspected, transfusion of platelets can rapidly restore normal hemostasis. The thienopyridines, ticlopidine and clopidogrel, are platelet adenosine diphosphate (ADP)-receptor antagonists that inhibit platelet function by disrupting interactions with ADP and fibrinogen. Similar to aspirin, these drugs produce a persistent antiplatelet effect that lasts for up to a week after the drug is discontinued.42 Therefore, these drugs usually should be discontinued 5 to 7 days before invasive procedures. Abciximab is the prototype of a class of antiplatelet drugs that directly block the binding of fibrinogen to its platelet receptor, glycoprotein (GP) IIb/IIIa (integrin αIIbβ3). Other drugs in this class include eptifibatide and tirofiban. These agents are indicated for use in acute coronary syndromes and to prevent thrombosis of intravascular stents. They are often given in conjunction with other antithrombotic agents such as heparin.44 Abciximab has an extended biological half-life because of its high affinity for GPIIb/IIIa; its antiplatelet effects can therefore persist for several days. In contrast, the antiplatelet effects of eptifibatide and tirofiban are transient, and usually resolve within a few hours of discontinuation in patients with normal renal function.45 Management of bleeding in patients receiving GPIIb/IIIa antagonists may be complicated by the development of acute thrombocytopenia (platelet count less than 50,000/µL), which occurs in up to 5% of patients.46 In cases of severe bleeding, platelet transfusions are an effective approach to control hemorrhage, either in the presence or absence of thrombocytopenia. Many other drugs can impair platelet function (Table 24-3). The antiplatelet effects of β-lactam antibiotics are generally apparent only in patients receiving large parenteral doses of penicillins or cephalosporins. The frequency of clinically important bleeding in patients taking β-lactam antibiotics appears to be low, and is not predicted by the bleeding time or other laboratory tests of platelet function. Prostacyclin and dipyridamole inhibit platelet aggregation by elevating the intracellular concentration of cyclic adenosine monophosphate. Infusion of dextran inhibits platelet aggregation and enhances fibrinolysis through multiple mechanisms. Nitrovasodilators (nitroglycerin, isosorbide dinitrate, and nitroprusside) inhibit platelet function through nitric-oxide-dependent mechanisms. Both tricyclic antidepressants and selective serotonin reuptake inhibitors can alter platelet function, but the risk of serious clinical bleeding appears to be low.47 Phenothiazines such as chlorpromazine, promethazine, or trifluoroperazine also have been reported to have mild antiplatelet effects. Consumption of ethanol can produce platelet

dysfunction as well as thrombocytopenia, and may contribute to clinical bleeding in patients with alcoholic liver disease.

Uremia Chronic renal insufficiency is associated with both hemorrhagic and thrombotic manifestations. Bleeding often manifests as ecchymoses, epistaxis, or gastrointestinal or genitourinary bleeding. The primary hemostatic abnormality in uremia is thought to be a defect in platelet function, but the pathophysiology of the platelet function defect remains poorly understood. Both dialyzable and nondialyzable substances contribute to the defect. As in many other acquired disorders of platelet function, the clinical importance of platelet dysfunction in uremia is uncertain. The bleeding time and PFA-100 closure time are prolonged, and platelet aggregation studies reveal defects in aggregation with ADP and epinephrine. In general, abnormalities of the bleeding time or platelet aggregation responses do not correlate with the severity of renal insufficiency or the degree of bleeding. Management of bleeding in uremia is directed by the clinical circumstances rather than the results of laboratory tests of platelet function. Many patients with chronic renal failure do not have significant problems with bleeding despite a prolonged bleeding time and abnormal platelet aggregation responses. If bleeding is encountered in a patient with uremia, assessment of hemoglobin and hematocrit, platelet count, PT, and aPTT is necessary to evaluate for other potential causes of defective hemostasis. Coexisting anemia contributes to a bleeding propensity, and patients with bleeding may benefit from transfusion of red cells, or treatment with erythropoietin or darbopoietin, to maintain the hematocrit above 30%. Treatment with DDAVP or estrogens improves the bleeding time and prevents clinical bleeding in some patients with uremia.48 Transfusion of cryoprecipitate may partially correct the hemorrhagic diathesis of uremia. Transfusion of platelets is usually not recommended in the absence of thrombocytopenia (platelet count less than 50,000/µL), because uremic plasma can induce dysfunction in transfused platelets. Patients with uremia may be unusually sensitive to anticoagulants or antiplatelet medications such as aspirin, which should be discontinued if possible. The benefit of intensive dialysis in correcting abnormal platelet function and diminishing bleeding is uncertain.48

Cardiopulmonary Bypass Abnormal platelet function, often in conjunction with thrombocytopenia, is a frequent cause of bleeding in patients undergoing cardiopulmonary bypass. The risk of perioperative bleeding varies depending on the type of surgical procedure, the age of the patient, preoperative renal function, and the duration of bypass. Prior exposure to antiplatelet agents and other antithrombotic medications increases the risk of bleeding. The platelet defect caused by cardiopulmonary bypass is thought to result from activation of platelets within the extracorporeal circulation. Platelet activation is stimulated by multiple mediators, including thrombin generated from activation of the intrinsic and extrinsic coagulation pathways, mechanical stress,

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complement activation, hypothermia, and exposure of platelets to the blood/air interface.49 Partial activation and degranulation leads to desensitization and decreased adhesiveness of residual circulating platelets. The severity of platelet dysfunction correlates with the duration of bypass. Platelet function abnormalities usually resolve within 5 hours, but can persist for 24 hours or longer following complicated surgery.50 Bleeding from platelet dysfunction is often exacerbated by thrombocytopenia caused by hemodilution and consumption of platelets. Consumption of coagulation factors, increased fibrinolytic activity, and inadequate neutralization of heparin with protamine sulfate also contribute to bleeding. Management of bleeding associated with cardiopulmonary bypass is generally based on clinical considerations rather than laboratory testing. Bleeding times and PFA-100 closure times are frequently prolonged but are not predictive of perioperative blood loss.50 Routine prophylactic administration of plasma or platelets is discouraged.51 In the setting of excessive perioperative or early (within 24 hours) postoperative bleeding, however, platelet transfusions may be indicated even in the absence of severe thrombocytopenia. Several drugs have been used to decrease the risk of perioperative bleeding during cardiopulmonary bypass. These include DDAVP, antifibrinolytic agents such as ε-aminocaproic acid or tranexemic acid, and the protease inhibitor aprotinin. Some recent clinical trials have raised concern that the use of aprotinin might be associated with an increased risk of long-term morbidity and mortality.52

Antithrombotic Therapy Use of antithrombotic drugs has increased in recent years because of growing recognition of the efficacy of these medications for the prevention and treatment of venous thromboembolism, embolic stroke, and other thrombotic disorders. Although warfarin, heparin, and aspirin continue to be the most commonly prescribed antithrombotic medications, several new antithrombotic drugs are making their way into routine clinical use. These new drugs include LMW heparins, direct thrombin inhibitors, fibrinolytic agents, pentasaccharides, clopidogrel, and platelet GPIIb/IIIa antagonists.53 The major complication of all these medications is bleeding. In this section, the management of bleeding associated with warfarin, heparin, LMW heparins, pentasaccharides, direct thrombin inhibitors, and fibrinolytic therapy is considered. Management of bleeding associated with antiplatelet medications is discussed above in the section on Drug-Induced Platelet Dysfunction.

Warfarin Warfarin and other vitamin K antagonists produce their antithrombotic effects by inhibiting the hepatic synthesis of vitaminK-dependent coagulation factors. Vitamin K becomes oxidized to vitamin K epoxide during the γ-carboxylation of coagulation

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Factors II, VII, IX, and X. Warfarin inhibits the cyclic regeneration of active vitamin K by blocking the action of vitamin K epoxide reductase and vitamin K reductase.54 In the absence of adequate concentrations of active vitamin K, hepatic production of normally carboxylated factors is limited, and the liver instead produces hypocarboxylated factors with reduced coagulant activity. Warfarin has a narrow therapeutic range with wide interindividual dosing requirements. Its metabolism is influenced by diet, patient compliance, genetic factors, liver dysfunction, and interactions with other medications. Numerous drugs influence the pharmacokinetics of warfarin by altering its absorption or metabolic clearance, or by altering the production of vitamin K by intestinal flora. As a general rule, any change in medication regimen, including nonprescription medications and dietary supplements that contain vitamin K, should be presumed to have a potential effect on the anticoagulant response to warfarin. The risk of bleeding is highest during the initial phase of anticoagulation, within the first few weeks after initiation of therapy. Dosing algorithms that consider multiple parameters, such as gender, age, weight, height, race, and use of other medications, offer promise for decreasing the risk of early bleeding. Newer dosing algorithms incorporate pharmacogenomic information derived from genotyping two polymorphic genes (CYP2C9 and VKORC1) that are involved in warfarin metabolism.55 The clinical utility of these pharmacogenomic approaches is being tested in several clinical trials.56 The most common laboratory method for monitoring the anticoagulant effect of warfarin is the PT, often reported as the INR. Because the response of the PT to depletion of vitamin-Kdependent coagulation factors is highly variable when measured in different laboratories, the INR has been widely adopted as a method to standardize monitoring of oral anticoagulant therapy. The INR compares the ratio of the patient’s PT to the mean PT of a group of normal individuals. The ratio is adjusted for the sensitivity of the PT reagent used in each laboratory. The target therapeutic INR for most indications is 2.0 to 3.0, although different therapeutic ranges are recommended for some indications.54 The risk of major bleeding in patients on chronic warfarin therapy has been estimated to be 1% to 5% per year.57 Intensity of anticoagulation is probably the most important risk factor for hemorrhage, particularly when the INR is greater than 5.0. Clinical conditions that increase risk of hemorrhage include advanced age, hypertension, cerebrovascular disease, heart disease, renal insufficiency, a history of gastrointestinal bleeding, and concomitant use of antiplatelet agents. The risk of hemorrhagic complications resulting from warfarin therapy is decreased when patients are enrolled in coordinated programs for management of anticoagulation.58 Reversal of the anticoagulant effects of warfarin can be achieved through discontinuation of warfarin, administration of vitamin K1, transfusion of plasma or prothrombin complex concentrates, or treatment with rFVIIa. Management should be

Chapter 24: Bleeding from Acquired Coagulation Defects and Antithrombotic Therapy

guided by the degree of elevation of the INR and the presence or absence of clinical bleeding (Table 24-4). Minor or moderate elevation of the INR (above the therapeutic target but less than 9.0) in the absence of bleeding can often be managed safely by decreasing or omitting several doses of warfarin until the INR approaches the therapeutic range. Major elevation of the INR (greater than 9.0), even in the absence of clinically evident bleeding, should be treated by temporary discontinuation of warfarin and administration of vitamin K1 (5 to 10 mg orally). Vitamin K1 also can be administered intravenously, but intravenous administration is rarely associated with anaphylaxis. A recent metaanalysis found that oral and intravenous vitamin K1 were equally effective in achieving a therapeutic INR at 24 hours in patients with excessive oral anticoagulation.59 Subcutaneous administration of vitamin K1 is associated with an unpredictable response and is therefore not recommended.59,60 In patients with active major bleeding, administration of plasma (15 mL/kg) or prothrombin complex concentrates10 may be lifesaving. Different preparations of prothrombin complex concentrates contain varying amounts of vitamin-K-dependent clotting factors and, therefore, differ in their clinical effectiveness for warfarin reversal.10 Recombinant Factor VIIa is also used in such patients, especially Table 24-4. Guidelines for Reversal of Warfarin Anticoagulation INR

Bleeding

Recommendation

5.0

None Minor

Decrease or omit warfarin dose Discontinue warfarin Consider vitamin K1 (2.5 mg orally) Discontinue warfarin Vitamin K1 (10 mg by slow intravenous infusion*) Fresh frozen plasma (15 mL/kg) or prothrombin complex concentrate Consider recombinant Factor VIIa

Major

5.0-9.0

None Minor Major

9.0

None Minor

Major

Omit one or two doses of warfarin; resume at lower dose Discontinue warfarin Vitamin K1 (2.5 to 5.0 mg orally) Discontinue warfarin Vitamin K1 (10 mg by slow intravenous infusion*) Fresh frozen plasma (15 mL/kg) or prothrombin complex concentrate Consider recombinant Factor VIIa Hold warfarin Vitamin K1 (5 to 10 mg orally) Discontinue warfarin Vitamin K1 (5 to 10 mg orally) Consider fresh frozen plasma (15 mL/kg) Discontinue warfarin Vitamin K1 (10 mg by slow intravenous infusion*) Fresh frozen plasma (15 mL/kg) or prothrombin complex concentrate Consider recombinant Factor VIIa

*Intravenous administration of vitamin K1 may produce (but rarely) an anaphylactic reaction. INR
International Normalized Ratio

if there is life-threatening hemorrhage.9,54 A dose of rFVIIa as low as 1.2 mg in an adult patient may be effective in normalizing the INR.9,61 The clinical effectiveness of rFVIIa or prothrombin complex concentrates in the setting of warfarin-associated hemorrhage has not been subjected to randomized, controlled trials, however, and both preparations may be associated with an elevated risk of thromboembolic complications.62 Several approaches can be used to manage warfarin anticoagulation in patients who require elective surgery.54 For many patients, a reasonable approach is to discontinue warfarin 4 to 5 days before surgery and begin heparin or a LMW heparin along with warfarin in the postoperative period. Heparin or LMW heparin can then be discontinued when the INR returns to the target therapeutic range. Patients who require major surgery urgently (within 24 hours) should receive plasma (15 mL/kg) and vitamin K1 (2.5 to 5 mg orally or parenterally).

Heparin The antithrombotic effect of heparin is mediated by its ability to potentiate the inhibition of activated coagulation factors by antithrombin. In the presence of heparin, antithrombin irreversibly inactivates thrombin, Factor Xa, and to a lesser extent, other coagulation factors. Heparin is often administered intravenously, although it also may be given subcutaneously. When administered by intravenous infusion, the onset of its anticoagulant action is rapid. Weight-based dosing of heparin appears to improve clinical outcomes in patients with venous thromboembolism.63 A common dosing schedule utilizes an intravenous bolus dose of 80 U/kg followed by 18 U/kg/hour by continuous infusion. The dosage is then adjusted to maintain a therapeutic aPTT. The therapeutic range may vary between different laboratories that utilize different reagents for measuring the aPTT. In many laboratories, prolongation of the aPTT to a value that is 1.5- to 3.0-fold higher than the control value corresponds to a therapeutic heparin level. For certain indications, such as cardiopulmonary bypass surgery, higher doses of heparin are required and monitoring is performed using the activated clotting time rather than the aPTT. The major side effect of heparin therapy is hemorrhage. In clinical studies of heparin administered for short-term treatment of venous thromboembolism, rates of major bleeding have ranged from 0% to 7%.57 In some patients, bleeding is exacerbated by thrombocytopenia or platelet dysfunction. Because heparin has a short biological half-life of about 1 hour, the primary approach to management of bleeding is to discontinue the drug. In patients with therapeutic levels of heparin, recovery from its anticoagulant effects can be expected within 2 hours after discontinuation of heparin infusion. In cases of major hemorrhage, or when very large doses of heparin have been given (eg, for cardiopulmonary bypass or through a medication error), protamine sulfate should be administered. Protamine sulfate is a strongly basic protein that neutralizes the anticoagulant effects of heparin within minutes. It is given by slow intravenous injection. The recommended dose of protamine

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sulfate is 1.0 mg for every 100 units of heparin remaining in the patient, which can be calculated based on the 60-minute halflife of heparin. For example, a patient receiving 1000 units of heparin per hour by continuous intravenous infusion should be given enough protamine to neutralize all of the heparin administered within the last hour (1000 units), plus half of the heparin administered in the preceding hour (500 units), plus a quarter of the heparin administered in the hour prior to that (250 units). Therefore, the total dose of protamine sulfate would be 17.5 mg. Protamine sulfate itself has a weak anticoagulant effect, and overdosage may exacerbate bleeding.

Low-Molecular-Weight Heparins LMW heparins are derived from heparin by chemical or enzymatic fragmentation.63 Several LMW heparins, including dalteparin, enoxaparin, and tinzaparin, are available for prevention or treatment of venous thromboembolism, unstable angina, and other indications. Danaparoid sodium is not derived from heparin, but it is a mixture of LMW heparin-like glycosaminoglycans. Although danaparoid sodium is considered to be a “heparinoid,” its effects are very similar to those of the LMW heparins. Compared with heparin, LMW heparins have less inhibitory activity against thrombin and greater relative inhibitory activity against Factor Xa. The LMW heparins produce a more predictable anticoagulant response than heparin, reflecting their better bioavailability, longer half-life, and dose-independent clearance. These properties allow LMW heparins to be given subcutaneously once or twice daily, usually without laboratory monitoring. Because LMW heparins are cleared through a renal route, dosages need to be adjusted in patients with renal insufficiency. A large number of randomized clinical trials have demonstrated that LMW heparins are at least as safe and effective as heparin for most indications.63 The incidence of heparininduced thrombocytopenia is lower with LMW heparins than with heparin (see Chapter 23). Despite the apparently favorable safety profile of LMW heparins, bleeding remains a major side effect. In clinical trials evaluating LMW heparins for treatment of venous thromboembolism, the rates of major bleeding have ranged from 0 to 3%.57 Epidural bleeding and spinal hematoma have been reported in patients receiving LMW heparins concurrently with spinal or epidural anesthesia.64 Management of bleeding is complicated by the long half-life of these medications. Unlike heparin, the anticoagulant effects of LMW heparins cannot be reversed completely by administration of protamine sulfate. Protamine sulfate can be administered in an attempt to control active bleeding in patients who have received LMW heparin within 8 hours, but large doses (20 to 50 mg) may be required.63

Pentasaccharides The pentasaccharides, fondaparinux and idraparinux, are synthetic analogs of the antithrombin-binding region of heparin.65 These drugs selectively inhibit Factor Xa without having any appreciable

388

inhibitory effect on thrombin. Fondaparinux has a half-life of 18 to 22 hours and is usually administered subcutaneously once daily. It is used for the prevention and treatment of venous thromboembolism and in acute coronary syndromes. Idraparinux is an investigational pentasaccharide that has a substantially longer half-life of 130 hours and can be administered in a fixed dose once weekly.65 The pentasaccharides have no specific antidotes, and their anticoagulant effects are not reversed by administration of plasma, prothrombin complex concentrates, or protamine sulfate.

Direct Thrombin Inhibitors The direct thrombin inhibitors include desirudin, lepirudin, bivalirudin, and argatroban. Because the direct thrombin inhibitors are structurally unrelated to heparin, one major indication for their use is in the treatment of heparin-induced thrombocytopenia. They also are being evaluated for use in prophylaxis of deep venous thrombosis, cardiopulmonary bypass surgery, and acute coronary syndromes.65 Most of the direct thrombin inhibitors are administered parenterally, and are monitored using the aPTT. They have short half-lives (less than 2 hours) when given by intravenous infusion. Because lepirudin, desirudin, and bivalirudin are cleared renally, their half-lives may be prolonged dramatically in renal failure. Clearance of argatroban is not influenced by renal impairment, but may be decreased in the presence of hepatic dysfunction. The risk of bleeding in patients receiving direct thrombin inhibitors appears to be dose-dependent.66 Management of bleeding relies on prompt discontinuation of the drug.

Fibrinolytic Agents Pharmacologic lysis of fibrin thrombi is a commonly used strategy for treatment of acute myocardial infarction and stroke, and also is used in selected cases of peripheral arterial occlusion, and venous thromboembolism. Most fibrinolytic agents in clinical use are plasminogen activators, which include streptokinase, urokinase, and recombinant forms of tissue plasminogen activator such as alteplase and reteplase. Plasminogen activators produce thrombolysis by converting plasminogen to plasmin, which then degrades fibrin into soluble FDPs. Fibrinolytic agents can be administered systemically by intravenous infusion, or delivered in proximity to sites of thrombi via catheter-directed approaches. Contraindications to the use of fibrinolytic therapy include recent hemorrhagic stroke or major surgery, prolonged cardiopulmonary resuscitation, uncontrolled hypertension, and active gastrointestinal bleeding.67 In addition to producing therapeutic lysis of pathologic thrombi, fibrinolytic agents generate plasmin in the systemic circulation, resulting in bleeding from lysis of hemostatic plugs at surgical sites and other locations.67 Bleeding, therefore, is a major complication of fibrinolytic therapy. Because circulating plasmin degrades fibrinogen, hypofibrinogenemia can contribute to bleeding. The extent of systemic fibrinogenolysis varies with different fibrinolytic agents and different routes of administration.68 Laboratory abnormalities associated with the use of

Chapter 24: Bleeding from Acquired Coagulation Defects and Antithrombotic Therapy

fibrinolytic agents include decreased fibrinogen, decreased plasminogen, prolonged thrombin time, and decreased euglobulin clot lysis time. These laboratory parameters are poorly predictive of bleeding risk, however.67 The risk of major hemorrhage associated with fibrinolytic therapy is influenced by the age of the patient and concomitant use of additional antithrombotic agents such as heparin, direct thrombin inhibitors, or antiplatelet agents. In the absence of invasive procedures or simultaneous heparin therapy, treatment of acute myocardial infarction with fibrinolytic agents generally is associated with a low incidence of major bleeding (less than 5%). Intracranial hemorrhage is a rare, but potentially devastating, complication of fibrinolytic therapy. In large clinical trials of streptokinase, or alteplase for treatment of acute coronary ischemia, the incidence of intracranial hemorrhage has ranged from 0.2% to 0.8%.67 Bleeding that occurs in association with fibrinolytic therapy can often be managed by discontinuing infusion of the plasminogen activator and replacing fibrinogen by transfusion of cryoprecipitate (0.2 unit/kg) or (in Europe) by infusion of fibrinogen conentrates. The goal of transfusion therapy with cryoprecipitate is to maintain a plasma fibrinogen level of greater than 100 mg/dL. Adjunctive treatment with antifibrinolytic agents such as ε-aminocaproic acid or aprotinin may be beneficial when rapid reversal of bleeding is desired.

Disclaimer The authors have disclosed no conflicts of interest.

References 1. Mammen EF, Comp PC, Gosselin R, et al. PFA-100 system: A new method for assessment of platelet dysfunction. Semin Thromb Hemost 1998;24:195-202. 2. Tien H, Nascimento B Jr, Callum J, Rizoli S. An approach to transfusion and hemorrhage in trauma: Current perspectives on restrictive transfusion strategies. Can J Surg 2007;50:202-9. 3. Papadopoulos V, Filippou D, Manolis E, Mimidis K. Haemostasis impairment in patients with obstructive jaundice. J Gastrointestin Liver Dis 2007;16:177-86. 4. Zheng X, Chung D, Takayama TK, et al. Structure of von Willebrand factor-cleaving protease (ADAMTS13), a metalloprotease involved in thrombotic thrombocytopenic purpura. J Biol Chem 2001;276: 41059-63. 5. Triulzi DJ. The art of plasma transfusion therapy. Transfusion 2006;46:1268-70. 6. Mannucci PM. Desmopressin (DDAVP) in the treatment of bleeding disorders: The first 20 years. Blood 1997;90:2515-21. 7. Caldwell SH, Chang C, Macik BG. Recombinant activated factor VII (rFVIIa) as a hemostatic agent in liver disease: A break from convention in need of controlled trials. Hepatology 2004;39:592-8. 8. Lorenz R, Kienast J, Otto U, et al. Efficacy and safety of a prothrombin complex concentrate with two virus-inactivation steps in patients with severe liver damage. Eur J Gastroenterol Hepatol 2003;15:15-20.

9. Deveras RA, Kessler CM. Reversal of warfarin-induced excessive anticoagulation with recombinant human factor VIIa concentrate. Ann Intern Med 2002;137:884-8. 10. Lankiewicz MW, Hays J, Friedman KD, et al. Urgent reversal of warfarin with prothrombin complex concentrate. J Thromb Haemost 2006;4:967-70. 11. Riess HB, Meier-Hellmann A, Motsch J, et al. Prothrombin complex concentrate (Octaplex) in patients requiring immediate reversal of oral anticoagulation. Thromb Res 2007;121:9-16. 12. Levi M. Disseminated intravascular coagulation. Crit Care Med 2007;35:2191-5. 13. Ono T, Mimuro J, Madoiwa S, et al. Severe secondary deficiency of von Willebrand factor-cleaving protease (ADAMTS13) in patients with sepsis-induced disseminated intravascular coagulation: Its correlation with development of renal failure. Blood 2006;107:528-34. 14. Levi M, de JE, van der PT. New treatment strategies for disseminated intravascular coagulation based on current understanding of the pathophysiology. Ann Med 2004;36:41-9. 15. Balk R, Emerson T, Fourrier F, et al. Therapeutic use of antithrombin concentrate in sepsis. Semin Thromb Hemost 1998;24:183-94. 16. Bernard GR, Vincent JL, Laterre PF, et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001;344:699-708. 17. Dhainaut JF, Yan SB, Joyce DE, et al. Treatment effects of drotrecogin alfa (activated) in patients with severe sepsis with or without overt disseminated intravascular coagulation. J Thromb Haemost 2004;2:1924-33. 18. Lusher JM. Inhibitor antibodies to factor VIII and IX: Management. Semin Thromb Hemost 2000;26:179-88. 19. Collins PW. Treatment of acquired hemophilia A. J Thromb Haemost 2007;5:893-900. 20. Brzoska M, Krause M, Geiger H, Betz C. Immunoadsorption with single-use columns for the management of bleeding in acquired hemophilia A: A series of nine cases. J Clin Apher 2007;22:233-40. 21. Brandt JT, Triplett DA, Alving B, Scharrer I, for the Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and Standardisation Committee of the ISTH. Criteria for the diagnosis of lupus anticoagulants: An update. Thromb Haemost 1995;74:1185-90. 22. Asherson RA, Cervera R, Piette JC, et al. Catastrophic antiphospholipid syndrome. Clinical and laboratory features of 50 patients. Medicine 1998;77:195-207. 23. de Groot PG, Derksen RH. Pathophysiology of the antiphospholipid syndrome. J Thromb Haemost 2005;3:1854-60. 24. de Laat B, Wu XX, van Lummel M, et al. Correlation between antiphospholipid antibodies that recognize domain I of beta2glycoprotein I and a reduction in the anticoagulant activity of annexin A5. Blood 2007;109:1490-4. 25. Miyakis S, Lockshin MD, Atsumi T, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006;4:295-306. 26. Bajaj SP, Rapaport SI, Barclay S, Herbst KD. Acquired hypoprothrombinemia due to non-neutralizing antibodies to prothrombin: Mechanism and management. Blood 1985;65:1538-43. 27. Pernod G, Arvieux J, Carpentier PH, et al. Successful treatment of lupus anticoagulant-hypoprothrombinemia syndrome using intravenous immunoglobulins. Thromb Haemost 1997;78:969-70. 28. Holm M, Andreasen R, Ingerslev J. Management of bleeding using recombinant factor VIIa in a patient suffering from bleeding

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49. George JN, Shattil SJ. The clinical importance of acquired abnormalities of platelet function. N Engl J Med 1991;324:27-39. 50. Lasne D, Fiemeyer A, Chatellier G, et al. A study of platelet functions with a new analyzer using high shear stress (PFA-100) in patients undergoing coronary artery bypass graft. Thromb Haemost 2000;84:794-9. 51. Despotis GJ, Goodnough LT. Management approaches to platelet-related microvascular bleeding in cardiothoracic surgery. Ann Thorac Surg 2000;70(Suppl):S20-32. 52. Mangano DT, Miao Y, Vuylsteke A, et al. Mortality associated with aprotinin during 5 years following coronary artery bypass graft surgery. JAMA 2007;297:471-9. 53. Weitz JI, Linkins LA. Beyond heparin and warfarin: The new generation of anticoagulants. Expert Opin Investig Drugs 2007;16: 271-82. 54. Ansell J, Hirsh J, Poller L, et al. The pharmacology and management of the vitamin K antagonists: The Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest 2004;126: 204S-33S. 55. Sconce EA, Khan TI, Wynne HA, et al. The impact of CYP2C9 and VKORC1 genetic polymorphism and patient characteristics upon warfarin dose requirements: Proposal for a new dosing regimen. Blood 2005;106:2329-33. 56. Gage BF. Pharmacogenetics-based coumarin therapy. Hematology Am Soc Hematol Educ Program 2006;467-73. 57. Levine MN, Raskob G, Beyth RJ, et al. Hemorrhagic complications of anticoagulant treatment: The Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest 2004;126: 287S-310. 58. Ansell JE. Anticoagulation management clinics for the outpatient control of oral anticoagulants. Curr Opin Pulm Med 1998;4:215-9. 59. Dezee KJ, Shimeall WT, Douglas KM, et al. Treatment of excessive anticoagulation with phytonadione (vitamin K): A meta-analysis. Arch Intern Med 2006;166:391-7. 60. Crowther MA, Douketis JD, Schnurr T, et al. Oral vitamin K lowers the international normalized ratio more rapidly than subcutaneous vitamin K in the treatment of warfarin-associated coagulopathy. A randomized, controlled trial. Ann Intern Med 2002;137:251-4. 61. Dager WE, King JH, Regalia RC, et al. Reversal of elevated international normalized ratios and bleeding with low-dose recombinant activated factor VII in patients receiving warfarin. Pharmacotherapy 2006;26:1091-8. 62. O’Connell KA, Wood JJ, Wise RP, et al. Thromboembolic adverse events after use of recombinant human coagulation factor VIIa. JAMA 2006;295:293-8. 63. Hirsh J, Raschke R. Heparin and low-molecular-weight heparin: The Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest 2004;126:188S-203. 64. Llau JV. Safety of neuraxial anesthesia in patients receiving perioperative low-molecular-weight heparin for thromboprophylaxis. Chest 1999;116:1843-4. 65. Weitz JI, Hirsh J, Samama MM. New anticoagulant drugs: The Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest 2004;126:265S-86S. 66. Anand S. Direct thrombin inhibitors. Haemostasis 1999;29(Suppl S1):76-8. 67. Marder VJ. Thrombolytic therapy: 2001. Blood Rev 2001;15:143-57. 68. Weitz JI, Stewart RJ, Fredenburgh JC. Mechanism of action of plasminogen activators. Thromb Haemost 1999;82:974-82.

PART II

25

Obstetric and Pediatric Patients

Fetal and Neonatal Hematopoiesis Robert D. Christensen1 & Martha C. Sola-Visner2 1 2

Director of Research, Department of Neonatology, Intermountain Health Care, Ogden, Utah, USA Assistant Professor of Pediatrics, Harvard Medical School, and Physician in Medicine, Childrens Hospital, Boston, Massachusetts, USA

The purpose of hematopoiesis in the fetus differs from that in the adult. The primary function of hematopoiesis in adults is to produce sufficient hemic cells to balance hemic cellular losses. In contrast, in the fetus, constant growth and dramatic physiologic changes necessitate a system with other functions. For example, the remarkable rate of somatic growth in the fetus and neonate and the resultant need to constantly increase blood volume necessitate an extraordinary hematopoietic effort, assessed as daily cell production per kilogram of body weight. In addition, the relatively low oxygen tensions but high metabolic rates of fetal tissues require a system of oxygen delivery fundamentally different from that in adults. Moreover, the sterile intraamniotic environment and consequently the low demand for the antimicrobial actions of neutrophils change markedly at birth. The extrauterine environment demands a constant and lifelong need for the antimicrobial actions of neutrophils. These issues are summarized in Table 25-1. The hematopoietic system of a fetus and neonate must be sufficiently plastic to accommodate many marked changes and dichotomies. It has been speculated that improved familiarity with developmental hematopoietic regulation improves interpretation of postnatal hematologic data and enhances appreciation of the granulocytopoietic, erythropoietic, and thrombopoietic capacities and limitations of prematurely delivered neonates. Cytopenia certainly is common in neonatal intensive care units (NICUs). Neutropenia occurs in 5% to 8% of patients in NICUs,1 thrombocytopenia in 25% to 30%,2 and anemia in perhaps as many as 50%3 at some time before discharge. The prevalence of cytopenia among patients in NICUs is related to gestational age. The prevalence is higher among those delivered at the earliest gestational ages. Transfusion is the only means of managing severe cytopenia among neonates, but alternatives to

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

Table 25-1. Inherent Differences in Hematopoietic Systems of the Fetus, Neonate, and Adult Fetus ● Because the fetus exists in a sterile environment, it does not generally need an antibacterial defense system until near term. ● In preparation for extrauterine life, the fetus must produce a neutrophil reserve. ● Fetal hematocrit and blood platelet concentration increase only slightly from 20 weeks to term, whereas blood volume increases approximately 10-fold. Erythrocyte and platelet production must be extremely rapid during this period to keep pace with the rapid expansion of blood volume. Neonate ● At birth, the fetus moves from a sterile into a nonsterile environment and must have a neutrophil reserve already developed to survive in this environment. ● At birth, oxygen delivery to tissues markedly increases, as PaO increases 2 from 27 to
90 mm Hg. This effectively shuts off erythropoietin production. Consequently, erythropoiesis temporarily ceases, resulting in the physiologic anemia of infancy. ● Rapid growth and blood volume expansion continue. However, platelet concentration does not change, and platelet production is very rapid during this period. Adult ● The rapid somatic growth and blood volume expansion of infancy and childhood cease. The previous need to accelerate hematopoiesis to keep pace with somatic growth ends. ● The neutrophil system must continue to be responsive to rapid increases in demand for cells. ● Blood platelet concentration and hematocrit remain relatively constant throughout healthy life.

repeated transfusions should emerge as more is learned about developmental hematopoiesis. Developmental hematopoiesis can be viewed as occurring in three anatomic stages—mesoblastic, hepatic, and myeloid. Mesoblastic hematopoiesis occurs in extraembryonic structures, principally the yolk sac, and begins between the 16th and 19th days of gestation. By about 6 weeks of gestation, the extraembryonic sites of hematopoiesis begin to ablate and hepatic hematopoiesis is initiated. By the 10th to 12th week,

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mesoblastic hematopoiesis ceases, and a small amount of hematopoiesis is evident in the marrow. In humans, it seems that the clavicle is the first bone to develop a marrow cavity.4 The first cells present in the developing marrow space have macrophage surface markers and phenotypes. These are followed by myeloperoxidase-positive cells with the characteristics of neutrophils. However, the liver remains the predominant hematopoietic organ until the last trimester of pregnancy. The anatomic site of hematopoiesis does not simply transfer from yolk sac to liver to marrow.5 Rather, each organ subsequently houses distinct hematopoietic populations. For example, at 18 to 20 weeks of gestation, more than 85% of the cells in the fetal liver are erythroid, and few neutrophils are present. At the same time, fewer than 40% of the cells within the marrow are erythroid, and most are neutrophils. The mechanisms responsible for the changing anatomic sites of hematopoiesis and for the differences in hemic cells produced in the mesoblastic, hepatic, and myeloid sites have not been determined. Regardless of gestational age or anatomic location, production of all hematopoietic tissues begins with pluripotent cells capable of both self-renewal and clonal maturation into all blood cell lineages. Progenitor cells differentiate under the influence of hematopoietic growth factors, which include those listed in Table 25-2.

Granulocytopoiesis Sites of Neutrophil Production in the Fetus A traditional view is that neutrophil production, similar to hematopoiesis in general, begins in the yolk sac and moves to the liver and spleen and finally to the marrow. However, recent results of studies of human fetuses do not support this concept. Specifically, the human yolk sac contains no neutrophils—its hematopoietic activity is limited to erythropoiesis and production of a small number of macrophages.5 Similarly, the human fetal liver produces few, if any, neutrophils. The few neutrophils found in photomicrographic sections of human fetal liver are not arranged in clusters of hematopoietic nests but are widely separated and found surrounding the blood vessels, as if they were carried in by the circulation, not produced in the organ. Moreover, the spleen is not a granulocytopoietic organ in human fetuses, as it is in rodents.6 No granulocytopoietic nests are found in human fetal spleen, and the neutrophils within it are mostly mature and evenly dispersed. This finding suggests they were carried there in the blood, not produced locally. Where, then, do neutrophils originate in the human fetus? The first neutrophils are present approximately 5 weeks after

Table 25-2. Hematopoietic Growth Factors Growth Factors

Molecular Mass (kD)

Chromosomal Location

Principal Target Cell

Erythropoietin

30.4

7q11-22

CFU-E, fetal BFU-E

Colony-stimulating factors G-CSF GM-CSF M-CSF SCF

18.8 14.4 26 (dimer) 15–20 (dimer)

17q11.2-21 5q23-31 1p13-21 12q2-24

CFU-G All CFC CFU-M Primitive CFC

Interleukins IL-1α IL-1β IL-2 IL-3 IL-4 IL-5 IL-6 IL-7 IL-8 IL-9 IL-10 IL-11 IL-12

17 17 15-20 14-15 16-20 13.2 (dimer) 20.8 25 8–10 16 23 22 70-75

2q13 2q13 4q26-27 5q23-31 5q23-31 5q23-31 7p21-24 8q12-13 4 5q31-32 1 19q13

Primitive CFC, hepatocyte, macrophage Primitive CFC, hepatocyte, macrophage T cell All CFCs T cell, B cell CFU-EOS Primitive CFC B cell Neutrophil, endothelial cell BFU-E, primitive CFC Macrophage, lymphocyte Primitive CFC, BFU-MK, CFU-MK T cell, NK cell, macrophage

Thrombopoietin

35

3q26-28

BFU-MK, CFU-MK

BFU-E  primitive erythroid progenitor; G-CSF  granulocyte colony-stimulating factor; CFU-G  colony-forming unit–granulocyte; GM-CSF  granulocyte-macrophage colony-stimulating factor; CFC  colony-forming cells; M-CSF  macrophage colony-stimulating factor; CFU-M  colony-forming unit–macrophage; SCF  stem cell factor; IL  interleukin; CFU-E  colony-forming unit–erythroid; CFU-EOS  colony-forming unit–eosinophil; BFU-MK  primitive megakaryocyte progenitor; CFU-MK  mature megakaryocyte progenitor; NK  natural killer.

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conception and are clustered in the periaortic tissue. These first fetal neutrophils contain myeloperoxidase, and they mature into cells with segmented and band nuclei. But beyond these features, any differences or similarities between them and the neutrophils of adults have not been reported.7 The function of these cells in the fetus and the explanation for this location of origin are unclear. The fetal marrow space begins to develop approximately 8 weeks after conception (Figs 25-1 and 25-2). The space is lined by osteoclast-like cells with cell-surface characteristics of macrophages. These cells appear to core out the space from the primitive cartilage. Eight to 10 weeks after conception, the marrow space progressively enlarges, but no neutrophils are present within the space until 10.5 to 11 weeks4 (Fig 25-3). The first neutrophils in the marrow do not have segmented or band nuclei, but they contain myeloperoxidase and express the cell surface characteristics of myeloblasts and promyelocytes. From 14 weeks to term, the most common cell type in the fetal marrow space is the neutrophil, although the marrow space is not nearly so densely packed with cells as it becomes in older children and adults.4,7 Macrophages are crucial to fetal morphogenesis, because they aid in shaping of organs and scavenging debris and apoptotic cells. Although neutrophils and macrophages have a

Figure 25-1. Clavicle approximately 7 weeks after conception, before any marrow space is present. In the human fetus, the first bone to contain a developing marrow space is the clavicle, followed closely by the other long bones. (Hematoxylin and eosin stain; original magnification, 100).

common progenitor cell, the discordant temporal appearance of neutrophils and macrophages in the fetus and the divergence of their anatomic locations are striking.8 Recent observations have cast doubt on long-held theories about the origins of macrophages in the human fetus. For example, it was believed that macrophages form from precursor cells in the marrow and mature through a progression of cell types from monoblasts to promonocytes to monocytes, which then migrate to various tissues and differentiate into macrophages. Observations of human fetuses do not support this origin of macrophages. First, macrophages appear in the yolk sac, liver, lung, and brain long before the marrow cavity has been formed. Second, promonocytes and monocytes are absent in the yolk sac and liver, but macrophages are present there nevertheless. Thus, the macrophages in the yolk sac may develop directly from stem cells without passing through a monocyte stage. It is not clear whether these primitive macrophages migrate from the yolk sac to populate the lungs, liver, brain, and other organs.9

Regulation of Neutrophil Production The mechanisms that regulate neutrophil production during human fetal development are not clear. Granulocyte colonystimulating factor (G-CSF) and macrophage colony-stimulating factor (M-CSF) are present in developing fetal bone as early as 6 weeks after conception and in the fetal liver as early as 8 weeks.10 Granulocyte-macrophage colony-stimulating factor (GM-CSF) is widely distributed in fetal tissues, including pulmonary epithelium, and is involved in pulmonary homeostasis. Stem cell factor (SCF) messenger RNA (mRNA) is present in the yolk sac, liver, and marrow at the earliest stages of their respective development.5 No changes in mRNA concentrations of these factors or of their specific receptors or in the concentrations of proteins, judged by immunohistochemical staining, appear to constitute the signal for production of neutrophils. The precise signals that initiate the production of macrophages and neutrophils in the embryo and fetus are not known. It is curious that the actions of G-CSF, M-CSF, GM-CSF, and SCF are not limited to hematopoiesis in the fetus and neonate. Receptors for all of these factors are located in distinct areas of the fetal central nervous system and gastrointestinal tract, where their patterns of expression change with development. Important undefined developmental roles clearly exist for these factors that are beyond those known for hematopoiesis. Although not fully characterized, these roles appear to be predominantly antiapoptotic, perhaps providing some degree of protection during adverse conditions such as hypoxemia or acidosis. Although neutrophil production in the marrow space clearly is present by 14 weeks of gestation, the blood of the fetus, even through 20 weeks, contains few neutrophils. Forestier et al.11 reported that fetuses at 20 weeks of gestation had a mean absolute blood neutrophil concentration of only 190/µL, a range of 0 to 490/µL, and a mode concentration of zero.11 Despite the near absence of circulating neutrophils in the first trimester, and the relative scarcity (by adult standards) of neutrophils in the second

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(A)

(B)

(C)

(D)

Figure 25-2. Photomicrographs of clavicles 6 to 14 weeks after conception (Hematoxylin and eosin stain; original magnification, 100). (A) Six weeks after conception. Clavicles at this stage consist of primitive cartilage and contain no marrow cavity and no myeloperoxidase positive cells. (B) Nine weeks after conception. Clavicles at this stage have the beginning of a marrow cavity (arrowhead) but

(A)

no myeloperoxidase positive cells. (C) Eleven weeks after conception. Clavicles at this stage have an elongated marrow cavity and small hematopoietic islands of myeloperoxidase positive cell in the marrow space. (D) Fourteen weeks after conception. Spiculation has begun, and the volume of hematopoietic marrow has begun to increase. (Modified with permission from Slayton et al.4)

(B)

Figure 25-3. Appearance and subsequent expansion of neutrophils within the clavicular marrow cavity 12 to 15 weeks after conception. (A) At 12 weeks of gestation a small number of myeloperoxidase-positive cells are present in the developing marrow cavity. Many of these cells have the morphologic appearance of neutrophils with

segmented or band nuclei and they appear in discrete clusters within the marrow cavity. (Myeloperoxidase stain; original magnification, 400.) (B) At 15 weeks, clavicles have a marrow cavity that contains numerous myeloperoxidase-positive cells. (Original magnification, 200.) (Modified with permission from Slayton et al.4)

trimester progenitor cells with the capacity to generate neutrophils in vitro [colony-forming units-granulocyte-macrophage (CFU-GM)] are abundant in the early human fetal liver, marrow, and blood.12-17 In rodents, the number of CFU-GMs per gram of body weight is far fewer in animals delivered prematurely than

in those delivered at term and is lower in term animals than in adults. The quantity of neutrophilic progenitor cells per gram of body weight in the developing human fetus has not been reported.18 It is not clear whether, as in experimental animals, preterm human infants have a relatively small supply of granulocytic progenitors.

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Chapter 25: Fetal and Neonatal Hematopoiesis

The concentration of CFU-GMs in the venous blood of adults is approximately 20 to 300/mL. In contrast, the concentration in term infants is approximately 2000/mL. Even higher concentrations are present in the blood of infants delivered prematurely. The high concentrations of CFU-GMs in fetal blood, however, do not necessarily indicate a large total body quantity of CFUGMs. It is likely that a significant percentage of fetal CFU-GMs are in the circulation.16 When fetal CFU-GMs are cultured in vitro in the presence of recombinant G-CSF, they undergo maturation into colonies of neutrophils. Fetal CFU-GMs often mature clonally into larger colonies and contain more cells than do CFU-GMs obtained from the marrow of adults.19 The physiologic role of G-CSF includes upregulation of neutrophil production. This appears to be the case for the fetus and neonate as well as for the adult. The low quantities of circulating and storage neutrophils in the midtrimester human fetus may be caused, in part, by low production of G-CSF. Supporting this hypothesis are observations of poor production of G-CSF by cells of human fetal origin.19,20 Monocytes isolated from the blood of adults produce G-CSF when stimulated with a variety of inflammatory mediators, such as bacterial lipopolysaccharide or interleukin-1. In contrast, monocytes isolated from the umbilical cord blood of preterm infants and from the liver and marrow of aborted fetuses up to 24 weeks of gestation generate only small quantities (10 to 100 times less per cell) of G-CSF protein and mRNA after lipopolysaccharide or interleukin-1 stimulation.19,20 Despite the poor capacity to generate G-CSF, it appears that G-CSF receptors on the surface of neutrophils of newborn infants are equal in number and affinity to those on adult neutrophils.

Neonatal Neutropenia Relatively few neutrophils are present in the human embryo and early fetus, and neutrophil production is a relatively minor component of hematopoiesis in the midtrimester fetus. On this basis, one might anticipate that neonates who are delivered extremely prematurely would be at high risk of serious bacterial infection. Indeed, of all the risk factors for neonatal infection analyzed in a national collaborative study21 on neonatal infections, premature birth had the strongest correlation. One might also anticipate that premature neonates, delivered at the limits of viability (22 to 25 weeks of gestation) would have a high likelihood of developing neutropenia. This propensity is indeed seen. For instance, if neutropenia is defined as a blood neutrophil concentration 1000/µL, 40% of neonates weighing 1000 g at birth develop neutropenia at some time during their hospital stay.21 Most such cases are detected on the first day after birth. Most are associated with maternal hypertension, or with a birthweight less than the 10th percentile for gestation, and most are relatively transient, with counts exceeding 1000/µL within a few days (Fig 25-4). When neutropenia first appears after the third day of life, the underlying cause is usually obscure.21 Neutropenia among extremely low birthweight neonates, although common, is viewed as problematic in only two situations—1) neutropenia

during an infectious illness among these patients is a very poor prognostic sign (see Chapter 30 for more information) and 2) neutropenia that is severe and prolonged is important to recognize, because it might indicate one of the syndromes or genetic abnormalities involving severe chronic neutropenia (see Chapter 14 for more information).

Erythropoiesis Erythropoietin in the Fetus and Neonate Production of erythrocytes requires a constant supply of amino acids, lipids, iron, specific vitamins, and trace nutrients.23 Limitations in any of these can thwart production. However, the rate of erythrocyte production is regulated not by any of these substances, but rather by the concentration of erythropoietin. Erythropoietin is an 18.4-kD glycoprotein that binds to specific receptors on the surface of erythroid precursors and various other cells and supports their clonal maturation.23,24 In the human fetus, erythropoietin is produced by a surprising variety of cells, including cells of monocyte-macrophage origin in the liver.25,26 The absence of erythropoietin or its receptor, as has been produced in murine knock-out models, leads to profound anemia and fetal death on approximately embryonic day 13.27,28 Postnatally, erythropoietin is produced almost exclusively by peritubular cells of the kidney, with a small amount produced by liver cells and neuronal and glial cells in the central nervous system. Although some erythropoietin production occurs in the fetal kidney, it is minimal. Anephric fetuses have normal serum erythropoietin concentrations and hematocrits.29 The factors that regulate the switch of erythropoietin production from the liver to the kidney are not known. However, some investigators have suggested that among preterm neonates this switch does not occur until term gestation or close to it; this physiologic delay is responsible for the relatively low concentrations of circulating erythropoietin in the common hyporegenerative anemia known as anemia of prematurity.30 It has become clear that the actions of erythropoietin in the human fetus are widespread. Erythropoietin receptors have been documented on the surface of cells within a surprising variety of nonhematopoietic fetal tissues, including intestinal villi, endothelium, mesangium, smooth muscle, placenta, and neurons.25,26,31,32 Erythropoietin has an antiapoptotic effect on several of these cells types in vitro. Erythropoietin also occurs in relatively high concentration in human amniotic fluid, colostrum, and milk—fluids that are swallowed in large amounts by the fetus and neonate.33 A healthy midtrimester human fetus may swallow 200 to 300 mL of amniotic fluid per kilogram of body weight per day. With an amniotic fluid erythropoietin concentration of 200 mU/mL, the fetus would swallow approximately 60,000 mU/kg a day—an amount that if given systemically to a neonate would have a marked erythropoietic effect. Erythropoietin receptors are present along the villous border of the fetal and neonatal intestine.25,33 It appears that

395

100%

80

80%

60

60%

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40%

20

20%

0

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Cummulative percent

100

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 50 4 9 –1
00 10 0

Number of patients

Section II: Part II

Day of life

Count

Cumulative percent

Figure 25-4. Day of life neutropenia (blood neutrophils 1000/µL) was first detected among 338 neutropenia neontes with birthweights 1000 g. (Used with permission from Christensen et al.21)

erythropoietin in amniotic fluid, colostrum, and human milk is not absorbed into the circulation and has no systemic effect. Rather, it acts locally in the intestine as a trophic and antiapoptotic factor. Erythropoietin in amniotic fluid, colostrum, and milk tends to resist digestive conditions present in the fetal and neonatal gastrointestinal tract.33,34 Erythropoietin is present, in concentrations of 20 to 50 mU/ mL, in the spinal fluid of neonates. Markedly greater spinal fluid concentrations of erythropoietin are seen after perinatal asphyxia, and this erythropoietin appears to be derived within the brain as opposed to crossing the blood-brain barrier.35,36 Further evidence that erythropoietin does not cross the bloodbrain barrier in neonatal humans includes the observation that neonates treated with recombinant erythropoietin do not have elevated erythropoietin concentrations in their spinal fluid.36 The spinal fluid of adults generally has an erythropoietin concentration lower than the lower limit of detectability.35,36 The biologic roles of erythropoietin and erythropoietin receptors in the fetal and neonatal intestinal tract and central nervous system are not known. One potential function for erythropoietin in the fetal brain is as a neuroprotectant.37 This

396

postulate is supported by the observations that erythropoietin production increases in the brain during fetal hypoxia, and that recombinant erythropoietin protects neurons in tissue culture from hypoxemic damage and does so by diminishing apoptosis, which is analogous to the function it has in erythropoiesis.

Fetal Erythroid Progenitors Culture of marrow cells in tissue culture has added to the understanding of erythropoietic regulation. When marrow cells are placed in semisolid media culture systems for 5 to 7 days, the erythropoietin-sensitive precursors [colony-forming units–erythroid (CFU-E)] mature clonally into clusters containing 30 to 100 normoblasts.38 Erythroid-specific progenitors that are less well differentiated than CFU-E, and hence more primitive cells, are called burst-forming units–erythroid (BFU-E). Twelve to 14 days after marrow cells are placed in semisolid culture systems, BFU-E have developed into large clusters of normoblasts, each containing 200 to more than 10,000 normoblasts. BFU-E from human fetuses respond in a slightly different manner than do BFU-E isolated from adults. Specifically, BFU-E of fetal origin generally develop into erythroid clones more rapidly and generally

Chapter 25: Fetal and Neonatal Hematopoiesis

develop substantially more normoblasts than do BFU-E of adult origin.39,40 Also, BFU-E from adult marrow require a combination of erythropoietin plus another factor, such as interleukin3 or GM-CSF, to mature clonally, whereas many fetal BFU-E mature in the presence of erythropoietin alone.39,40

Embryonic, Fetal, and Adult Hemoglobins Tissues must receive a constant supply of oxygen. The development of oxygen-carrying proteins increases the ability of blood to transport oxygen. The binding of oxygen and its dissociation from hemoglobin are accomplished without expenditure of metabolic energy.41 Hemoglobin consists of iron-containing heme groups and globin, a protein moiety. An interaction between heme, globin, and 2,3-diphosphoglycerate (also called 2,3bisphosphoglycerate) gives hemoglobin its unique properties in the reversible transport of oxygen. Hemoglobin is a tetrameric molecule composed of two pairs of polypeptide chains, each encoded by a different family of genes; the α-like globin genes on chromosome 16 and the β-like globin genes on chromosome 11. The main hemoglobin of normal adults (HbA) is made up of one pair each of α and β chains (α2β2). Six distinct hemoglobins can be detected within the erythrocytes of the human embryo, fetus, child, and adult—Gower-1, Gower-2, Portland, fetal hemoglobin (HbF), and the adult hemoglobins HbA and HbA2. The time of appearance and quantitative relations among the hemoglobins are determined by complex developmental processes that are not well defined. Human embryos have the slowly migrating hemoglobins Gower1, Gower-2, and Portland. The ζ chains of Portland and Gower-1 hemoglobins are structurally similar to α chains. Both Gower hemoglobins contain a unique polypeptide chain, the ε chain. Gower-1 hemoglobin has the structure ζ2ε2 and Gower-2, α2ε2. Portland hemoglobin has the structure ζ2γ2. Four to 8 weeks after conception, the Gower hemoglobins predominate, but by 12 to 14 weeks, they are no longer detected. Fetal hemoglobin contains γ chains in place of the β chains of HbA and are represented as α2γ2. The resistance of HbF to denaturation by strong alkali usually is used in its quantitation. After the eighth postconceptional week, HbF is the predominant hemoglobin. At 24 weeks of gestation, it constitutes 90% of total hemoglobin. Thereafter, a gradual decline in HbF occurs, so that at birth the average is 70% of the total. Synthesis of HbF decreases rapidly postnatally, and by 6 to 12 months of age only a trace is present. Fetal hemoglobin is heterogeneous because of two types of γ chains, synthesis of which is directed by two sets of genes. The chains differ at position 136 in the presence of either a glycine (Gγ) or an alanine (Aγ) residue. In the neonate, the relative proportion of Gγ to Aγ chain is 3:1. Trace quantities of HbA can be detected in embryos. Thus, it is possible to make an early prenatal diagnosis of major β-chain hemoglobinopathy. Prenatal diagnosis is based on techniques used to examine the rates of synthesis of β chains or the structure of newly synthesized β chains or on molecular techniques from sampling the chorionic villus tissue or amniotic fluid. Gene

deletion disorders, such as α-thalassemia, can be detected with the same methods. At 24 weeks of gestation, approximately 5% to 10% of hemoglobin in a fetus is HbA. A steady increase follows, so that at term, the proportion of HbA averages 30%. By 1 year of age, the normal adult hemoglobin pattern appears. The minor adult hemoglobin component (HbA2) contains δ chains and has the structure α2δ2. It is seen only when significant amounts of HbA are present. At birth, less than 1% of HbA2 is present, but by 12 months, the normal level of 2% to 4% is attained. Throughout life the normal ratio of HbA to HbA2 is approximately 30:1. In the fetus and neonate, the rates of synthesis of γ and β chains and the amounts of HbA and HbF are inversely related. This has been attributed to a switch mechanism, but the developmental processes that direct the switch from predominantly γchain synthesis in utero to predominantly β-chain synthesis after birth are unclear. Primitive erythrocyte progenitors undergoing clonal maturation in culture (BFU-E) predominantly generate HbF. This may be the basis for the increased levels of HbF that occur in anemia with severe erythropoietic stress. Alternative explanations involve more basic genetic regulators in the DNA sequences that flank the hemoglobin gene complexes. Because hemoglobins containing ε chains are normally present only very early in intrauterine life, they are largely of theoretic interest. Small amounts of the Gower hemoglobins have been detectable in a few neonates with trisomy 13. Increased levels of Portland hemoglobin have been found in cord blood of stillborn infants with homozygous α-thalassemia. The normal adult level of HbA2 (2.4% to 3.4%) is seldom altered. Levels of HbA2 exceeding 3.4% are found in most persons with the β-thalassemia trait and in those with megaloblastic anemia secondary to vitamin B12 and folic acid deficiency. Decreased HbA2 levels are found in those with iron deficiency anemia and thalassemia major.42 Erythrocytes of the midtrimester fetus and preterm infant are extremely large and laden with hemoglobin (Fig 25-5). At 22 to 23 weeks of gestation, the mean corpuscular volume (MCV) can be 135 fL or more, compared with a value of 88 8 fL in adults. Similarly, at 22 to 23 weeks of gestation, the mean corpuscular hemoglobin (MCH) can be over 45 pg, compared with 29 2 pg in adults.43 Even at term, the MCV and MCH are strikingly higher than the upper limit of normal among healthy adults. However, throughout gestation the mean cell hemoglobin concentration is essentially the same as in adults.43 Presumably, these very large and hemoglobin-laden erythrocytes are advantageous to the early fetus, although it is not clear what those advantages are. Perhaps the very high content of hemoglobin carried by these large cells somehow constitutes a fetal advantage. Whether large erythrocytes are also advantageous for prematurely delivered neonates ex utero is not known. Many preterm neonates have large phlebotomy losses in the first week of NICU care and are transfused with much smaller erythrocytes from adult donors. The consequences of this change in erythrocyte size are not known.

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endomitosis, a process in which the ploidy of the cell increases without cellular division.

Thrombopoiesis Platelets were described in the early part of the 19th century, but it was not until 1882 that their role in hemostasis was recognized. The origin of platelets from megakaryocytes was reported in 1906,44 and since then the study of mechanisms responsible for platelet production has focused on megakaryocytes. The megakaryocyte compartment in the marrow consists of two pools of cells.45 One is composed of cells that are morphologically unrecognizable but are committed to the megakaryocyte lineage. These cells, the megakaryocyte progenitors, retain a high proliferative capacity and ultimately determine the number of megakaryocytes in the marrow. The other pool consists of nondividing cells that are morphologically recognizable as megakaryocytes and undergo endoreduplication or

Fetal and Neonatal Megakaryocyte Progenitors Progenitor cells committed to the megakaryocyte lineage can be identified by two methods: a culture system, in which they are identified by their ability to form megakaryocyte colonies, and immunologic staining, which allows characterization according to the specific antigens expressed on their membranes. With these methods, two different megakaryocyte progenitor cells have been identified; the burst-forming unit–megakaryocyte (BFUMK), which is a more primitive megakaryocyte progenitor,46,47 and a later progenitor known as the colony-forming unit–megakaryocyte (CFU-MK).45,48 In culture, CFU-MK-derived colonies are smaller (3 to 50 cells per colony) and primarily unifocal

(A) 140 135 130

MCV (fL)

125 120 115 110 105 100 95 90 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 Weeks gestation (B) 48 46 44

MCH (pg)

42 40 38 36 34 32 30 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 Weeks gestation

398

Figure 25-5. The expected range of values for (A) mean corpuscular volume (MCV) and for (B) mean corpuscular hemoglobin (MCH) for neonates of 22 weeks through 42 weeks of gestation, as measured on the first day after birth. The upper and lower boundaries incorporate 95% of the measured values. (Used with permission from Christensen et al.43)

Chapter 25: Fetal and Neonatal Hematopoiesis

(Fig 25-6), whereas BFU-MK-derived colonies are larger (
50 cells per colony) and have multiple foci of development. When immunologically phenotyped, CFU-MKs express CD34 and HLA-DR, whereas BFU-MKs express CD34 but not HLA-DR. There are no differences between fetal and adult megakaryocyte progenitors in regard to the expression profile of CD34 and HLA-DR.49 However, fetal BFU-MK-derived colonies are significantly larger than adult BFU-MK-derived colonies and are usually composed of only one or two foci of development. Adult BFU-MK-derived colonies are typically multifocal.49 In addition, a unique megakaryocyte progenitor present in fetal marrow has an unusually high proliferative potential and gives

Figure 25-6. Photomicrograph showing a megakaryocyte colony derived from a megakaryocyte progenitor (CFU-MK) from the marrow of a thrombocytopenic preterm neonate. (Original magnification, 400). Light-density mononuclear cells isolated from the marrow were cultured in collagen-based serum-free media with 50 ng/mL of recombinant human thrombopoietin. After fixation, the colonies were stained with a monoclonal antibody against glycoprotein IIb/IIIa to allow accurate identification of megakaryocytes.

rise to very large unifocal colonies (
300 cells).50 This cell, not found in adult marrow cultures, may represent a more primitive megakaryocyte progenitor. The development of miniaturized assay systems to study megakaryocyte progenitors has made it possible to study these cells in the peripheral blood of neonates. Using these techniques, it has been shown that preterm neonates (24 to 36 weeks) have higher circulating concentrations of all megakaryocyte progenitors than do term neonates.51,52

Fetal and Neonatal Megakaryocytes Unlike their progenitors, megakaryocytes have no proliferative abilities but undergo a complex maturational process. Through this process, they evolve from small, mononuclear cells to very large, polyploid cells easily recognized in the marrow as mature megakaryocytes.53,54 The modal ploidy is 16N in normal adult marrow.55 In the human fetus, megakaryocytes are first detected in the circulatory system at 8 weeks of gestation, and the first platelets appear at 5 weeks.56 Compared with the megakaryocytes of adults, fetal megakaryocytes are significantly smaller at all stages of maturation. Their ploidy distribution is also shifted to the left, with a higher proportion of immature megakaryocytes.57–59 The modal ploidy in the marrow of near-term fetuses is 8N.60 Umbilical cord blood has higher concentrations of circulating megakaryocytes than adult blood. As in the fetus, cord blood megakaryocytes from term infants are considerably smaller than adult circulating megakaryocytes,61 although they are otherwise phenotypically mature megakaryocytes. “Adult-size” megakaryocytes appear by 2 years of age, but the evolution of the process of megakaryocytopoiesis in the first year of life is poorly understood because of the lack of normal marrow specimens. Because cord-blood-derived megakaryocytes have been shown to generate fewer platelets than adult-derived megakaryocytes,62 it has been postulated that the normal platelet counts of fetuses and neonates are maintained by the increased proliferative potential of the fetal/neonatal megakaryocyte progenitors (Table 25-3).63 The process of platelet production and release is one of the less well understood steps of thrombopoiesis. However, recent work has substantially enhanced our understanding of this process, and has painted a graphic picture of how megakaryocytes—in their

Table 25-3. Differences in Megakaryocytopoiesis Between Neonates and Adults Adults

Neonates

Thrombopoietin (TPO) concentrations

Very high in hyporegenerative thrombocytopenia

Not as high in thrombocytopenic neonates (mostly small for gestational age) as in thrombocytopenic adults

Megakaryocyte progenitors

Sparse in the blood Give rise to small colonies Less sensitive to TPO

Abundant in the blood Give rise to large colonies More sensitive to TPO

Megakaryocytes

Large High ploidy levels

Small Low ploidy levels

Effects of recombinant TPO

Stimulates megakaryocyte proliferation Stimulates megakaryocyte maturation

Stimulates megakaryocyte proliferation Inhibits megakaryocyte maturation

Used with permission from Sola-Visner.63

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Section II: Part II

final hours—convert their cytoplasm into branched proplatelet extensions that elongate based on a process of microtubule sliding and polymerization.64 Organelles and platelet-specific granules are delivered to the nascent platelets (at the tips of each proplatelet extension) by a mechanism involving both traveling of the organelles along the microtubules and movement of the linked microtubules relative to each other.65 The role of blood-flowinduced shear stress on platelet release has also been evidenced in a recent study using intravital microscopy to observe in-vivo platelet release in intact murine marrow.66 In this study, marrow megakaryocytes were observed to extend dynamic proplatelet-like protrusions into microvessels. These intravascular extensions were then sheared from their transendothelial stems by flowing blood, resulting in the appearance of proplatelets in peripheral blood.

Thrombopoietin and Thrombopoietin Mimetics Thrombopoietin (TPO), the main physiologic regulator of platelet production, was first isolated in 1994.67 The gene that encodes thrombopoietin has been localized to the long arm of human chromosome 3.68 Thrombopoietin mRNA is expressed primarily in the liver, and to a lesser extent in other tissues, including kidney and marrow stromal cells.69 In vitro, TPO acts as a potent stimulator of all stages of megakaryocyte growth and development, except platelet release.70 It also plays roles in platelet activation, mostly by priming the platelets to the effects of other agonists, and in release of platelets from megakaryocytes.71 Thrombopoietin and TPO receptor knock-out mice models have been generated.72,73 These mice have megakaryocyte and blood platelet concentrations of only 10% to 15% those of control mice. These studies confirmed that TPO is the primary regulator of platelet production, but also proved that alternative pathways exist for megakaryocytopoiesis. Much attention has also been directed to the effects of TPO on other hematologic cell types. In vitro, TPO alone stimulates the proliferation and survival of erythroid, myeloid, and multipotential progenitors.74 Thrombopoietin also enhances erythropoietin-induced erythroid burst formation, an effect mediated by its ability to inhibit apoptosis of erythroid progenitors.75 Further studies have shown that TPO acts on early hematopoietic progenitors (including hematopoietic stem cells), thus disclosing an important role for this cytokine in hematopoiesis in general, in addition to its megakaryopoietic functions.76,77 These findings have significant clinical implications for patients with mutations in the TPO receptor that render it unresponsive to TPO. These patients, such as those with congenital amegakaryocytic thrombocytopenia, frequently progress to aplastic anemia. Results of several animal studies have confirmed the role of TPO as a potent stimulator of platelet production. The recombinant human full-length TPO molecule (rTPO) and a recombinant human polypeptide that contains the receptorbinding N-terminal domain of TPO (rHuMGDF) were the subjects of several studies. When injected into normal animals, rTPO and rHuMGDF induced marked thrombocytosis.78,79

400

Administered to animals exposed to myelosuppressive chemotherapy, rHuMGDF not only ameliorated the associated thrombocytopenia but also accelerated red cell and neutrophil recovery.80,81 Several Phase I and II trials in human subjects also demonstrated the efficacy of rTPO and rHuMGDF as stimulators of platelet production in adults without thrombocytopenia82 and in patients with chemotherapy-induced thrombocytopenia.83,84 However, the appearance of thrombocytopenia and ultimately aplastic anemia secondary to the generation of cross-reactive anti-TPO in a small number of patients receiving rHuMGDF85 led to the discontinuation of all clinical trials involving any of the forms of rTPO. As an alternative, much interest has been directed recently to the development of TPO-mimetic molecules. These are small molecules that have no sequence homology to TPO, but bind to the TPO receptor and have biologically comparable effects. The lack of homology represents a significant advantage over recombinant forms of TPO, because it should preclude the development of cross-reactive neutralizing antibodies against endogenous TPO. At least five different TPO receptor agonists have been described, and at least two of them have been studied in human subjects: AMG 531, an engineered peptibody composed of a recombinant protein carrier FC domain linked to multiple c-mpl-binding domains,86,87 and Eltrombopag (SB-497115), an oral, nonpeptide TPO receptor agonist.88 Both compounds showed very promising results in patients with refractory immune thrombocytopenic purpura, and had a favorable safety profile.

Thrombopoietin in the Fetus and Neonate Little is known about the role of TPO or the theoretical benefits of administration of rTPO or TPO-mimetic compounds to neonates. Recombinant TPO certainly supports the growth of megakaryocyte colonies from the blood or marrow of neonates (Fig 25-6). In fact, marrow progenitors from neonates are more sensitive to rTPO than progenitors from adults in vitro (Fig 25-7).89 Similarly, newborn rhesus monkeys are highly sensitive to rTPO in vivo (Fig 25-8) and respond to lower doses (per kilogram body weight) than those required in adult rhesus monkeys to achieve a similar effect.90 Megakaryocyte progenitors from preterm neonates with or without thrombocytopenia also seem to be more sensitive to rTPO than progenitors from term neonates.91 However, there are substantial qualitative differences in the response of neonatal and adult megakaryocytes to TPO, as demonstrated by a recent study evaluating the maturation of megakaryocytes cultured in either in TPO alone or in adult marrow stromal conditioned media.92 In that study, adult megakaryocytes reached the highest ploidy levels when cultured in serum-free medium with maximal concentrations of rTPO, while neonatal megakaryocytes reached their highest ploidy when cultured in marrow stromal cell conditioned media in the absence of rTPO. In fact, the addition of supraphysiologic concentrations of this cytokine (0.1 ng/mL) inhibited the maturation of neonatal megakaryocytes (Fig 25-9). Whether rTPO or

Chapter 25: Fetal and Neonatal Hematopoiesis

(A)

(B)

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Colony count

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150

100

50

110 100

Percent of maximum colony count

Figure 25-7. (A) Dose response to recombinant thrombopoietin of megakaryocyte progenitors obtained from the marrow of neonates with thrombocytopenia (T), neonates without thrombocytopenia (NT), and healthy adults. The marrow obtained from neonates generated approximately three times more colonies (per 105 low-density cells) than the marrow obtained from adults. (B) In a percentage of maximal colony count vs recombinant thrombopoietin concentration curve, the curves for the neonates with thrombocytopenia and those without thrombocytopenia reached a plateau at 10 ng/mL, compared with 50 ng/mL for the adults. This indicates that megakaryocyte progenitors from neonates are more sensitive to recombinant thrombopoietin in vitro than are their adult counterparts. (Used with permission from Sola et al.89)

90 80 70 60 50 40 30 20 NT neonates T neonates Adults

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0 10 20 30 40 50 60 70 80 90 100 TPO concentration (ng/mL)

TPO concentration (ng/mL)

Figure 25-8. Dose-response to polyethylene glycol (PEG)-rHuMGDF (a truncated form of recombinant thrombopoietin) of the blood platelet concentration in newborn rhesus monkeys, demonstrating the sensitivity of megakaryocyte progenitors of neonates to recombinant thrombopoietin in vivo. The monkeys received daily subcutaneous injections of placebo or PEG-rHuMGDF at doses of 0.25, 1.00, or 2.50 µg/kg/day for 7 days. Each line represents the average platelet counts of the two monkeys in each treatment group sampled on study Days 2, 4, and 6 and then twice weekly until Day 28. The peripheral platelet count increased on Day 6 of treatment, peaked on Day 11, and returned to baseline by Day 23. The two higher doses generated similar increases in platelet count. (Used with permission from Sola et al.90)

Platelet count (100/L)

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Placebo 0.25 µg/kg/day 1.0 µg/kg/day 2.5 µg/kg/day

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PB CB

10 5 0 0

0.1

1

10

100

TPO (ng/mL) Figure 25-9. The percentage of megakaryocytes with ploidy levels 8N in peripheral blood (PB)- and cord blood (CB)-derived megakaryocyte cultures differed depending on media source and recombinant thrombopoietin (rTPO) concentration. PB- and CB-CD34 cells were cultured for 14 days in serum-free unconditioned media (A) and in adult marrow stromal conditioned media (B), with varying rTPO concentrations. PB-derived megakaryocytes (solid lines) cultured in unconditioned

15

PB CB

10 5 0 0

0.1

1

10

100

TPO (ng/mL) media (A) exhibited a rTPO dose-dependent increase in ploidy levels, an effect inhibited by the presence of conditioned media (B). CB-derived megakaryocytes (dashed lines) reached highest ploidy levels when cultured in conditioned media with no rTPO, and effect that was reversed by rTPO concentrations 1 ng/mL (B). Data shown represent the means and standard error of the mean (SEM) of four separate experiments. (Used with permission from Pastos et al.92)

401

Section II: Part II

Figure 25-10. Cumulative distribution plots of megakaryocyte diameters in the marrow of thrombocytopenic neonates (dark circles), nonthrombocytopenic neonates (clear circles), thrombocytopenic adults (dark diamonds), and nonthrombocytopenic adults (clear diamonds). The megakaryocyte diameters are displayed on the X-axis, and their cumulative distribution on the Y-axis. Both adult curves are shifted to the right compared to the neonatal curves, indicating the predominance of larger megakaryocytes in these samples. Furthermore, the curve for the thrombocytopenic adults is also shifted to the right compared to that for the nonthrombocytopenic adults, indicating a higher percentage of large megakaryocytes. In contrast, the curves of thrombocytopenic and nonthrombocytopenic neonates are overlapping, indicating no change in the megakaryocyte size distribution in this cohort of thrombocytopenic neonates compared to controls. (Used with permission from Sola-Visner et al.96)

100 90 80 70

Percentile

60 50 40 30 20 10 0 0

10

20

30 Diameter

40

any of the TPO-mimetic peptides will be clinically useful as an alternative to platelet transfusions in the care of neonates with thrombocytopenia remains to be determined. However, it is clear that there are substantial developmental differences in megakaryocyte regulation, which will have to be taken into account when considering both the possible efficacy and the potential toxicity of thrombopoietic growth factors in this population.

Neonatal Thrombocytopenia When a fetus is delivered prematurely, the thrombopoietic system can be taxed by new demands. For instance, endothelial damage from infection or from the presence of intravascular catheters can accelerate platelet usage well beyond that normally experienced by the fetus. Moreover, disorders that tend to shorten gestation, such as pregnancy-induced hypertension and placental insufficiency, appear to have suppressive effects on fetal platelet production.93 Because of these and other issues, thrombocytopenia is a very common problem in the neonatal intensive care unit, manifested in 20% to 30% of all NICU patients, and in 75% or more of the extremely low birthweight population.94 Recent work has suggested that when fetuses or preterm neonates require an increase in platelet production, as a compensation for accelerated platelet usage or destruction, they might face developmental limitations in their ability to upregulate thrombopoiesis. Specifically, TPO concentrations in small-for-gestational-age neonates with hyporegenerative thrombocytopenia are less elevated than those reported in adults

402

50

60

with hyporegenerative thrombocytopenia, suggesting that—at least in this group of neonates—lower than expected TPO concentrations might contribute to the thrombocytopenia.91,95 Furthermore, thrombocytopenic neonates in general do not seem to increase the size of their megakaryocytes as compared to their nonthrombocytopenic counterparts, thus blunting one of the mechanisms by which adults increase platelet production in response to platelet demand96 (Fig 25-10). The mechanisms underlying the small size of neonatal megakaryocytes are not clearly understood, but recent studies suggest that they involve a combination of cell-intrinsic factors and factors in the fetal/neonatal microenvironment. Indeed, as demonstrated by stem cell transplant experiments, the adult environment is more conducive to megakaryocyte maturation than the fetal environment, but cell-intrinsic factors (such as the response to TPO) nevertheless limit the ultimate size and ploidy that neonatal megakaryocytes can achieve.97,98

Summary Neutropenia, anemia, and thrombocytopenia are relatively common problems in the NICU. These forms of cytopenia stem from multiple etiologic factors, and they involve many different kinetic mechanisms. However, all types of cytopenia in the NICU are more prevalent among those of shortest gestation and smallest birthweight. It is proposed that this relationship is

Chapter 25: Fetal and Neonatal Hematopoiesis

the result of gestational age-linked developmental differences in hematopoietic regulation. Transfusion is currently the main therapy available for NICU patients who have severe and prolonged cytopenia (see Chapter 30 for more information). As more is learned about human fetal hematopoiesis, perhaps alternatives to transfusion will emerge. To a certain extent, this process has already begun, because treatments such as recombinant erythropoietin and administration of recombinant G-CSF have been used in neonatology. However, much remains to be accomplished, such as more precise definitions of which neonates with cytopenias will benefit from treatment with recombinant hematopoietic growth factors, better methods for dosing and monitoring such treatments, and more precise means of identifying their risks and benefits.

Disclaimer The authors have disclosed no conflicts of interest.

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55. Tomer A, Harker LA, Burstein SA. Flow cytometric analysis of normal human megakaryocytes. Blood 1988;71:1244-52. 56. Kelemen E, Calvo W, Fliedner TM. Atlas of human hemopoietic development. Berlin: Springer-Verlag, 1979:51. 57. Allen Graeve JL, de Alarcon PA. Megakaryocytopoiesis in the human fetus. Arch Dis Child 1989;64:481-4. 58. Hegyi E, Nakazawa M, Debili N, et al. Developmental changes in human megakaryocyte ploidy. Exp Hematol 1991;19:87-94. 59. De Alarcon PA, Graeve JLA. Analysis of megakaryocyte ploidy in fetal bone marrow biopsies using a new adaptation of the Feulgen technique to measure DNA content and estimate megakaryocyte ploidy from biopsy specimens. Pediatr Res 1996;39:166-70. 60. Ma DC, Sun YH, Chang KZ, et al. Developmental change of megakaryocyte maturation and DNA ploidy in human fetus. Eur J Haematol 1996;57:121-7. 61. Levine RF, Olson TA, Shoff PK, et al. Mature micromegakaryocytes: An unusual developmental pattern in term infants. Br J Haematol 1996;94:391-9. 62. Mattia G, Vulcano F, Milazzo L, et al. Different ploidy levels of megakaryocytes generated from peripheral or cord blood CD34 cells are correlated with different levels of platelet release. Blood 2002;99:888-97. 63. Sola-Visner MC. Thrombocytopenia in the NICU: New insights into causative mechanisms and treatment. Haematologica Reports 2006;2:65-9. 64. Patel SR, Richardson JL, Schulze H, et al. Differential roles of microtubule assembly and sliding in proplatelet formation by megakaryocytes. Blood 2005;106:4076-85. 65. Richardson JL, Shivdasani RA, Boers C, et al. Mechanisms of organelle transport and capture along proplatelets during platelet production. Blood 2005;106:4066-75. 66. Junt T, Schulze H, Chen Z, et al. Dynamic visualization of thrombopoiesis within bone marrow. Science 2007;317:1689-91. 67. Lok S, Kaushansky K, Holly RD, et al. Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo. Nature 1994;369:565-8. 68. Foster DC, Sprecher CA, Grant FJ, et al. Human thrombopoietin: Gene structure, cDNA sequence, expression, and chromosomal localization. Proc Natl Acad Sci U S A 1994;91:13023-7. 69. Sungaran R, Markovic B, Chong BH. Localization and regulation of thrombopoietin mRNA expression in human kidney, liver, bone marrow and spleen using in situ hybridization. Blood 1997;89:101-7. 70. Debili N, Wendling F, Katz A, et al. The Mpl-ligand or thrombopoietin or megakaryocyte growth and differentiative factor has both direct proliferative and differentiative activities on human megakaryocyte progenitors. Blood 1995;86:2516-25. 71. Chen J, Herceg-Harjacek L, Groopman JE, et al. Regulation of platelet activation in vitro by the c-Mpl ligand, thrombopoietin. Blood 1995;86:4054-62. 72. De Sauvage F, Carver-Moore K, Luoh S, et al. Physiological regulation of early and late stages of megakaryocytopoiesis by thrombopoietin. J Exp Med 1996;183:651-6. 73. Gurney AL, Carver-Moore K, de Sauvage FJ, et al. Thrombocytopenia in c-mpl-deficient mice. Science 1994;265:1445-7. 74. Yoshida M, Tsuji K, Ebihara Y, et al. Thrombopoietin alone stimulates the early proliferation and survival of human erythroid, myeloid and multipotential progenitors in serum-free culture. Br J Haematol 1997;98:254-64.

Chapter 25: Fetal and Neonatal Hematopoiesis

75. Ratajczak MZ, Ratajczak J, Marlicz W, et al. Recombinant human thrombopoietin (TPO) stimulates erythropoiesis by inhibiting erythroid progenitor cell apoptosis. Br J Haematol 1997;98:8-17. 76. Young JC, Bruno E, Luens KM, et al. Thrombopoietin stimulates megakaryocytopoiesis, myelopoiesis, and expansion of CD34 progenitor cells from single CD34 Thy-1 Lin primitive progenitor cells. Blood 1996;88:1619-31. 77. Kaushansky K. Thrombopoietin and the hematopoietic stem cell. Ann N Y Acad Sci 2005;1044:139-41. 78. Harker LA, Hunt P, Marzec UM, et al. Regulation of platelet production and function by megakaryocyte growth and development factor in nonhuman primates. Blood 1996;87:1833-44. 79. Harker LA, Marzec UM, Hunt P, et al. Dose-response effects of pegylated human megakaryocyte growth and development factor on platelet production and function in nonhuman primates. Blood 1996;88:511-21. 80. Grossmann A, Lenox J, Ren HP, et al. Thrombopoietin accelerates platelet, red blood cell, and neutrophil recovery in myelosuppressed mice. Exp Hematol 1996;24:1238-46. 81. Neelis KJ, Qingliang L, Thomas GR, et al. Prevention of thrombocytopenia by thrombopoietin in myelosuppressed rhesus monkeys accompanied by prominent erythropoietic stimulation and iron depletion. Blood 1997;90:58-63. 82. Vadhan-Raj S, Murray LJ, Bueso-Ramos C, et al. Stimulation of megakaryocyte and platelet production by a single dose of recombinant human thrombopoietin in patients with cancer. Ann Intern Med 1997;126:673-81. 83. Basser RL, Rasko JEJ, Clarke K, et al. Randomized, blinded, placebocontrolled phase I trial of pegylated recombinant human megakaryocyte growth and development factor with filgrastim after dose-intensive chemotherapy in patients with advanced cancer. Blood 1997;89:3118-28. 84. Vadhan-Raj S, Verschraegen CF, Bueso-Ramos C, et al. Recombinant human thrombopoietin attenuates carboplatin-induced severe thrombocytopenia and the need for platelet transfusions in patients with gynecologic cancer. Ann Intern Med 2000;132:364-8. 85. Li J, Yang C, Xia Y, et al. Thrombocytopenia caused by the development of antibodies to thrombopoietin. Blood 2001;98:3241-8. 86. Bussel JB, Kuter DJ, George JN, et al. AMG 531, a thrombopoiesisstimulating protein, for chronic ITP. N Engl J Med 2006;355:1672-81.

87. Newland A, Caulier MT, Kappers-Klunne M, et al. An open-label, unit dose-finding study of AMG 531, a novel thrombopoiesisstimulating peptibody, in patients with immune thrombocytopenic purpura. Br J Haematol 2006;135:547-53. 88. Jenkins JM, Williams D, Deng Y, et al. Phase 1 clinical study of eltrombopag, and oral, nonpeptide thrombopoietin receptor agonist. Blood 2007;109:4739-41. 89. Sola MC, Du Y, Hutson AD, et al. Dose-response relationship of megakaryocyte progenitors from the bone marrow of thrombocytopenic and non-thrombocytopenic neonates to recombinant thrombopoietin. Br J Haematol 2000;110:449-53. 90. Sola MC, Christensen RD, Hutson AD, et al. Pharmacodynamics, pharmacokinetics and safety of administering pegylated recombinant megakaryocyte growth and development factor to newborn rhesus monkeys. Pediatr Res 2000;47:208-14. 91. Murray NA, Watts TL, Roberts IAG. Endogenous thrombopoietin levels and effect of recombinant human thrombopoietin on megakaryocyte precursors in term and preterm babies. Pediatr Res 1998;43:148-51. 92. Pastos KM, Slayton WB, Rimsza LM, et al. Differential effects of recombinant thrombopoietin and bone marrow stromal-conditioned media on neonatal versus adult megakaryocytes. Blood 2006;108:3360-2. 93. Murray NA, Roberts IA. Circulating megakaryocytes and their progenitors in early thrombocytopenia in preterm neonates. Pediatr Res 1996;40:112-19. 94. Christensen RD, Henry E, Wiedmeier SE, et al. Thrombocytopenia among extremely low birth weight neonates: Data from a multihospital healthcare system. J Perinatol 2006;26:348-53. 95. Sola MC, Calhoun DA, Hutson AD, Christensen RD. Plasma thrombopoietin concentrations in thrombocytopenic and non-thrombocytopenic patients in a neonatal intensive care unit. Br J Haematol 1999;104:90-2. 96. Sola-Visner MC, Christensen RD, Hutson AD, Rimsza LM. Megakaryocyte size and concentration in the bone marrow of thrombocytopenic and nonthrombocytopenic neonates. Pediatr Res 2007;61:479-84. 97. Slayton WB, Wainman DA, Li XM, et al. Developmental differences in megakaryocyte maturation are determined by the microenvironment. Stem Cells 2005;23:1400-8. 98. Ignatz M, Sola-Visner MC, Rimsza LM, et al. Umbilical cord blood produces small megakaryocytes after transplantation. Biol Blood Marrow Transplant 2007;13:145-50.

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26

Obstetric Transfusion Practice Humphrey H.H. Kanhai,1 Jos J.M. van Roosmalen,2 & Anneke Brand3 1

Professor, Department of Obstetrics, Leiden University Medical Center, Leiden, The Netherlands Professor, Department of Obstetrics, Leiden University Medical Center, Leiden, and Section of Health Care and Culture, VU University Medical Center, Amsterdam, The Netherlands 3 Professor, Sanquin Division Southwest and Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands 2

Because of physiologic hypervolemia in a healthy pregnant woman, a certain amount of blood loss after the birth of a child is considered normal. Postpartum hemorrhage (PPH) up to 1000 mL in a healthy mother, therefore, does not lead to disturbances in hemodynamics. PPH of more than 1000 mL in 24 hours occurs in 5% of deliveries. In women with anemia or poor increase in blood volume during pregnancy (eg, in preeclampsia), PPH less than 1000 mL can be hazardous to the mother. Blood loss in pregnancy, with consequences in oxygencarrying capacity, does lead to additional danger for the fetus. A worldwide survey shows huge variation in the lifetime risk of maternal mortality—from one in six in developing countries with a low human development index (HDI) to one in 30,000 in Northern European countries.1 Obstetric hemorrhage and preeclampsia together account for 50% of maternal deaths in low HDI as well as high HDI countries.1,2 Pregnancy complications and management differ in various parts of the world because of differences in blood groups, genetic and environmentally acquired diseases, socioeconomic status, and logistic and cultural factors.

Blood Loss in Pregnancy Obstetric Causes

women, 1606 needed 4 or more RBC units, indicating that 70% of severe pregnancy complications are associated with severe hemorrhage. Data on the cause of bleeding were available for 1590 of the 1606 women. Table 26-1 shows the most frequent etiologies of the obstetric hemorrhages. In 1480 women (93%), blood loss occurred during the postpartum period. Antepartum hemorrhage occurred in 135 women (8.5%) and major bleeding in early pregnancy occurred in 51 women (3.2%). These

Table 26-1. Primary Diagnosis in Major Obstetric Hemorrhage Defined as the Need for at Least 4 Units of Blood 3 Timing

Diagnosis*

Early pregnancy (n⫽51)

Ectopic pregnancy Spontaneous abortion Termination of pregnancy Miscellaneous†

29 (56.9%) 10 (19.6%) 10 (19.6%) 2 (3.9%)

Antepartum (n⫽135)‡

Abruptio placentae Placenta previa Miscellaneous§ Unknown diagnosis

61 (45.5%) 54 (40.3%) 7 (5.2%) 12 (9.0%)

Postpartum (n⫽1480)*

Retained placenta or placental rests Uterine atonia Hemorrhage following cesarean section Perineal tears/episiotomy Clotting disorders Placenta acreta/increta/percreta Rupture of cervix Uterine rupture Uterine inversion Miscellaneous Unknown diagnosis

Delivery poses the highest risk for blood loss during pregnancy. If administration of at least 4 Red Blood Cell (RBC) units is used as a surrogate definition for major obstetric hemorrhage, then a national survey on severe maternal morbidity in the Netherlands has identified major obstetric hemorrhage in 4.5 per 1000 births.3 Of a total of 358,874 births during the study period from August 2004 until August 2006, overall severe maternal morbidity occurred in 2552 women (7.1 per 1000 births). Of these *

406

703 (47.8%) 567 (38.5%) 183 (12.4%) 148 (10.1%) 116 (7.9%) 109 (7.4%) 58 (3.9%) 44 (3.0%) 13 (0.9%) 65 (4.4%) 10 (0.7%)

Up to three postpartum diagnoses could be coded. Molar pregnancy and placenta percreta. ‡ In 76 cases both antepartum and postpartum diagnoses were coded. § Rupture of uterine/ovarian artery, rupture of ovarian cyst, placenta percreta, vasa previa, retroplacental hematoma, rupture of uterine vein. †

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

No. of Patients

Chapter 26: Obstetric Transfusion Practice

percentages total more than 100, because 76 women experienced both antepartum and postpartum bleeding. Because only those women in need of at least 4 RBC units were included in the survey, the most frequent causes of obstetric blood loss before birth (such as miscarriage and unexplained minor antepartum hemorrhage) are relatively underrepresented in these figures. Assuming that the 239 women who received 10 or more units of blood would have died if they had not received those transfusions, the maternal mortality ratio (MMR) in the Netherlands would stand at 70 instead of the actual 12 per 100,000 live births. This underscores the impact of a functioning blood transfusion service on the reduction of maternal mortality. Lack of access to sufficient blood leads to a higher frequency of hysterectomy to stop the bleeding. Although hysterectomy may be a lifesaving procedure for some women, it adds to the risk of dying for others. The MMR for Jehovah’s Witnesses in the Netherlands was recently estimated to be 68 per 100,000 live births (van Roosmalen JJM, personal communication). This is strikingly similar to the likely MMR if blood had not been available for those women who received 10 or more RBC units.

Hematologic Diseases Maternal diseases such as diabetes, renal failure, hemoglobinopathies, systemic lupus erythematosus, and antiphospholipid antibody syndrome (APAS) are not associated with primary bleeding. Rather, they are associated with increased maternal and fetal mortality resulting from thrombotic and vascular complications leading to preeclampsia, thromboembolism, and placental insufficiency. A few maternal diseases, such as maternal thrombocytopenia and (iatrogenic) coagulopathies, can pose primary bleeding problems during pregnancy and delivery; however, in the absence of obstetric complications, these problems rarely lead to maternal mortality.

Maternal Thrombocytopenia A platelet count below 150,000/µL is a rather frequent finding in pregnancy. The possible causes are listed in Table 26-2. Spurious thrombocytopenia caused by laboratory errors or EDTA agglutination is excluded by examination of a blood film or platelet counting in citrated blood. Table 26-2. Causes of Thrombocytopenia in Pregnancy ● ● ● ● ● ● ● ●

Spurious thrombocytopenia Gestational thrombocytopenia Preeclampsia and HELLP Thrombotic microangiopathies (HELLP, TTP, HUS) Disseminated intravascular coagulation Idiopathic/immune thrombocytopenic purpura Antiphospholipid antibody syndrome Congenital thrombocytopenia and/or platelet function disorders

HELLP ⫽ hemolysis, elevated liver enzymes, and low platelet count; TTP ⫽ thrombotic thrombocytopenic purpura; HUS ⫽ hemolytic uremic syndrome.

Thrombocytopenia (platelet count between 80,000 and 150,000/µL) of unknown origin, often referred to as gestational thrombocytopenia (GT), occurs in approximately 6% of third trimesters.4 Its cause and significance is often unknown. GT has been considered as subclinical immune thrombocytopenic purpura (ITP) or as a precursor of HELLP (hemolysis, elevated liver enzymes, low platelet count) syndrome.5,6 GT is a diagnosis of exclusion. If, in uncomplicated pregnancy, the blood film is normal in an otherwise healthy female without a history of ITP or antiphospholipid antibodies, no diagnostic tests are needed. However, such women should be monitored carefully for aggravation of thrombocytopenia and symptoms of HELLP after delivery, because they may be at higher risk for postpartum HELLP syndrome. HELLP syndrome belongs to the microangiopathic hemolytic anemias, also referred to as thrombotic microangiopathic anemias (TMAs), which can complicate pregnancy and puerperium.7,8 The peak incidence has a relationship with the weeks of gestation (Fig 26-1), but there is considerable overlap with other syndromes (see below). HELLP complicates 4% to 15% of cases of preeclampsia (hypertension and proteinuria). Its peak incidence is at 36 weeks, although in one-third of cases HELLP presents up to 7 days after a normal delivery not complicated by preeclampsia and in 10% it presents before the 27th week of gestation.9 Typical symptoms include right-upper-quadrant abdominal pain, nausea, and vomiting. HELLP is not associated with overt disseminated intravascular coagulation (DIC), although in 70% the antithrombin level is decreased in contrast to thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS).10 Because of vasoconstriction in preeclampsia, severe bleeding rarely occurs. Severe neglected HELLP, often resulting from delayed delivery, leads to DIC. Other complications are pulmonary edema, acute renal failure, abruptio placentae, and liver hematoma. Maternal mortality is as high as 20% and neonatal death ranges from 10% to 60% depending on intervention with elective preterm deliveries, which reduce the risk of death to both mother and fetus. Mild thrombocytopenia and schistocytes are found in 50% of the neonates and resolve without treatment or transfusions. Because termination of pregnancy is the definitive treatment, elective birth should be considered. Severe preeclampsia and HELLP will subsequently resolve 1 to 10 days after termination of pregnancy. If recovery is delayed beyond 3 days or HELLP after delivery progresses to (multi)-organ failure, plasma exchange may support recovery.11 The ranges of laboratory values that can be found in HELLP are shown in Table 26-3. HUS typically presents from 2 days after delivery onwards (mean 26 days), although in 5% to 10% of cases antepartum HUS has been described. At presentation 50% of females have thrombocytopenia (platelet counts less than 100,000/µL).8 Despite plasma exchange, approximately 25% of the patients experience persistent renal failure. TTP results from an antibody-mediated reduction in the level of von Willebrand factor (vWF)-cleaving protease (ADAMTS13).

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Pregnancy

Puerperium 10%

60%

30%

HELLP 10%

5% HUS

TTP

15

20

25

30 Weeks

35

40

45

50

Figure 26-1. Pregnancy-associated microangiopathic thrombocytopenias. HELLP ⫽ hemolysis, elevated liver enzymes, and low platelet count; HUS ⫽ hemolytic uremic syndrome; TTP ⫽ thrombotic thrombocytopenic purpura.

Table 26-3. Laboratory Values in 442 Cases of HELLP7 Test Platelets (⫻1000/µL)

Median

57.00

Range

7-99

Alanine aminotransferase (U/L)

249.00

70-6193

Lactate dehydrogenase (U/L)

853.00

564-23.584

Bilirubin (µmol/L) (mg/dL)

26.00 1.53

8.6-436 0.51-25.65

Creatinine (µmol/L) (mg/dL)

97.00 1.10

53-1414 0.6-16.1

Urine acid (µmol/L) (mg/dL)

462.00 7.70

174-900 2.9-15

TTP is most often seen in the second and third trimesters and requires plasma exchange similar to treatment of TTP in nonobstetric situations. TTP has no relationship with preeclampsia and there is no need to terminate pregnancy. Patients with a history of TTP can have a relapse in pregnancy, but after remission pregnancy is not contraindicated, although close monitoring of the ADAMTS13 level is recommended.12 DIC is seen in far-advanced TMA syndromes, predominantly caused by primary obstetric or septic pathology, such as abruptio placentae and severe intrauterine infection. Between 20% and 40% of obstetric patients with critical illness have symptoms of DIC. In pregnancy and puerperium the use of a DIC score (based on decreased platelet count, increased D-dimers, decreased fibrinogen, and prolonged prothrombin time) is less reliable because D-dimers start to increase in the first trimester

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and are always increased after the 35th week. Fibrin deposition in DIC not only contributes to multiple organ failure, but also (because of consumption coagulopathy with massive and ongoing activation of coagulation) results in depletion of platelets and coagulation factors with severe bleeding. With an incidence of 6/100,000 new cases per year, ITP mostly affects young women.13,14 Consequently, a history or current evidence of ITP is frequently encountered in pregnancy (estimated to be 1-5 cases per 10,000 pregnancies).15 Often the mother has a history of ITP with or without a low platelet count, although first presentation of ITP or accidentally discovered ITP in pregnancy also occurs. During pregnancy platelets decrease with a nadir in the third trimester.16,17 Antenatal treatment is given exclusively on maternal indication and aims to maintain the platelet count above 20,000 to 30,000/µL or higher in case of bleeding symptoms, using corticosteroids as first-line treatment.13,17 Intravenous immunoglobulin (IVIG) is reserved as second-line treatment. IVIG is used to prepare for delivery, if necessary, combined with platelet transfusions. There is no demonstrated effect of maternal treatment with corticosteroids or IVIG on the fetal platelet count.18,19 Perinatal mortality in ITP is 0.6%.20 The risk for neonatal thrombocytopenia below 50,000/µL ranges from 8.9% to 14.7%.19-22 Intracranial hemorrhage (ICH) or other severe bleeding is not increased in the fetus of a mother with ITP. The nadir of the neonatal platelet count and the highest risk for bleeding is 4 (range, 1-7) days after delivery.15-17 In large series, ICH in the neonate is reported to be 0% to 1.5%.13,17,23 Most studies have reported only weak correlations between the fetal platelet count at birth and the maternal platelet counts, the presence of antibodies in the maternal serum, or a history of splenectomy.13,17 In individual cases, the risk for fetal thrombocytopenia below 50,000/µL cannot be predicted from the maternal platelet count. However, in women who are refractory to corticosteroids and splenectomy and who cannot maintain a platelet count above 30,000/µL despite immunosuppressive treatment, the risk for bleeding and neonatal thrombocytopenia

Chapter 26: Obstetric Transfusion Practice

is higher.16,17,24 In asymptomatic women, severe fetal thrombocytopenia is rare and approaches the background rate of approximately 1% in term newborns. In approximately 70% of multigravidae, the absence of thrombocytopenia in a previous child predicts a similar good outcome in the next child.25 Antiphospholipid antibody syndrome is an acquired condition of hypercoagulability, often associated with systemic lupus erythematosus (SLE). APAS is characterized by arterial and venous thrombosis and gestational vascular complications leading to recurrent fetal loss, growth retardation, and prematurity. In patients with suspected clinical symptoms, antibodies against β2-glycoprotein I, anticardiolipin autoantibodies, and/or prolongation of plasma clotting time (lupus anticoagulant) are present.26 On the basis of randomized studies, women with recurrent abortion are treated with a combination of low dosages of aspirin and low-molecular-weight heparin.23,26 There is no risk for fetal bleeding but the mother has a slight increased risk for bleeding during delivery. Treatment of inherited disorders of hemostasis and platelet function disorders (such as von Willebrand disease, hemophilia, or Glanzmann’s thrombasthenia) aims to avoid blood components when possible, at least during pregnancy, because of the risk of alloimmunization. Before and after delivery, blood transfusion may be unavoidable; this can be problematic if the patient is extensively immunized. Planned delivery and an individualized treatment plan are needed for these rare diseases.

Chronic Maternal Anemia Physiologic anemia is caused by plasma volume expansion and is maximal around the 25th week of pregnancy. The World Health Organization defines anemia in pregnancy as a hemoglobin level ⬍6.8 mmol/L (11 g/dL), which is most often caused by nutritional deficiencies. Below a hemoglobin level of 5.5 to 6.2 mmol/L (9-10 g/dL) growth retardation and prematurity are reported, although it is not known whether these are related to the underlying disease or the lower hemoglobin level itself.27-29 For chronic hemolytic diseases, such as thalassemia and paroxysmal nocturnal hemoglobulinuria, hypertransfusion is applied to maintain hemoglobin levels above 10 g/dL in order to depress hemolysis of autologous red cells.30 Randomized studies have involved only pregnant patients with sickle cell disease and have showed that maintenance of a hematocrit above 33% with less than 35% sickled cells resulted in no better pregnancy outcome, although painful crises were significantly reduced.31

Assessment of Amount of Blood Loss Visual estimation is frequently used in routine practice to assess the amount of blood loss. However, this technique is inaccurate and leads to significant underestimates of blood loss. In a nicely researched experiment using pictorial guidelines to facilitate visual estimation (Fig 26-2), all professionals involved in obstetric care underestimated blood loss.32 In fact, obstetricians tended to underestimate blood loss to a higher degree than anesthetists, surgical nurses, and midwives. The guidelines used in this study (available at http://www.bmfms.org.uk) can be used

as an effective tool to train health-care professionals to improve their abilities to estimate blood loss. Other methods of estimating blood loss include measuring actual blood loss with a weighing scale or measuring the peripartum hemoglobin change. Measurement with a scale, however, must be corrected for the amount of amniotic fluid and is cumbersome. Peripartum hemoglobin change has been shown to be unrelated to visually estimated blood loss.33 In other studies the need for additional therapeutic uterotonic drugs or blood transfusion is taken as an indication of the amount of blood loss.34 This is not relevant for clinical practice, but is important for audit purposes.

Maternal Transfusions Indications The indications for maternal transfusion can be categorized in two groups—those occurring after delivery and those occurring during pregnancy or delivery.

Transfusion Indications for Postpartum Bleeding Forty percent of women lose more than 500 mL of blood after vaginal delivery and 30% lose more than 1000 mL after cesarean section. As discussed above, the blood loss is difficult to estimate.33 Although guidelines suggest a transfusion threshold at a hemoglobin concentration of 7.0 to 8.0 g/dL, concentrations of 5.0 g/dL or more are usually well tolerated if isovolemia is maintained. In a study of healthy individuals, Weiskopf et al35 found that acute isovolumetric reduction of hemoglobin concentration to 5.0 g/dL does not appear to cause inadequate tissue oxygenation. In healthy women with a median hemoglobin level of 10.5 g/dL (range, 7-15 g/dL) 3 to 6 weeks after delivery, no relationship between hemoglobin level and the quality of life was observed.36 Studies of the effects of blood transfusion after PPH on quality of life are not available thus far. Transfusion is required only for symptomatic anemia and should not be given solely on the basis of the hemoglobin level. Limited data exist on outcomes at concentrations below 5.0 g/dL. Two retrospective studies involving patients who declined blood transfusion, mostly Jehovah’s Witnesses, found that morbidity and mortality rates were extremely high below this hemoglobin level.37,38 However, survival has been reported at hemoglobin levels below 2.0 g/dL—even as low as 1.4 g/dL.39,40 Life-threatening hemorrhages after delivery are often associated with complex coagulopathies. Therefore, a management protocol must be in place.41-43 Recent protocols advocate that when massive bleeding in complex trauma is anticipated, the patient should be transfused earlier without waiting for consumption and dilutional coagulopathies. This has been shown to reduce total blood use and mortality.44,45 This seems to hold for obstetric bleeding as well. Control of bleeding, early transfusion, and correction of coagulopathy must be carried out during the window of opportunity, which may be less than 2 hours, before

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Section II: Part II

(A)

(B)

Soiled sanitary towel (30 mL)

(D)

(C)

(E)

Incontinence pad (250 mL)

(G)

(F)

Saturated large swab 45 ⫻ 45 cm (350 mL)

(H)

PPH on bed only (1000 mL)

Saturated small swab 10 ⫻ 10 cm (60 mL)

Soiled sanitary towel (100 mL)

100-cm diameter floor spill (1500 mL)

(I)

PPH spilling to floor (2000 mL)

Full kidney dish (500 mL)

Figure 26-2. Pictorial guidelines to facilitate visual estimation of blood loss from obstetric hemorrhage.32

the vicious circle initiated by hypothermia, acidosis, and irreversible coagulopathy leads to death.

Platelet Transfusions during Pregnancy and for Delivery During pregnancy, thrombocytopenias result from enhanced platelet phagocytosis in ITP or consumption in TMA, both disorders in which platelet transfusions are reserved for therapeutic indications. Reduced marrow platelet production

410

is rare unless a hematologic or oncologic condition is present. Whatever the cause of thrombocytopenia, most guidelines recommend a platelet count above 50,000/µL for delivery and 80,000/µL for cesarean section, allowing all forms of anesthesia.13 Because of different pathophysiology, an individual treatment plan for every pregnant patient with thrombocytopenia is required. Except in cases of TTP, platelet transfusions are not contraindicated in TMA, ITP, or DIC. However, because platelet survival is

Chapter 26: Obstetric Transfusion Practice

shortened, other supportive treatment intended to improve the cause of thrombocytopenia is required. (See Chapter 23.)

Table 26-4. Risk Factors for Postpartum Hemorrhage ● ●

Blood Products Used and Special Precautions Industrialized Countries with a High HDI Red Cell Transfusions Pregnancy and transfusions both stimulate red cell antibody formation. A survey of the transfused population in developed countries shows that women have three or four times more alloantibodies than men and children.46,47 Females with a history of transfusion and pregnancy must be screened early in a new pregnancy to identify alloantibodies as a potential cause for hemolytic disease of the fetus and newborn (HDFN) or availability of blood in case an indication for maternal transfusion should arise. In particular, antibodies against c and K1 antigens can cause HDFN as severe as Rh(D) HDFN.48 In cases of HDFN caused by c or K1 antigens, more than 40% of the women had received prior transfusions.49 Transfusions of Rh- and Kcompatible components to females of childbearing potential can reduce non-D HDFN. Most national guidelines recommend the use of K-negative donors for women ⭐45 years of age.

● ● ● ● ●

Overdistended uterus (multiple pregnancy, large fetus, and polyhydramnious) Prolonged labor Induction of augmentation of labor Postpartum hemorrhage in previous pregnancy Chorioamnionitis High parity Coagulation disorders (placental abruption, syndrome of hemolysis, elevated liver enzymes, and low platelet count)

components selected to reduce CMV and parvovirus B19 risk should be administered. Filtration of cellular blood components virtually abolishes CMV transmission. Pooled plasma products (eg, IVIG) must contain less than 104 copies of parvovirus B19, a load that is neutralized by antibodies present in the product. However, this does not protect against parvovirus B19 in individual cellular products.

Immunoglobulin Immunoglobulin preparations are used during pregnancy for immunomodulatory purposes to prevent formation of anti-D, increase the maternal platelet count in ITP, and reduce severe fetal bleeding in fetal and neonatal thrombocytopenia (FNAIT). To prevent Rh HDFN, D-negative women are given RhIG between the 28th and 34th weeks of gestation and also within 72 hours after delivery of a D-positive infant.54 This reduces Rh HDFN to pregnancies of less than 1% of D-negative women. Nevertheless, Rh HDFN is still the most frequent and severe type of HDFN. The residual immunization results from medical, laboratory, and clerical errors, or an inadequate dose of RhIG after larger fetomaternal hemorrhages during delivery, after trauma, and after obstetric interventions.55,56

Indications and Practice Prevention of PPH can be achieved by active management of the third stage of labor.34 Although active management combines the use of oxytocic drugs, early cord clamping, and active efforts to deliver the placenta, actual practice differs, sometimes including selective radiologic embolization of uterine vessels, and sometimes consisting only of oxytocic drugs.57 Postpartum hemorrhage may occur in any woman in labor. Risk factors, however, will help the clinician anticipate the problem (Table 26-4). The indications for transfusion are guided in the Netherlands by the 4-5-6 Flexinorm.58 In the United States, guidelines are promulgated by the American Society of Anesthesiologists (ASA). When the hemoglobin levels is ⬍4 mmol/L (hematocrit 20%) in ASA category I patients (healthy persons) with acute blood loss, transfusion is indicated. In ASA category II patients (mild systemic disease without disability) and category III patients (systemic disease and serious disability), the threshold for transfusion lies at a hemoglobin level ⬍5 mmol/L (hematocrit 25%). In ASA category IV patients (constant lifethreatening condition) the transfusion threshold is a hemoglobin level ⬍6 mmol/L (hematocrit 30%). In massive hemorrhage, Fresh Frozen Plasma is used to counteract clotting disorders. Preeclampsia, placental abruption, and amniotic fluid embolism are the most important obstetric disorders that may be accompanied by clotting problems. A woman with preeclampsia is already in a hypovolemic condition before hemorrhage starts and this needs special attention. Urinary output is the best indicator of renal perfusion in a bleeding or preclamptic woman and should be at least 30 mL/hour.

General Aspects of Transfusion Products Blood components can transmit cytomegalovirus (CMV) and parvovirus B19—infections that may harm the fetus. Blood

Countries with a Low HDI In developing countries, blood transfusion services are generally not able to deliver the number of units needed to save the lives of

Platelet Transfusions Platelet transfusions can contain minimal numbers of erythrocytes, sufficient to immunize against the D antigen. If D-positive platelets are given to D-negative females of childbearing potential, prophylaxis with 375 IU intravenous Rh Immune Globulin (RhIG) is indicated.50 With increasing numbers of pregnancies, females produce leukocyte antibodies against paternal antigens, which may impair platelet recovery after transfusions from randomly selected donors.51,52 HLA antibodies must be excluded if a need for platelet transfusion during delivery is anticipated. Because pregnancy stimulates immune anti-A and B, major ABO-incompatible platelet transfusions also show reduced recovery.53

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Section II: Part II

women with obstetric blood loss. In many places blood donation “on the spot” by relatives after the adverse event has taken place is the only available option. Transmission of hepatitis and human immunodeficiency virus (HIV) is a serious risk. In Tanzania, for instance, a National Blood Transfusion Service was developed in response to the HIV epidemic and a serious train crash in 2002. In 2006, 67,000 units of blood had been donated by volunteer donors. These nonremunerated donors have proven to be safer donors than relatives. However, there are not enough safe donors, and patients remain dependent on relatives as important sources for blood. It is tragic that these relatives have high rates of hepatitis and HIV infections. Because anemia resulting from malaria, dietary deficiencies (iron and folic acid), sickle cell disease, and infectious disease is rampant in low HDI countries, a relatively small amount of blood loss is often disastrous. Thus, the World Health Organization defines PPH as a blood loss of ⬎500 mL, while in many high HDI countries the threshold is ⬎1000 mL. Anemia should be treated rigorously during pregnancy in order to reduce the consequences of PPH.59 The use of bednets, nutritional supplements, and antihelmintics; presumptive treatment of malaria; folic acid and ferrous medication during antenatal care; and active management of the third stage of labor all help women to overcome the problems created by hemorrhage during childbirth. These strategies may reduce the need for blood transfusion. Physicians should remember that unnecessary blood transfusion in a setting of limited resources is not only a waste but also likely to increase exposure to transfusion-related risks and infections.60 Transfusion or injection of unsafe blood is responsible for 8 to 16 million hepatitis B virus infections, 2.3 to 4.7 million hepatitis C virus infections, and 80,000 to 160,000 HIV infections annually. At present, 80% of the world’s population has access to only 20% of the world’s safe blood supply.60 A recent paper from Nigeria reports a blood transfusion rate of 25.2% during cesarean section, a high rate that demonstrates the risk of surgery when resources are not available.61 A strategy that safely reduces the number of cesarean sections also reduces the need for blood transfusion.

Fetal Transfusions This section discusses aspects of fetal transfusion focusing on transfusion for red cell immunization and the management of FNAIT.

Alloimmune Hemolytic Disease The first fetal transfusion was performed in 1963 by Liley.62 After amniography and fetography, red cells were transfused under X-ray guidance in the intraperitoneal cavity of the fetus. This method of treatment of fetal anemia is based on the fact that red cells are absorbed from the peritoneal cavity and enter the circulation. Limitations of this technique include less uptake of the red cells in cases of severe fetal hydrops. Reported survival rates at the time were approximately 50%.63

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The advent of real-time ultrasound ushered in a new era of fetal management. Since the 1980s fetal transfusions have been performed with the use of ultrasound guidance—specifically, ultrasound-guided puncture of the umbilical vein.64 Intravascular transfusions have been performed in thousands of pregnancies worldwide. Although randomized trials comparing intraperitoneal and intravascular transfusions have not been performed, results of the latter technique, which is feasible from 16 weeks of gestation onwards, are widely considered as superior. Fetal blood sampling (FBS) by cordocentesis or puncture of the intrahepatic umbilical vein is indicated when fetal anemia is suspected (by use of Doppler measurements of the fetal vessels65) or in cases of hydrops. Intrauterine transfusion is performed directly after the diagnosis of anemia is established by FBS. The most frequent indication for fetal blood transfusion is anemia caused by red cell alloimmunization of the mother (see Chapter 27). Other indications for fetal blood transfusion include: human parvovirus B19 infection,66,67 severe fetomaternal hemorrhage,68 placental chorioangiomas,69 and homozygous α-thalassemia.70 Placental chorioangioma and thalassemia are very rare indications and are not discussed further. Fetal blood sampling is also applied for the management of other conditions such as thrombocytopenia or severe tachyarrhythmia of the unborn.

The Technique The principle of fetal transfusion is to access the fetal circulation with a needle under real-time ultrasound guidance, aspirate blood for diagnosis, and deliver red cells, platelets, or drugs to the fetal circulation. The most frequently used site for transfusion is the umbilical vein when the placenta is inserted anteriorly. When the umbilical vein at the placental insertion cannot be reached safely, the fetus is punctured in the intrahepatic vein. The umbilical vein is preferred for several reasons. First, the vein has a larger diameter than the arteries. Second, because of the eccentric location of the vein in the cord, an overshoot or slip of the needle does not in general lead to a hematoma in the Wharton’s jelly, but to a leakage in the amniotic cavity. A puncture in the artery may lead to leakage in the Wharton’s jelly, inducing spasm of the artery and subsequent fetal bradycardia. Third, fetal transfusion in the vein allows the operating team to monitor the flow of the transfused blood sonographically during the procedure. In some centers intravascular transfusion is combined with an intraperitoneal approach. The procedure is performed with a 20- or 22-gauge spinal needle. Premedication to the mother is used for relief of maternal anxiety and sedation of the fetus. Routine intravenous or intramuscular administration of muscle relaxants such as atracurium or pancuronium is advocated to achieve fetal paralysis and thus prevent needle displacement caused by fetal movements.71 Donor Blood, Volume, and Rate of Transfusion The blood selected for fetal transfusion is group O, RhD-negative and compatible with any maternal antibodies that are present.

Chapter 26: Obstetric Transfusion Practice

Because intrauterine transfusions easily stimulate new maternal antibodies against fetal or donor red cell antigens, a freshly derived maternal blood sample should be crossmatched before every subsequent transfusion.72 The selected blood component should not carry the risk of transmitting CMV and parvovirus B19 or causing graft-vs-host disease (GVHD). To promote optimal survival, the red cells are stored as briefly as possible. Before transfusion, supernatant solutions containing potassium, ABO antibodies, and red cell preservation solutions are often removed and replaced by saline. A leukocyte-reduced red cell concentrate in 0.9% saline with a hematocrit of 80% irradiated with 25 Gy just before transfusion can correct extremely low hemoglobin values without causing or transmitting disease. After access to the fetal circulation, 1 to 2 mL blood is aspirated and promptly examined for hemoglobin, hematocrit, and mean corpuscular volume. Some facilities prefer to determine these values immediately, using a cell counter in the operating room. The volume of blood transfused is calculated by using a formula in which pretransfusion fetal hematocrit, the estimated fetal placental blood volume, and the hematocrit of the donor blood are included.73 The blood is transfused at a rate of 5 to 10 mL per minute.73 During transfusion, the blood flow and the fetal heart rate are monitored by ultrasound and Doppler. The second transfusion is given 1 to 2 weeks after the first. Following subsequent transfusions, fetal erythropoiesis is depressed and the fetus is completely dependent on transfusions every 3 to 4 weeks. At the end of treatment, more than 70% of women with HDFN requiring intrauterine transfusions have antibodies to multiple red cell antigens.72 Selection of donors compatible with the mother for clinically relevant antigens could reduce this alloimmunization. In approximately half of the cases, additional antibody formation cannot be circumvented because the antibodies are evoked by fetal red cells.72

Outcome of Treatment At Leiden University Medical Center, 593 ultrasound-guided intravascular transfusions have been performed in 210 fetuses between 1988 and 1999. The transfusions were given between 17 and 35 weeks of gestation and resulted in an overall survival rate of 86%.48 In the period from 1999 to 2006 the survival rate improved to 95% (unpublished data). The improvement in the survival rate results from fewer cases presenting with severe irreversible hydrops, after the 1997 implementation of routine screening for red cell antibodies early in pregnancy.50 There has also been a reduction of procedure-related complications. Follow-up of the infants thus far has revealed no adverse effects of the treatment.

Fetal and Neonatal Alloimmune Thrombocytopenia In 0.3% of newborns, thrombocytopenia with an immunologic origin is encountered. Fetal and neonatal alloimmune thrombocytopenia (also known as neonatal alloimmune thrombocytopenia) and autoimmune thrombocytopenia (also known as ITP or Morbus Werlhof disease) are immune-mediated thrombocytopenias. In cases with severe red cell alloimmunization,

thrombocytopenia (platelet count ⬍50,000/µL) in the fetus is found before the first intrauterine transfusion in 3% of the cases. In severely hydropic fetuses this percentage is 10% to 30%.74 In these cases the mechanism causing thrombocytopenia is not known. Because severe in-utero bleeding is not documented in ITP19 and the risk of ICH in the neonate is estimated at 0% to 1.5%,13,17,23 intrauterine platelet transfusions are not indicated; the risk of complications exceed the risk of bleeding. Approximately 2% of pregnant women of European ancestry are negative for HPA-1a. Anti-HPA-1a is responsible for the majority (75-95%) of cases of FNAIT.75 About 5% to 25% of FNAIT is caused by other HPA antibodies, most frequently anti-HPA-5b, anti-HPA-3a, and anti-HPA-1b. In 15% to 20% of FNAIT cases no antibodies can be detected. Antibodies against private antigens and new antigens causing FNAIT are still being discovered.76 In platelet-alloimmunized pregnant women carrying a fetus that is positive for the offending antigen, the risk for thrombocytopenia can be as high as 85%. The incidence of ICH varies from 7% (in alloimmunized pregnancies and a sibling without ICH) to 85% (in alloimmunized pregnancies and a sibling with ICH).77 It is assumed that most ICH occurs antenatally between 30 to 35 weeks of gestation. For antenatal management, it is important to know whether the father is homozygous or heterozygous for the offending antigen. In case of heterozygosity of the father, genotyping of the fetus is indicated. Although genotyping is possible by using polymerase chain reaction on either chorionic villi or amniocytes, amniocentesis in the second trimester is preferable because of its lower risk of boosting antibodies. Currently, methods are being developed to assess the fetal HPA type from fetal DNA in maternal plasma. When the fetus is positive for the offending antigen, the pregnancy should be managed in, or in collaboration with, a specialized fetal care center. Because HPA screening is not routine, FNAIT is recognized most often only after an affected sibling is identified. The antenatal management protocols in FNAIT are intended to prevent ICH. Unfortunately, monitoring HPA antibodies by titration and quantification has not accurately predicted the severity of fetal thrombocytopenia. However, recent studies indicate that it is helpful in predicting the severity of fetal thrombocytopenia.78-80 In order to diagnose fetal thrombocytopenia as early as possible, various centers have designed protocols for routine FBS beginning as early as 20 weeks.81,82 In cases of fetal thrombocytopenia and platelet transfusion, the procedure must be repeated frequently, given the fact that the half-life of transfused platelets is only 5 days. Although FBS is a reliable tool for direct monitoring of the fetal thrombocytopenia, the cumulative procedure-related risk for fetal loss is high and approximates 6% per pregnancy.83,84 The cumulative risk for emergency delivery due to FBS is 13% to 17%.85,86 Currently, most centers have adopted the use of weekly high-dose maternal IVIG. A few centers use corticosteroids as first-line treatment. Although corticosteroids do not enhance the effect of IVIG,85,86 combinations of these options are also in use. Bussel et al87 were the first who reported in 1988 that

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weekly maternal IVIG (1.0 g/kg maternal weight) was effective at elevating the fetal platelet count. Later studies showed that not all fetuses showed a substantial increase in platelet count with this treatment. The reported response rate in the literature varies between 30% and 85%. In addition, observational studies have suggested that IVIG reduces the risk of ICH even in nonresponders to IVIG,86,88,89 possibly by downregulation of endothelial activation by platelet antibodies that cross-react with endothelial epithelial cells.90 In the past decade there has been a gradual change in antenatal treatment of platelet alloimmunized pregnancies, from an invasive approach with repetitive FBS to a less invasive approach (treatment with IVIG and less FBS or platelet transfusion) to a completely noninvasive approach (IVIG only). At Leiden University Medical Center, treatment of 52 pregnancies (53 neonates) at risk for fetal ICH (5 with and 48 without a sibling with ICH) resulted in term live births without ICH in all neonates.89 Long-term follow-up of the infants, treated antenatally with IVIG, showed no adverse effects.91 The mechanism of action of IVIG in FNAIT is still unclear. There are several possible explanations. First, in the maternal circulation the IVIG will dilute the HPA antibodies, resulting in a lower proportion of HPA antibodies among the IgG transferred via the Fcγ receptors (FcγRs) in the placenta. Second, in the placenta, IVIG may block the placenta receptor and decrease the placental transmission of maternal antibodies including anti-HPA. Third, in the fetus, IVIG may block the FcγRs on the macrophages and prohibit the destruction of antibody-covered platelets. Recently, Ni et al92 presented a murine model of FNAIT, measuring response to IVIG therapy. This model demonstrates that maternal IVIG administration has multiple effects on the amelioration of FNAIT, including decreased maternal platelet antibody, decreased fetal platelet clearance, reduced bleeding disorders, and increased fetal survival.92 In a cost-effectiveness analysis, Thung et al94 compared the use of noninvasive empiric IVIG with FBS treatment. They concluded that noninvasive IVIG is a cost-effective strategy.93 In the antenatal management of FNAIT, it is important to realize that the majority of at-risk patients are those who have a sibling with a history of severe thrombocytopenia but without internal bleeding. The risk of ICH in this category of patients is low and approximates the risk of fetal loss with invasive management; therefore, a noninvasive approach is preferred. Considering the high cost of IVIG, and that most of the ICH in these patients occurs later in pregnancy, treatment of FNAIT pregnancies without a history of ICH may be started between 28 and 32 weeks. In pregnancies with a previous sibling with an ICH, IVIG should be started at 12 to 18 weeks. To evaluate the response, FBS can be performed at 24 to 28 weeks. When the anticipated risk of the procedure is high (eg, less experienced center or a posteriorly located cord insertion), IVIG should be continued until delivery without FBS. A recent study examined this less invasive approach in seven pregnancies with a prior sibling with ICH.88 IVIG (1 g/kg/week) was administered from a median of 16 weeks (range, 16-29 weeks)

414

without initial or follow-up FBS. Although all neonates had a birth platelet count of less than 50,000/µL, none of them showed signs of internal or external bleeding. The neonates needed only one platelet transfusion and did well. The results demonstrate that the primary goal in the management of pregnancies at risk for FNAIT is the prevention of severe bleeding complications and not thrombocytopenia per se. The very rare case of a patient with a history of ICH very early in pregnancy might benefit from a more aggressive approach (eg, high-dose IVIG, followed by repeated FBS and, when necessary, rescue high-dose corticosteroids). In FNAIT patients, safety precautions may lower the risk of hemorrhage during FBS. The Leiden University Medical Center protocol for FBS includes the following: 1. FBS in FNAIT pregnancies is performed by the most experienced members of the fetal medicine unit. 2. The use of a cell counter in the operating room enables platelet count and histogram to be obtained within 2 minutes. 3. Before each FBS, compatible platelets are made available, and infused during the procedure, if necessary, to achieve a platelet count of ⬎300,000/µL. Platelet transfusions for fetal indications are generally blood group O RhD-negative (HPA compatible in case of FNAIT) units that have a reduced risk of CMV and parvovirus B19 transmission. The intent is to increase the platelet count to normal levels, preferentially higher than 300,000/µL to achieve a fetal platelet count ⬎20,000/µL for 7 to 10 days. The recovery in the fetal circulation is approximately 25%, because 50% of the platelets remain in the placental bed and 50% of the platelets in the child are pooled in the spleen. This implies that approximately 100,000 to 200,000/µL platelets (depending on gestational agerelated fetoplacental volume) are concentrated in 20 to 50 mL for intrauterine transfusion after irradiation with 25 Gy to prevent GVHD.

Fetal Transfusions for Other Indications Parvovirus B19 infection during pregnancy may lead to an arrest in maturation of hematopoietic stem cells and thus anemia and thrombocytopenia. Parvovirus B19 is a frequent cause of nonimmune hydrops fetalis and 46% of hydropic fetuses have thrombocytopenia below 50,000/µL. The platelet count will further decrease after transfusion of red cells alone.94 After serologic and DNA diagnosis of infection in the mother and elevated middle cerebral artery peak velocity at Doppler investigation, intrauterine transfusion is indicated. Usually one intrauterine blood transfusion is sufficient.67 Because a low platelet count (⬍50,000/ µL) may lead to bleeding complications from the FBS, platelets should be available for transfusion. Massive fetomaternal (transplacental) hemorrhage is a rare, unexpected, and serious complication in pregnancy resulting in severe fetal anemia, hydrops, and death. Reduced fetal movement is the only cognizant sign for the pregnant woman. The diagnosis can be confirmed when a sinusoidal pattern is observed at cardiotocogram registration and/or when an increased flow in the middle cerebral artery is measured.95 Fetal red cells in the

Chapter 26: Obstetric Transfusion Practice

maternal circulation can be confirmed with the Kleihauer-Betke test.93 In the preterm period, fetal red cell transfusion is a realistic option. After the introduction of laser coagulation of placental anastomosis as treatment for severe twin-to-twin transfusion syndrome (TTTS), TAPS (twin anemia-polycythemia sequence) has been described as a new syndrome.96 After laser coagulation, very small residual anastomosis may lead to chronic anemia and polycythemia in one of the twins.97 Robyr et al98 described 13 cases of TAPS (13% of their group of TTTS cases). In 12 of the 13 cases, between one and five fetal red cell transfusions were performed. Because expectant management can lead to a favorable outcome, further research in this field is needed.

Conclusion Postpartum hemorrhage is a serious risk factor in obstetrics. Protocols for both prevention and management should be available in all labor and delivery wards. In high HDI countries, most maternal mortality can be prevented using oxytocics, embolization of uterine vessels, and blood transfusion. Unfortunately, both control of bleeding and early transfusion are difficult to achieve in most low HDI countries. In these countries, blood transfusion services are generally unable to help women who need red cells. Blood donation “on the spot” is often the only available option, and bears a serious risk for transmission of infections such as hepatitis and HIV. The option of fetal transfusion, a widely used treatment of the unborn in developed countries, is not available in many other countries.

Acknowledgment We are grateful to Mrs. Ivanka Bekker for expert secretarial assistance in the preparation of this chapter.

Disclaimer The authors have disclosed no conflicts of interest.

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5. Ajzenberg N, Dreyfus M, Kaplan C, et al. Pregnancy-associated thrombocytopenia revisited: Assessment of follow-up of 50 cases. Blood 1998;92:4573-80. 6. Minakami H, Kohmura Y, Izumi A, et al. Relation between gestational thrombocytopenia and the syndrome of hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome. Gynecol Obstet Invest 1998;46:41-5. 7. Sibai BM. The HELLP syndrome: Much ado about nothing? Am J Obstet Gynecol 1990;162:311-6. 8. Miller JM, Pastorek. Thrombotic thrombocytopenic purpura and hemolytic uremic syndrome in pregnancy. Clin Obstet Gynecol 1991;34:64-71. 9. Sibai BM, Kustermann L, Velasco J. Current understanding of different clinical syndromes or just different names? Curr Opin Nephrol Hypertens 1994;3:436-45. 10. Greer IA, Cameron AD, Walker JJ. HELLP syndrome: Pathologic entity or technical inadequacy? Am J Obstet Gynecol 1985;152:113-4. 11. Martin JN, Files JC, Blake PG, et al. Postpartum plasma exchange for atypical (pre)eclampsia as HELLP syndrome. Am J Obstet Gynecol 1995;172:1107-27. 12. Scully M, Starke R, Lee R, et al. Successful management of pregnancy in women with a history of thrombotic thrombocytopenic purpura. Blood Coagul Fibrinolysis 2006;17:459-63. 13. British Committee for Standards in Haematology, General Haematology Task Force. Guidelines for the investigation and management of idiopathic thrombocytopenic purpura in adults, children and in pregnancy. Br J Haematol 2003;120:574-96. 14. McMillan R. Therapy for adults with refractory chronic immune thrombocytopenic purpura. Ann Intern Med 1997;126:307-14. 15. Kessler I, Lancet M, Borenstein R, et al. The obstetrical management of patients with immunologic thrombocytopenic purpura. Int J Gynecol Obstet 1982;20:23-8. 16. Burrows RF, Kelton JG. Fetal thrombocytopenia and its relation to maternal thrombocytopenia. N Engl J Med 1993;329:1463-6. 17. George JN, Woolf SH, Raskob GE, et al. Idiopathic thrombocytopenic purpura: A practice guideline developed by explicit methods for the American Society of Hematology. Blood 1996;88:3-40. 18. Christiaens GCML, Nieuwenhuis HK, von dem Borne AEGK, et al. Idiopathic thrombocytopenic purpura in pregnancy: A randomized trial on the effect of antenatal low dose corticosteroids on neonatal platelet count. Br J Obstet Gynaecol 1991;98:334-6. 19. Samuels P, Bussel JB, Braitman LE, et al. Estimation of thrombocytopenia in the offspring of pregnant women with presumed immune thrombocytopenic purpura. N Engl J Med 1990;323:229-35. 20. Burrows RF, Kelton JG. Thrombocytopenia during pregnancy. In: Greer IA, Turpie AGG, Forbes CD, eds. Haemostasis and thrombosis in obstetrics and gynaecology. London: Chapman and Hall, 1992:407-29. 21. Kaplan C, Daffos F, Forestier F, et al. Fetal platelet counts in thrombocytopenic pregnancy. Lancet 1990;336:979-82. 22. Payne SD, Resnik R, Moore TR, et al. Maternal characteristics and risk of severe neonatal thrombocytopenia and intracranial hemorrhage in pregnancies complicated by autoimmune thrombocytopenia. Am J Obstet Gynecol 1997;717:149-55. 23. Cook RL, Miller R, Katz VL, Cefalo RC. Immune thrombocytopenic purpura in pregnancy: A reappraisal of management. Obstet Gynecol 1991;78:578-83. 24. Valat AS, Caulier MT, Devos P, et al. Relationships between severe neonatal thrombocytopenia and maternal characteristics in

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27.

28. 29. 30. 31.

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34.

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37.

38.

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46. Wells AW, Mounter PJ, Chapman CE, et al. Where does blood go? Prospective observational study of red cell transfusion in North England. Br Med J 2007;325:803-4. 47. Schonewille H, Brand A. Alloimmunization to red blood cell antigens after universal leucodepletion. A regional multicenter retrospective study. Br J Haematol 2005;129:151-6. 48. van Kamp IL, Klumper FJCM, Meerman RH, et al. Treatment of fetal anemia due to red cell alloimmunization with intra uterine transfusions in The Netherlands, 1988-1999. Acta Obstet Gynecol Scand 2004;83:731-7. 49. Kamphuis MM, Lindenburg I, van Kamp IL, et al. Implementation of routine screening for Kell antibodies: Does it improve perinatal survival? Transfusion 2008;48:953-7. 50. Guideline for the use of platelet transfusions. Br J Haematol 2003;122:10-23. 51. Schnaidt M, Wernet D. Platelet specific antibodies in female blood donors after pregnancy. Transfus Med 2000;10:77-80. 52. Murphy MF, Waters AH. Immunological aspects of platelet transfusions. Br J Haematol 1985;60:409-14. 53. Brand A, Sintnicolaas K, Claas FHJ, Eernisse JG. ABH-antibodies causing platelet transfusion refractoriness. Transfusion 1986;26:463-6. 54. Urbaniak SJ, Greiss MA. RhD haemolytic disease of the fetus and the newborn. Blood Rev 2000;14:44-61. 55. Howard HL, Martlew VJ, McFayden IR, Clarke CA. Preventing rhesus(D) haemolytic disease of the newborn by giving anti-D immunoglobulin: Are the guidelines being adequately followed? Br J Obstet Gynaecol 1997;104:34-41. 56. Letsky E, de Silva M. Preventing Rh immunisation: Much scope for improvement. Br Med J 1994;309:213-4. 57. Winter C, Macfarlane A, Deneux-Tharaux C, et al. Variations in policies for management of the third stage of labor and the immediate management of postpartum hemorrhage in Europe. Br J Obstet Gynaecol 2007;114:845-54. 58. Blood transfusion in pregnancy. Utrecht, The Netherlands: Dutch Society of Obstetrics and Gynaecology, 2002. 59. Geelhoed D, Agadzi F, Visser L, et al. Maternal and fetal outcome after severe anemia in pregnancy in rural Ghana. Acta Obstet Gynecol Scand 2006;85:49-55. 60. Bates I. Blood transfusion. In: Cook GC, Zumla AI, eds. Manson’s tropical diseases. 21st ed. Amsterdam: Elsevier, 2003:245-51. 61. Ozumba BC, Ezegwui HU. Blood transfusion and cesarean section in a developing country. J Obstet Gynaecol 2006;26:746-8. 62. Liley AW. Intrauterine transfusion of foetus in haemolytic disease. Br Med J 1963;5365:1107-9. 63. van Kamp IL, Kanhai HHH. Management of red cell alloimmunization in pregnancy. In: Smit Sibinga CT, Luban N, eds. Neonatology and blood transfusion. New York: Springer, 2005:71-114. 64. Daffos F, Capella-Pavlovsky M, Forestier FA. A new procedure for fetal blood sampling in utero: Preliminary results of fifty-three cases. Am J Obstet Gynecol 1983;146:985-7. 65. Oepkes D, Seaward PG, Vandenbussche FP, et al. Doppler ultrasonography versus amniocentesis to predict fetal anemia. N Engl J Med 2006;355:156-64. 66. Rodis JF, Borgida AF, Wilson M, et al. Management of parvovirus infection in pregnancy and outcomes of hydrops: A survey of members of the Society of Perinatal Obstetricians. Am J Obstet Gynecol 1998;179:985-8. 67. Nagel HT, de Haan TR, Vandenbussche FP, et al. Long-term outcome after fetal transfusion for hydrops associated with parvovirus B19 infection. Obstet Gynecol 2007;109:42-7.

Chapter 26: Obstetric Transfusion Practice

68. Giacoia GP. Severe fetomaternal hemorrhage: A review. Obstet Gynecol Surv 1997;52:372-80. 69. Haak MC, Oosterhof H, Mouw RJ, et al. Pathophysiology and treatment of fetal anemia due to placental chorioangioma. Ultrasound Obstet Gynecol 1999;14:68-70. 70. Carr S, Rubin L, Dixon D, et al. Intrauterine therapy for homozygous alpha-thalassemia. Obstet Gynecol 1995;85:876-9. 71. Kamp IL, Klumper FJ, Oepkes D, et al. Complications of intrauterine intravasular transfusion for fetal anemia due to maternal red-cell alloimmunization. Am J Obstet Gynecol 2005;192:171-7. 72. Schonewille H, Klumper FJCM, van de Watering LMG, et al. High additional maternal alloimmunization after Rh and K-matched intrauterine intravascular transfusions for hemolytic disease of the fetus. Am J Obstet Gynecol 2007;196:143.e1-6. 73. Rodeck CH, Deans A. Red cell alloimmunization. In: Rodeck CH, Whittle MJ, eds. Fetal medicine. Basic science and clinical practice. New York: Churchill Livingstone, 1999:785-804. 74. Saade GR, Moise KJ Jr, Copel JA, et al. Platelet counts correlate with the severity of the anemia in red-cell alloimmunization. Obstet Gynecol 1993;82:987-91. 75. Porcelijn L, Kanhai HHH. Diagnosis and management of fetal platelet disorders. In: Rodeck CH, Whittle MJ, eds. Fetal medicine. Basic science and clinical practice. New York: Churchill Livingstone, 1999:805-15. 76. Feng ML, Liu DZ, Shen W, et al. Establishment of an HPA-1- to -16typed platelet donor registry in China. Transfus Med 2006;16:369-74. 77. Radder CM, Brand A, Kanhai HHH. Will it ever be possible to balance the risk of intracranial hemorrhage in fetal or neonatal alloimmune thrombocytopenia against the risk of treatment strategies to prevent it? Vox Sang 2003;84:318-25. 78. Bertrand C, Martageix C, Jallu V, et al. Predictive value of sequential anti-HPA-1a antibody concentrations for the severity of fetal alloimmune thrombocytopenia. J Thromb Haemost 2006;4:628-37. 79. Proulx C, Filion MM, Goldman M, et al. Analysis of immunoglobulin class IgG subclass and titre of HPA-1a antibodies in alloimmunized mothers giving birth to babies with or without neonatal alloimmune thrombocytopenia. Br J Haematol 1994;87:813-17. 80. Killie MK, Husebekk A, Kaplan C, et al. Maternal human platelet antigen-1a antibody level correlates with the platelet count in the newborns: A retrospective study. Transfusion 2007;47:55-8. 81. Lynch L, Bussel JB, McFarland JG, et al. Antenatal treatment of alloimmune thrombocytopenia. Obstet Gynecol 1992;80:67-71. 82. Berkowitz RL, Bussel JB, McFarland JG. Alloimmune thrombocytopenia: State of the art 2006. Am J Obstet Gynecol 2006;195:907-13. 83. Overton TG, Duncan KR, Jolly M, et al. Serial aggressive platelet transfusion for fetal alloimmune thrombocytopenia: Platelet dynamics and perinatal outcome. Am J Obstet Gynecol 2002;186:826-31. 84. Birchall JE, Murphy MF, Kaplan C, et al. European collaborative study of the antenatal management of feto-maternal alloimmune thrombocytopenia. Br J Haematol 2003;122:275-88.

85. Berkowitz RL, Kolb EA, McFarland JG, et al. Parallel randomized trials of risk-based therapy for fetal alloimmune thrombocytopenia. Obstet Gynecol 2006;107:91-6. 86. Bussel JB, Berkowitz RL, Lynch L, et al. Antenatal management of alloimmune thrombocytopenia with intravenous gamma-globulin: A randomized trial of the addition of low-dose steroid to intravenous gamma-globulin. Am J Obstet Gynecol 1996;174:1414-23. 87. Bussel JB, Berkowitz RL, McFarland JG, et al. Antenatal treatment of neonatal alloimmune thrombocytopenia. N Engl J Med 1988;319:1374-8. 88. Kanhai HHH, van den Akker ESA, Walther FJ, et al. Intravenous immunoglobulins without initial and follow-up cordocentesis in alloimmune fetal and neonatal thrombocytopenia at high risk for intracranial hemorrhage. Fetal Diagn Ther 2006;21:55-60. 89. van den Akker E, Oepkes D, Lopriore E, et al. Noninvasive antenatal management of fetal and neonatal alloimmune thrombocytopenia: Safe and effective. Br J Obstet Gynaecol 2007;114:469-73. 90. Radder CM, Beekhuizen H, Kanhai HH, Brand A. Effect of maternal anti-HPA-1a antibodies and polyclonal IVIG on the activation status of vascular endothelial cells. Clin Exp Immunol 2004;137:216-22. 91. Radder CM, de Haan MJ, Brand A, et al. Follow up of children after antenatal treatment for alloimmune thrombocytopenia. Early Hum Dev 2004;80:65-76. 92. Ni H, Chen P, Spring CM, et al. A novel murine model of fetal and neonatal alloimmune thrombocytopenia: Response to intravenous IgG therapy. Blood 2006;107:2976-83. 93. Thung SF, Grobman WA. The cost effectiveness of empiric intravenous immunoglobulin for the antepartum treatment of fetal and neonatal alloimmune thrombocytopenia. Am J Obstet Gynecol 2005;193:1094-9. 94. Haan TR de, van den Akker ESA, Porcelijn L, et al. Thrombocytopenia in hydropic fetuses with parvovirus B19 infection: Incidence, treatment and correlation with fetal B19 viral load. Br J Obstet Gynaecol 2008;115:76-81. 95. Sueters M, Arabin B, Oepkes D. Doppler sonography for predicting fetal anemia caused by massive fetomaternal hemorrhage. Ultrasound Obstet Gynecol 2003;22:186-9. 96. Lopriore E, Middeldorp JM, Oepkes D, et al. Twin anemiapolycythemia sequence in two monochorionic twin pairs without oligo-polyhydramnios sequence. Placenta 2007;28:47-51. 97. Lopriore E, Middeldorp JM, Oepkes D, et al. Residual anastomoses after fetoscopic laser surgery in twin-to-twin transfusion syndrome: Frequency, associated risks and outcome. Placenta 2007;28:204-8. 98. Robyr R, Lewi L, Salomon LJ, et al. Prevalence and management of late fetal complications following successful selective laser coagulation of chorionic plate anastomoses in twin-to-twin transfusion syndrome. Am J Obstet Gynecol 2006;194:796-803.

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27

Hemolytic Disease of the Fetus and Newborn Bjarte G. Solheim1 & Morten Grönn2 1 2

Professor Emeritus, Institute of Immunology, Rikshospitalet University Hospital and University of Oslo, Oslo, Norway Head Physician, Division of Paediatrics, Rikshospitalet University Hospital, Oslo, Norway

Hemolytic disease of the fetus and newborn (HDFN) is caused by increased destruction of red cells in the fetus and newborn. The disease is associated with immune-mediated destruction (alloor autoantibodies), intrinsic red cell abnormalities (hemoglobinopathies, enzyme defects, and membrane defects), and acquired red cell defects (related to infections, see Chapter 26). This chapter focuses on fetomaternal alloimmune-mediated HDFN, and only briefly mentions important intrinsic abnormalities.

The Changing Spectrum of HDFN Status in Developed Countries During the past 30 to 40 years, the spectrum of HDFN has changed profoundly. In the late 1960s and early 1970s, HDFN was a common neonatal problem dominated by Rh(D) alloimmunization. Overt fetal or neonatal hemolysis, with marked anemia and hyperbilirubinemia in the neonate, were commonly observed symptoms. The neonates were often severely affected and unstable at birth, and required multiple exchange transfusions. HDFN was associated with considerable neonatal morbidity and mortality. Introduction in the 1970s of routine postpartum prophylactic Rh Immune Globulin (RhIG or anti-D) to D-negative women (with D-positive infants) and after pregnancy termination or abortion, has changed the picture dramatically and reduced D alloimmunization by about 90%.1 After introduction of additional antepartum prophylactic RhIG, the frequency of D alloimmunization is now well below 0.2%.1 The introduction of prophylactic RhIG ranks as one of the great success stories of modern perinatal care and immunology. Programs for antibody screening and ABO/Rh typing of pregnant women allows focus on fetuses and neonates at risk, and has paved the way for the vastly improved antepartum surveillance and therapy outlined in Chapter 26. Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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Finally, postpartum therapy has improved considerably.2 Forty years ago, exchange transfusion and early versions of phototherapy were the only therapeutic alternatives for serious HDFN. Today modern phototherapy, if necessary combined with high-dose intravenous immunoglobulin (IVIG), has reduced the need for exchange transfusion to single figures even in large centers. This raises serious challenges in order to keep the necessary competence in the practical execution of exchange transfusion.2 Alloantibody frequencies in populations of mostly European ancestry before and after introduction of postpartum prophylactic RhIG treatment are indicated in Table 27-1.

Status in Developing Countries Programs for antibody screening and ABO/Rh typing of pregnant women and introduction of postpartum routine prophylactic RhIG treatment have posed both organizational and economic challenges in countries with a low human development index (HDI). Improved neonatal surveillance and therapy has been Table 27-1. Red Cell Antibodies in 1967 and 19953,4 Antibody D E C Cw c e K1 Duffy MNSs Kidd Lutheran P1 Lewis I Others Total Blood samples

1967 (Minnesota, 7 years) 1864 (63.1%) 80 (2.7%) 448 (15.2%) 4 (0.14%) 68 (2.3%) 2 (0.07%) 93 (3.1%) 17 (0.6%) 45 (1.5%) 7 (0.2%) — 27 (0.9%) 94 (3.2%) 13 (0.4%) 194 (6.6%) 2,956 43,000

1995 (Sweden, 12 years) 159 (19.0%) 5 (6.1%) 36 (4.3%) 10 (1.2%) 38 (4.5%) 1 (0.1%) 48 (5.7%) 26 (3.1%) 35 (4.2%) 10 (1.2%) 13 (1.6%) 48 (5.7%) 241 (28.8%) — 120 (14.4%) 836 110,765

Chapter 27: Hemolytic Disease of the Fetus and Newborn

introduced only to a limited degree, and antepartum treatment is often nonexistent. HDFN is, therefore, still a major cause of morbidity and mortality in these countries.

Serologic and Clinical Features Development of immune HDFN requires maternal IgG antibodies directed against paternal antigens on fetal red cells. Immunization can occur when fetal red cells pass through the placenta to the mother’s circulation or the antigens were present in previous transfusions of the mother. Only maternal IgG antibodies can cross the placenta. The transport is an active process dependent upon interaction between maternal IgG and synciotrophoblast Fc receptors, and is most active in the third trimester.5 IgG antibodies can be elicited by most blood group antigens (Tables 27-1 and 27-2, and Chapter 5). The immune response to peptide antigens (eg, D) typically requires T-cell help, and is dominated by IgG antibodies. Because the D antigen is extraordinarily immunogenic and induces high affinity anti-D IgG, it has been a major cause for HDFN in White and Black populations where 10% to15% of people lack the D antigen. This is not the case in East Asia (particularly China), where the population is D-positive (see Chapter 7). In addition, IgG autoantibodies causing maternal hemolytic anemia can result in HDFN. The common occurrence of antibodies reacting with carbohydrate antigens of the ABO, Hh, Ii, Lewis, and P systems without prior exposure to allogeneic red cells is a result of the wide distribution of these antigens in nature. IgM Table 27-2. Blood Group Antigens and Hemolytic Disease in the Neonate6 in Populations of European and African Ancestry Antigen

Antibody Frequency (per 1000 pregnant women)

Severity of HDFN in Infants with the Antigen None/Mild

Moderate

Severe

Not HDFN, but antibody in 90%

⬍10%

⬍1%

A, B, AB

Not relevant

D

2.6

51%

30%

19%

c, cE

0.9

70%

23%

7%

E

2.0

Almost all

-

Rare

C, Ce, Cw, e

0.7

86%

14%

Rare

a

Le , Le

b

Kell (K1) a

3.0

Not HDFN

3.2

30-50%

30-37%

13-38%

16%

6-16%

Fy

0.8

67-94%

Fyb

Rare

Rare cause of mild HDFN

Kidd (Jka)

0.2

Rare

M

0.5

Rare

N

0.1

Rare

U

Rare

Rare

N P (P1)

0.03 0.03

Not HDFN Not HDFN

is generally the dominating antibody, but IgG anti-A, anti-B, and more rarely anti-H are also present. Because the I antigen and antigens of the Lewis and P systems are almost lacking on fetal cells, they are not associated with HDFN.7 Table 27-2 summarizes antibody frequencies and severity of HDFN caused by relevant blood group antigens in populations of European and African ancestry. The direct antiglobulin test (DAT) (see Chapter 5) is one of the cornerstones for the diagnosis of HDFN. It detects IgG antibodies on the surface of red cells, and the number of IgG molecules bound to the erythrocyte surface determines the strength of the reaction. Using the spin antiglobulin test, the detection limit is 100 to 150 IgG molecules per red cell; when over 1000 are bound, the DAT is strongly positive (all cells are agglutinated).8 Transplacental passage of maternal IgG results in antibody coating of the fetal red cells. High alloantibody concentrations lead to a strong DAT, whereas low concentrations or binding affinity result in a weak or, by traditional methods, often negative DAT. A strong positive DAT is observed particularly with anti-D; however, the reaction is more variable with alloantibodies against other Rh antigens and antigens such as Kell, Duffy, Kidd, and MNSs, which are well expressed on fetal red cells.5 Because ABO antigens are not fully developed on fetal red cells and A and B substances are expressed by most tissues, the DAT in ABO incompatibility is often negative. However, with very sensitive techniques (eg, automated analyzer with lowionic-strength medium with enhancing agents) IgG can almost always be demonstrated on the red cells in ABO incompatibility.9 As a basic test, the DAT has poor predictive value in identifying neonates requiring treatment. Only 23% of newborns found to be DAT positive on neonatal screening have been reported to develop hyperbilirubinemia requiring treatment, but when the DAT was strongly positive (generally because of anti-D) all required treatment.10 It should also be borne in mind that after antepartum treatment of D-negative women with prophylactic RhIG, neonates may demonstrate a positive DAT without signs of hemolysis.11 In an affected fetus, HDFN is caused by the hypoxic effects of anemia, while in the newborn, HDFN is caused by toxic effects of bilirubin and anemia. Bilirubin crosses the placenta during pregnancy and is removed by the mother’s liver. However, because of insufficient capacity or failure of the neonate’s liver, plasma levels of unconjugated bilirubin can quickly become high enough to cause serious central nervous system damage in the newborn. The anemia causes increased fetal erythropoiesis, which in severe cases leads to erythroblastosis with widespread extramedullary hematopoiesis and many nucleated red cells (erythroblasts) in the circulation. The most severe state of erythroblastosis, hydrops fetalis, can lead to stillbirth and is characterized by severe anemia, massive edema, and hypoproteinemia. In severe erythroblastosis, blood film examination will show massive presence of nucleated red cells, but only a few spherocytes due to the rapid destruction of the red cells (eg, serious Rh HDFN). In less severe HDFN, spherocytes will dominate the blood film, and only a few nucleated red cells are observed (eg, ABO HDFN). This emphasizes the importance of blood film examination in neonates with nonphysiologic jaundice.2

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Persistent or prolonged jaundice in neonates is a common feature of HDFN, and can be observed in virtually all conditions mentioned in this chapter. Achieving the correct diagnosis is important for the neonate and can have implications also for other family members.

Hemolytic Disease Predicted by Antepartum Maternal Antibody Screening Although the majority of cases of acute neonatal hyperbilirubinemia/hemolysis currently occur without antepartum warning in high HDI countries, there remain a number of neonates for whom HDFN is predicted by antepartum screening. Almost all blood group antibodies of IgG type can cause HDFN. However, the severity and frequency of HDFN varies, and anti-D still remains the most common cause of severe HDFN.2 The most frequent alloantibodies are directed against: Rh antigens (anti-D, anti-E, anti-c, anti-C), Kell antigens (anti-K1), Duffy antigens (anti-Fya), Kidd antigens (anti-Jka), and MNSs antigens (Table 27-2). Anti-D, anti-c, anti-E, and anti-K are found in about 1 per 1000 pregnant women. Anti-K is mostly caused by transfusion, and the frequency of HDFN due to anti-K is much lower than that due to anti-D.5 However, anti-Kell can cause severe fetal anemia because the antibody also inhibits fetal erythropoiesis.12 The frequency and severity of HDFN caused by the other antibodies mentioned above is low (Table 27-2). Positive antepartum maternal antibody screening results in frequent antepartum monitoring and makes treatment with intrauterine transfusions possible (see Chapter 26). Antepartum diagnosis and postpartum management of neonates affected by HDFN require close collaboration among obstetric, neonatal, and transfusion medical teams. Delivery is generally induced around 36 weeks of gestation. All neonates at risk should have cord blood taken for measurement of hemoglobin, DAT, and bilirubin (see Chapter 26). Because of the use of intrauterine transfusions, many neonates have normal hemoglobin at birth and develop only modest hyperbilirubinemia. In these neonates, the DAT demonstrates few antibody-covered red cells, and typing barely reveals the neonate’s original blood type. However, all potentially affected neonates should remain in the hospital until hyperbilirubinemia and/or anemia is under control and appropriate follow-up is organized. Phototherapy, highdose IVIG, and exchange transfusions are often not required. Red cell transfusions can be indicated when anemia develops during the first couple of weeks after birth. Severe anemia (hemoglobin ⬍10 g/dL) at birth or rapidly increasing hyperbilirubinemia should be treated as detailed later in this chapter.

Early-Onset/Rapidly Progressive Hemolytic Disease not Predicted by Maternal Antibody Screening Today neonatal pediatricians in developed countries are often alerted to HDFN in a relatively full-term infant whose mother’s

420

antepartum screening was negative. Within the first 48 hours of life such a newborn develops early-onset jaundice with a total bilirubin already in excess of 20 mg/dL (342 µmol/L).2 Because the problem in such cases has not been predicted by antepartum screening for maternal antibodies, therapy would not have been started. The most common causes of HDFN presenting in this way are as follows: ● ABO incompatibility. ● Hemoglobinopathies. ● Erythrocyte enzyme defects. ● Disorders of the red cell membrane. The reader is referred to Chapter 29 for detailed information on hemoglobinopathies, erythrocyte enzyme defects, and disorders of the red cell membrane.

ABO Incompatibility ABO incompatibility is now the leading cause of HDFN in high HDI countries.10 HDFN resulting from ABO incompatibility generally occurs only in offspring of women of blood group O because IgG anti-A and anti-B is far more common in group O than in group A or B mothers, and is confined to the 1% with antepartum high-titer IgG antibodies.13 Unlike in nonABO HDFN, the first ABO-incompatible infant is at risk for significant hemolysis, because ABO antibodies are developed before pregnancy. Minor degrees of red cell destruction are common, as shown by the increased incidence of neonatal jaundice and slightly lower hemoglobin values in ABO-incompatible infants compared to ABO-compatible infants.5 With respect to ethnic groups, the prevalence of maternal-newborn ABO incompatibility ranges from 31% in those of European ancestry to 50% in Asians. Generally, ABO HDFN causes mild postpartum jaundice, which is detected 24 hours after delivery; in very rare cases, however, hemolysis is severe enough to cause hydrops.14 The frequency of HDFN caused by anti-A is only about 1 in 150 births, and even lower for anti-B.2 Higher frequencies that have been reported from Africa and Saudi Arabia are probably related to more potent anti-A and anti-B (caused by environmental factors) and a relatively strong expression of A and B antigens.5 Antepartum and postpartum serologic tests are poor predictors of ABO hemolytic disease. The results of the DAT in HDFN caused by ABO incompatibility are very different from those observed with HDFN caused by anti-D (not treated antepartum). In the latter, a positive DAT can be observed even in cases showing minor clinical symptoms. However, in ABO incompatibility only 20% to 40% of incompatible pairs demonstrate a positive reaction and significant hemolysis can be observed with a negative or only weakly a positive DAT. In infants with a positive DAT result, the test differentiates poorly between newborns with clinical hemolysis and unaffected newborns. Only about 10% of the DAT-positive, ABO-incompatible maternal-fetal pairs develop clinically significant HDFN. However, in infants with clinically significant hemolysis, ⬎80% have a positive DAT.10,15 Also, the clinical symptoms differ in that anti-A and anti-B HDFN result mainly in hyperbilirubinemia detected 24 hours after delivery

Chapter 27: Hemolytic Disease of the Fetus and Newborn

without the significant fetal or neonatal anemia observed in antiD HDFN. This is explained by the few and unbranched A and B antigen sites on fetal red cells, allowing antibody-coated cells to remain in the circulation longer than in anti-D HDFN. Another important factor is that ABO antigens are expressed on virtually all cells in the body, whereas D antigen is restricted to red cells. Thus, anti-D is “focused” to red cells, while anti-A and anti-B are absorbed by tissues and neutralized by soluble A and B substances in plasma. As a reflection of this, the blood film in anti-A or antiB HDFN shows a large number of spherocytes, with little or no increase in nucleated red cells. In anti-D HDFN there are few spherocytes but large numbers of nucleated red cells. In ethnic groups expressing strong (many and branched) A and B antigen sites, severe anemia, nucleated red cells, and significant antepartum morbidity (even with hydrops fetalis) can be observed.2 Management of ABO HDFN is usually successful with modern phototherapy alone. Close monitoring of affected neonates is important, as high-dose IVIG adjuvant therapy and occasionally even exchange transfusion can be required. The latter is particularly relevant in individuals from certain ethnic groups who express strong A and B sites.2

Red Cell Enzyme Defects The important enzyme defects in the neonatal period are glucose6-phosphate dehydrogenase (G6PD) deficiency and pyruvate kinase deficiency. They usually present with early onset, often severe, hyperbilirubinemia. Blood smear is normal, and anemia rare. Family medical history and measurement of the relevant pretransfusion enzyme activities are of diagnostic importance. G6PD deficiency is transmitted in an X-linked recessive form, and seen in all ethnic groups; however, its highest prevalence is in central Africa (20%) and the Mediterranean region (10%). It is not clear why only some neonates develop jaundice. However, because these patients can develop severe hyperbilirubinemia, close monitoring is important. Interaction with other risk factors for hyperbilirubinemia in the newborn, as well as some medicines, chemicals, and foods, can severely aggravate the clinical picture. Pyruvate kinase deficiency is autosomal recessive, and the second most common red cell enzyme defect in neonates. The severity varies from hydrops fetalis to severe early-onset neonatal hyperbilirubinemia, and mild hyperbilirubinemia mimicking physiologic jaundice. For further details and information see Chapter 29.

Red Cell Membrane Defects Hemoglobinopathies Except for α-thalassemia major, hemoglobinopathies generally do not cause antepartum or neonatal problems. Some occasional structural mutations in α and γ genes that give no symptoms in adults, however, can result in transient hemolytic anemia in neonates. Mutations in the β genes (eg, sickle cell disease or β-thalassemia) give no symptoms at birth because of the predominance of fetal hemoglobin. α-thalassemia major occurs when all four α-globin genes on the homologous chromosome 16 are deleted (αo), and is predominantly seen in some Mediterranean island populations and in Southeast Asia. The lack of α-globin production leads to the dominating production of fetal γ4 hemoglobin (hemoglobin Bart’s), which has a very high oxygen affinity and is physiologically useless. Infants are usually stillborn between 28 and 40 weeks, demonstrating the typical picture of hydrops fetalis with pallor, generalized edema, and massive hepatosplenomegaly. The few that are live-born expire within the first hours after birth. Postpartum management has no impact on survival unless combined with carefully planned antepartum therapy, and has to be followed up with lifelong red cell transfusions or hematopoietic stem cell transplantation. Checking blood samples from the parents will show whether they are carriers of the αo gene (hypochromic, microcytic red cells). The diagnosis is confirmed by hemoglobin electrophoresis. α- and γ-globin chain structural abnormalities are generally clinical silent, but can result in occasional neonatal hemolytic anemia due to formation of unstable αγ-hemoglobin. When the fetal γ-globin is replaced by adult β-globin, the hemolytic anemia resolves. Diagnosis is confirmed by hemoglobin high-performance liquid chromatography. For further details and information see Chapter 29.

The main clues that a neonate has a red cell membrane disorder are a family history, DAT-negative hemolysis (with a sensitive method), and an abnormal blood film. Red cell membrane electrophoresis from pretransfusion samples is diagnostic. Hereditary spherocytosis is the most common red cell membrane defect. It occurs in 1 in 5000 live births in couples of North European ethnicity, but is less frequent in other population groups. It is inherited as an autosomal dominant trait.2 Blood films have a similar appearance to those in ABO HDFN; the DAT is negative. Some neonates require 1 or 2 red cell transfusions during the first months of life. Hereditary eliptocytosis is a more complex autosomal dominant disorder. In heterozygotes the only findings are eliptocytes in the blood smear. Neonates who have more than one mutation in a red cell membrane protein (homozygotes or compound homozygotes) have severe, transfusion-dependent, hemolytic anemia. The blood film is characteristic, showing a high proportion of bizarre fragmented red cells and microcytes (mean corpuscular volume ⬍60 fL). Red cell transfusion is often necessary until splenectomy can be carried out. For further details and information see Chapter 29.

Management of Immune-Mediated Hemolytic Disease of the Fetus and Newborn Surveillance and Tests Antepartum surveillance and treatment are detailed in Chapter 26. After adequate intrauterine transfusion treatment, fetal erythropoiesis is suppressed, and the red cell mass maintained by compatible transfused cells. As a result, most of these neonates present neither neonatal jaundice nor anemia of clinical importance

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after birth, and very few require postpartum exchange transfusion. In contrast, late anemia is common in the newborn because of suppression of erythropoiesis caused by intrauterine transfusions and continued presence of hemolyzing maternal antibodies. Therefore, the neonates need follow-up with monitoring of hemoglobin, blood film, reticulocyte count, and bilirubin in order to determine when hemolysis has stopped and endogenous red cell production takes over.2 In any consideration of red cell transfusions, the neonate’s general condition, growth, and development are of major importance for defining the appropriate hemoglobin level for intervention (see Chapter 30). The Apgar score can be useful because it reflects important signs and symptoms associated with postpartum anemia (eg, color, tachypnea, tachycardia, lethargy). In developing countries, antepartum treatment is mostly unavailable, and even antibody screening and ABO/Rh typing are often not performed in the pregnant mother. In such areas, the clinical picture can be more like that seen in developed countries in the 1960s, with serious anti-D HDFN (erythroblastosis, hyperbilirubinemia with grave anemia on the first postpartum day, and, in serious cases, hydrops fetalis or even stillbirth).

treatment of HDFN. The efficacy of phototherapy is dependent upon the following factors16: ● Spectral qualities of the delivered light (wavelength range 400-520 nm, with peak emissions of 460 nm). ● Intensity of light (irradiance). ● Surface area receiving phototherapy. ● Skin pigmentation. ● Total serum bilirubin concentration at the start of phototherapy. ● Duration of exposure. Modern phototherapy devices with high-intensity gallium nitride light-emitting diodes (LEDs) are small, efficient, easy to use, and ensure the correct distance between the neonate and the device.16 The total radiation applied to a neonate has vastly increased and made “intensive phototherapy” an important alternative to exchange transfusion, even in the treatment of severe HDFN. “Intensive phototherapy” implies radiation in the blue-green spectrum (wavelengths ⬃ 430-490 nm) of at least 30 µW/cm2/nm (measured at the infant’s skin) and delivered to as much as possible of the infant’s body surface area.17 Figure 27-1 shows guidelines for “intense phototherapy” in neonates of 35 weeks or more with hyperbilirubinemia. The levels shown are approximations adapted to the infant’s age and risk factors. Infants are designated as “higher risk” because of the potential negative effects of the conditions listed in the figure on albumin binding of bilirubin, the blood-brain barrier, and the susceptibility of brain cells to damage by bilirubin.17 Low body

Phototherapy

25

428

20

342

15

257

10

171

␮mol/L

Total serum bilirubin (mg/dL)

Improved delivery and understanding of phototherapy since its introduction in the late 1950s has had major effect on the clinical

5

85

Infants at lower risk (⭓38 wk. and well) Infants at medium risk (⭓38 wk. ⫹ risk factors or 35-37 6/7 wk. and well) Infants at higher risk (35-37 6/7 wk. ⫹ risk factors)

0 Birth

24 h

48 h

72 h

96 h

5 Days

0 6 Days

7 Days

Age ● ●

●

●

Use total bilirubin. Do not subtract direct reacting or conjugated bilirubin. Risk factors are isoimmune hemolytic disease, G6PD deficiency, asphyxia, significant lethargy, temperature instability, sepsis, acidosis, or albumin ⬍3.0 g/dL (if measured). For well infants 35-37 weeks and 6/7 days, TSB levels can be adjusted for intervention around the medium risk line. It is an option to intervene at lower TSB levels for infants closer to 35 weeks and at higher TSB levels for those closer to 37 weeks and 6/7 days. It is an option to provide conventional phototherapy in hospital or at home at TSB levels 2 to 3 mg/dL (35-50 µmol/L) below those shown, but home therapy should not be used in any infant with risk factors.

Figure 27-1. Guidelines for phototherapy in hospitalized infants of 35 weeks or more of gestation with hyperbilirubinemia, including that caused by HDFN. The guidelines refer to the use of “intensive phototherapy,” which should be used if total serum bilirubin (TSB) exceeds the line indicated for each category. Used with permission from American Academy of Pediatrics.17

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Chapter 27: Hemolytic Disease of the Fetus and Newborn

weight, particularly ⬍1000 g, is an additional risk factor to be considered. Today the “typical” neonate with HDFN presents in countries of high HDI as a well, term neonate with ABO incompatibility and a total serum bilirubin often in excess of 20 mg/dL but without significant anemia. “Intensive phototherapy” during the 4 to 6 hours it generally takes to organize an exchange transfusion most often results in a significant decrease in total serum bilirubin, thus frequently removing the indication for exchange transfusion.2 When high-dose IVIG is given as adjuvant to phototherapy the indication for exchange transfusion is even further reduced.

bilirubin (TSB) is rising despite intensive phototherapy or the TSB level is within 2 to 3 mg/dL (34-51 µmol/L) of exchange level. If necessary, this dose can be repeated in 12 hours.”

The exact action of IVIG in HDFN is still unclear, but following mechanisms are probably involved (see also Chapter 18): ● Prevention of phagocytosis of IgG-coated red cells by blocking of Fc receptors on cells in the reticuloendothelial system. ● Neutralization of maternal antibodies by anti-iodiotypic antibodies in IVIG. ● Cytokine modifying actions of IVIG.

Exchange Transfusions High-Dose IVIG Since the beginning of the 1990s, several pilot studies have reported the effect of high-dose IVIG as adjuvant to standard therapy for HDFN. Two systematic reviews have performed a metaanalysis of the impact of high-dose IVIG on the use of exchange transfusion, comparing IVIG plus phototherapy with phototherapy alone.18,19 The conclusion of both reviews was that IVIG significantly reduced the risk of exchange transfusion. However, the authors of the two systematic reviews differ with respect to their recommendation as to the use of high-dose IVIG. The Cochrane review18 concludes that the number and quality of studies, and the number of infants included is too small to recommend the use of adjuvant IVIG in HDFN, and suggests further well-designed studies. However, the other review19 supports the use of high-dose IVIG, and also shows that when IVIG is used in addition to phototherapy in HDFN, both duration of phototherapy and hospital stay are reduced to a point that equals the cost of the IVIG in the United Kingdom. With the decline in the number of exchange transfusions, the opportunity for further large, well designed studies is limited. Although exchange transfusions are associated with a mortality of 2% and serious complications in 12% of the patients (Table 27-3), no serious side effects have been reported in neonates treated with phototherapy and high-dose IVIG. It must, however, be remembered that IVIG is a pooled plasma product, which before the introduction of adequate pathogen inactivation, was involved in several cases of viral transmission. Since 1994 there has been no transmission of blood-borne infections with pathogen-inactivated IVIG prepared from selected donors20 (see also Chapter 19). Thus, further randomized studies as suggested by the authors of the Cochrane meta-analysis18 may even be considered unethical. Recently the IVIG Hematology and Neurology Expert Panels have recommended that high-dose IVIG be offered to neonates with HDFN as treatment for severe hyperbilirubinemia20 and endorsed the recommendations outlined in the American Academy for Pediatrics guideline on the treatment of hyperbilirubinemia.17 That guideline states: “In isoimmune hemolytic disease, administration of IVIG (0.5- 1.0 g/kg over 2 hours) is recommended if the total serum

Criteria used to assess the need for exchange transfusion include total bilirubin level, hemoglobin level, and clinical symptoms. Traditional guidelines suggest exchange transfusion in the following circumstances: Within 12 hours of birth if ● Cord blood bilirubin concentration exceeds 3 to 5 mg/dL for preterm infants, 5 to 7 mg/dL for term infants, or the rate of increase is ⬎0.5 mg/dL/hour. ● Severe anemia: hemoglobin ⬍10 g/dl combined with hyperbilirubinemia. After 24 hours of birth if ● Total bilirubin concentration ⬎20 mg/dL or a bilirubin increase of ⬎0.5 mg/dL/hour or hemoglobin ⬍10 g/dL combined with hyperbilirubinemia. Today, the effect of “intensive phototherapy” and reduction of hemolysis by high-dose IVIG treatment almost always results in a considerable decrease in bilirubin levels during the 4 to 6 hours

Table 27-3. Complications of Exchange Transfusion Vascular ● Embolization of air or thrombi ● Thrombosis Cardiac ● Arrhythmias ● Volume overload ● Cardiac arrest Electrolytes ● Hyperkalemia ● Hypernatremia ● Hypocalcemia ● Acidosis Coagulation ● Overheparinization ● Thrombocytopenia Infectious ● Bacteremia ● Hepatitis ● Cytomegalovirus

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30

513 Infants at medium risk (⭓38 wk. ⫹risk factors or 35-37 6/7 wk. and well) Infants at higher risk (35-37 6/7 wk.⫹risk factors)

25

428

20

342

15

257

␮mol/L

Total serum bilirubin (mg/dL)

Infants at lower risk (⭓38 wk. and well)

171

10 Birth

24 h

48 h

72 h

96 h

5 Days

6 Days

7 Days

Age ●

●

●

● ●

The dashed lines for the first 24 hours indicate uncertainty due to a wide range of clinical circumstances and a range of responses to phototherapy and highdose IVIG. Immediate exchange transfusion is recommended if infant shows signs of acute bilirubin encephalopathy (hypertonia, arching, retrocollis, opisthotonos, fever, high pitched cry) or if TSB is ⭓5 mg/dL (85 µmol/L) above these lines. Risk factors are isoimmune hemolytic disease, G6PD deficiency, asphyxia, significant lethargy, temperature instability, sepsis, acidosis, or albumin ⬍3.0 mg/dL (if measured). Use total bilirubin. Do not subtract direct reacting or conjugated bilirubin. If infant is well at 35-37 weeks and 6/7 days (median risk), TSB levels can be individualized for exchange based on actual gestation age.

Figure 27-2. Guidelines for exchange transfusion in infants 35 weeks or more of gestation with hyperbilirubinemia, including that caused by HDFN. The guidelines refer to the use of exchange transfusions, which should be used if total serum bilirubin (TSB) exceeds the line indicated for each category. During birth hospitalization, exchange transfusion is recommended if TSB rises to these levels

despite “intensive phototherapy.” For readmitted infants, if TSB level is above the exchange level, repeat TSB measurements every 2 to 3 hours and consider exchange transfusion if the TSB remains above the levels indicated after “intensive phototherapy” for 6 hours. Used with permission from American Academy of Pediatrics.17

needed to set up an exchange transfusion. Thus, the clinical indication for exchange transfusion may be removed, but the need for simple transfusion increases.2 Exchange transfusions are considered indicated when “intensive phototherapy,” with or without IVIG, fails to adequately reduce bilirubin concentration or when the initial serum bilirubin places the infant at risk for kernicterus. Factors potentiating bilirubin toxicity include immune-mediated hemolysis, acid-base disturbances, asphyxia, free heme groups, and other byproducts of hemolysis or drugs that displace bilirubin from albumin and other plasma binding proteins.6 Figure 27-2 shows current guidelines for exchange transfusion in neonates of 35 weeks or more with hyperbilirubinemia.17 The dashed lines for the first 24 hours on the figure indicate uncertainty due to the range of responses to phototherapy and to differences in clinical circumstances, thus emphasizing the importance of individualized treatment decisions. Treatment decisions after the first 24 hours are dependent upon gestational age, bilirubin concentration, the rate of its increase (⬎0.5 mg/ mL/hour) and risk factors that have a potential negative effect on albumin binding of bilirubin, the blood brain barrier, and the susceptibility of brain cells to damage by bilirubin (see Fig 27-2). Immediate exchange transfusion is recommended if the

infant shows signs of acute bilirubin encephalopathy as detailed in Fig 27-2, or if total bilirubin is 5 mg/dL above the lines. Exchange transfusions supply the neonate with compatible red cells and fresh plasma, while incompatible red cells, bilirubin, and maternal antibodies in plasma are removed. A standard exchange transfusion of twice the infant’s blood volume reduces incompatible fetal red cells by about 85%; bilirubin and maternal antibody concentrations are reduced by 25% to 45%.6 The amount of blood needed for an exchange transfusion is 170 mL/ kg (200 mL/kg in preterm infants). An exchange transfusion consists of hemoglobin S-negative red cells that are compatible with maternal serum and/or plasma and are reconstituted with fresh frozen group AB plasma to obtain a hematocrit of 45% to 50%. The red cells should be leukocyte-reduced (to prevent transmission of cytomegalovirus), irradiated, and preferably ⬍5 days old. Platelet addition is to be considered only if an infant’s platelet count is seriously compromised or after multiple exchange transfusions. In view of the declining number of exchange transfusions, and the risks involved with the procedure (Table 27-3) it is important that maternity and neonatal services develop, maintain, and teach appropriate written guidelines for the use of exchange transfusion in HDFN and the performance of the procedure itself.

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Conclusion In immune-mediated HDFN, maternal IgG antibodies— usually related to the ABO, Rh, or Kell blood groups—cross the placental barrier, causing hemolysis of fetal red cells. HDFN caused by hereditary red cell disorders are less frequent. Although HDFN can be fatal, advances in diagnosis, treatment, and prevention have made immune-mediated HDFN increasingly controllable in developed countries. Diagnostic modalities include maternal assays, antepartum treatment, and routine prophylactic RhIG given to D-negative women at risk. Postpartum management includes treatment of hyperbilirubinemia and anemia. Hyperbilirubinemia is usually treated by means of “intensive phototherapy,” while adjuvant highdose IVIG is given in order to reduce hemolysis. In addition, anemia is corrected by simple transfusion of red cells. These approaches have reduced the indications for exchange transfusion to very severe HDFN or when “intensive phototherapy” combined with IVIG fails to reduce hyperbilirubinemia to acceptable levels. Further improvement of phototherapy equipment, IVIG treatment, and possible administration of D-penicillamine and/or metalloporphyrins in order to reduce bilirubin formation21,22 could reduce the need of exchange transfusions and change the spectrum of HDFN treatment even more. While mortality and morbidity of HDFN have been greatly reduced in developed countries, HDFN still poses a serious challenge in many developing countries. In these countries with low HDI, introduction of maternal assays, routine prophylactic RhIG treatment, and affordable phototherapy should be encouraged. Because high-dose IVIG often will be beyond economic reach, phototherapy combined with drugs to reduce the postpartum formation of bilirubin could be of interest, and further studies with the administration of D-penicillamine21 or metalloporphyrins22 are to be encouraged.

Disclaimer The authors have disclosed no conflicts of interest.

References 1. Fung Kee Fung K, Eason E, Crane J, et al. Prevention of Rh alloimmunization. J Obstet Gynaecol Can 2003;25:765-73. 2. Murray NA, Roberts IAG. Hemolytic disease of the newborn. Arch Dis Child Fetal Neonatal Ed 2007;92:F83-8. 3. Polesky HF. Blood group antibodies in prenatal sera. Results of screening 43,000 individuals. Minn Med 1967;50:601-3.

4. Filbey D, Hanson U, Wesström G. The prevalence of red cell antibodies in pregnancy correlated to the outcome of the newborn: A 12 year study in central Sweden. Acta Obstet Gynecol Scand 1995;74:687-92. 5. Haemolytic disease of the fetus and newborn. In: Klein HG, Anstee DJ. Mollison’s blood transfusion in clinical medicine. 11th ed. Oxford: Blackwell, 2005:496-545. 6. Roseff SD, ed. Pediatric transfusion: A physician’s handbook. 2nd ed. Bethesda, MD: AABB, 2006:53-71. 7. Hemolytic disease in the newborn. In: Marsh WL, Reid ME, Kuriyan M, Marsh NJ. A handbook of clinical and laboratory practices in the transfusion of red blood cells. Moneta, VA: Moneta Medical Press, 1993:61-2. 8. Blood grouping techniques. In: Klein HG, Anstee DJ. Mollison’s blood transfusion in clinical medicine. 11th ed. Oxford: Blackwell, 2005:311-20. 9. Hsu TC, Rosenfield RE, Rubinstein P. Instrumented PVP-augmented antiglobulin tests. 3. IgG-coated cells in ABO incompatible babies; depressed hemoglobin levels in type A babies of type O mothers. Vox Sang 1974;26:326-33. 10. Dinesh D. Review of positive direct antiglobulin tests found on cord blood sampling. J Paediatr Child Health 2005;41:504-7. 11. Cortey A, Brossard Y. Prevention of fetomaternal rhesus-D alloimmunization. Practical aspects. J Gynecol Obstet Biol Reprod (Paris) 2006;35(1 Suppl):1S123-30. 12. Vaughan JI, Manning M, Warwick RM. Inhibition of erythroid progenitor cells by anti-Kell antibodies in fetal alloimmune anemia. N Engl J Med 1998;338:793-803. 13. Chen Y, Ling UP. Prediction of the development of neonatal hyperbilirubinemia in ABO incompatibility. Zhonghua Yi Xue Za Zhi (Taipei) 1994;53:13-8. 14. Gilja BK, Shah VP. Hydrops fetalis due to ABO incompatibility. Clin Pediatr 1988;27:210-2. 15. Desjardins L, Blajchman MA, Chintu C, et al. The spectrum of ABO hemolytic disease of the newborn infant. J Pediatr 1979;95:447-9. 16. Vreman HJ, Wong RJ, Stevenson DK. Phototherapy: Current methods and future directions. Semin Perinatol 2004;28:326-33. 17. American Academy of Pediatrics Subcommittee on Hyperbilirubinemia. Management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation. Pediatrics 2004;114: 297-316. 18. Alcock GS, Liley H. Immunoglobulin infusion for isoimmune haemolytic jaundice in neonates. Cochrane Database Syst Rev 2002;3:CD003313. 19. Gottstein R, Cooke RWI. Systematic review of intravenous immunoglobulin in haemolytic disease of the newborn. Arch Dis Child Fetal Neonatal Ed 2003;88:F6-10. 20. Anderson D, Ali K, Blanchette V, et al. Guidelines on the use of intravenous immune globulin for hematologic conditions. Transfus Med Rev 2007;21:S9-56. 21. Lakatos L. Bloodless treatment of infants with haemolytic disease (letter). Arch Dis Child 2004;89:1076. 22. Dennery PA. Metalloporphyrins for the treatment of neonatal jaundice. Curr Opin Pediatr 2005;17:167-9.

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Congenital Disorders of Clotting Proteins and Hypercoagulable States in Pediatrics Linda J. Butros1 & Prasad Mathew2 1 2

Assistant Professor of Pediatrics, Pediatric Hematology/Oncology, University of New Mexico, Albuquerque, New Mexico, USA Professor of Pediatrics, Pediatric Hematology/Oncology, University of New Mexico, Albuquerque, New Mexico, USA

Historically, inherited disorders of coagulation lead to severe, debilitating disability and early demise. Even 20 to 30 years ago, children afflicted with hemophilia and thrombosis suffered much with few safe and effective treatment options. Today, the treatment of pediatric coagulopathies consists of effective modalities that are primarily administered on an outpatient basis. There are life-saving treatment options exist even for some of the most difficult-to-treat hemophilia patients with inhibitor or children with inherited thrombophilia. Multicenter prospective clinical trials involving children with bleeding disorders, as well as those with thrombophilia, are being performed in order to make accurate recommendations of care. Although studies in children with thrombotic conditions are few, evolving treatments contribute to an exciting era of growth in pediatric coagulopathy.

Hemophilia The hemophilias form a group of inherited lifelong bleeding disorders caused by deficiency of Factor VIII (hemophilia A) or Factor IX (hemophilia B). Hemophilia A and hemophilia B have many features in common. Both are inherited as X-linked recessive traits and thus affect males almost exclusively. The incidence of hemophilia A is 1 in 5000 male live births, and that of hemophilia B is 1 in 30,000.1,2 Hemophilia affects at least 20,000 persons in the United States alone. Results of a US epidemiologic survey indicated that 43% of hemophiliacs are classified as severe, with 26% classified as moderate, and 31% as mild.3 There are, however, some reports of hemophilia A and B in females, which could be caused by inheritance of a gene for hemophilia from both parents or a new mutation. The genes for Factor VIII and Factor IX are on the long arm of the X chromosome in band Xq28 and Xq27, respectively.

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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Hemophilia A and B are clinically indistinguishable. Bleeding into joints and soft tissues is the hallmark of both conditions. Laboratory screening tests are the same in both disorders as well. Persons with hemophilia A or B characteristically have normal values for prothrombin time, platelet count, and platelet function analyzer testing (which has replaced the template bleeding time in many institutions), but have a greatly prolonged activated partial thromboplastin time (aPTT). The two disorders can be distinguished by performance of assays for Factor VIII and Factor IX coagulant activity. Hemophilia is worldwide in distribution and affects all racial groups.

Clinical Features In hemophilia A, Factor VIII coagulant activity is deficient or abnormal, whereas other components of the Factor VIII system—von Willebrand factor (vWF) and vWF antigen (vWF: Ag)—are normal. In general, clinical severity is correlated with the degree of Factor VIII deficiency. Persons with severe hemophilia have ⬍1% (⬍0.01 IU/mL) Factor VIII activity. Severely affected persons have spontaneous bleeding into joints and soft tissues. Those with 1% to 5% of normal Factor VIII activity are classified as moderate, and have little, if any, spontaneous bleeding. Those with Factor VIII values of ⬎5% to 40% (⬎0.05 to 0.40 IU/mL) have clinically mild disease, usually bleeding only with surgery or trauma.1 The degree of Factor VIII deficiency is fairly consistent in individuals, and in affected members in the same kindred. “Normal” is 1 IU/mL of Factor VIII (100%), as defined by the current World Health Organization International Standard for Plasma Factor VIII [as distributed by the National Institute for Biological Standards and Control (NIBSC) in the United Kingdom].1 Female carriers of the disease have variable factor levels; bleeding does not occur in those with near normal levels, but those with ⬍50% levels may bleed more often than unaffected relatives or matched controls.4 Hemophilia B (Christmas disease) is characterized by subnormal Factor IX activity. Depending on the genetic defect, Factor IX deficiency may reflect a quantitative or qualitative abnormality in the Factor IX molecule. Several different subtypes of hemophilia B

Chapter 28: Congenital Disorders of Clotting Proteins and Hypercoagulable States in Pediatrics

have been described. In fact, genotype heterogeneity is quite marked; almost every family with hemophilia B has its own unique mutation.5 Hemophilia B is far less common than hemophilia A, accounting for approximately 15% to 20% of cases of hemophilia. As in hemophilia A, clinical severity is correlated with the degree of Factor IX deficiency, with those with Factor IX levels of ⭐1% generally having spontaneous bleeding into joints and soft tissues. The Leyden phenotype of hemophilia B is characterized by severe hemophilia in childhood that becomes mild after puberty.6,7 Joints are the classical bleeding sites in patients with severe hemophilia. Those with severe hemophilia have an annual average of 20 to 30 episodes of spontaneous or excessive bleeding after minor trauma. Some patients with severe disease have a milder clinical course. Coinheritance of the Factor V Leiden mutation (see below) occurs in a small proportion (4.4% in one series) of patients with severe hemophilia A.8 The presence of this and other prothrombotic conditions partially counteracts the bleeding tendency of hemophilia, resulting in fewer bleeding episodes and a later onset of first bleeding.8-10 Clinical presentations may vary depending on the age of the patient. In the perinatal period, approximately 5% of such patients develop significant subgaleal or intracranial hemorrhage (ICH).11-13 In a multicenter survey, symptomatic ICH occurred in 4% of 744 patients, with 40% occurring within the first week of life due to trauma as the major precipitating factor.11 These hemorrhages may be complicated by seizures, psychomotor retardation and cerebral palsy.12 In approximately 50% of undiagnosed hemophiliacs early bleeding occurs in association with circumcision.14 Children become symptomatic more often after the newborn period but within the first 2 years of life.15 In one study, the first symptomatic bleeding leading to the diagnosis of severe hemophilia occurred at a median age of 0.9 year in children not carrying any prothrombotic risk factors, and 1.6 years in those carrying prothrombotic risk factors (eg, Factor V Leiden) (p ⫽ 0.01).8-10 The onset of disease occurs later in patients with moderate and mild hemophilia, sometimes as late as 22 months.16 Mild hemophilia, in the absence of an informative family history, may go undetected for significant periods (age 14 to 62 years in one report).17,18 With ambulation, bleeding episodes occur more often and in any location. The factors that initiate hemorrhage are not known, and the onset of hemorrhage often is a random event, occurring either spontaneously or after minimal injury. Bleeding into a joint (hemarthrosis) is the commonest bleeding manifestation; the ankles are most commonly affected in children, and the knees, elbows, and ankles are more affected in adolescents and adults. Spontaneous hemarthroses are so characteristic of severe hemophilia as to be almost diagnostic.1,19 Hemarthrosis is painful and physically debilitating, and is the initiating event in hemophilic arthropathy. One joint is usually affected at a time, often leading to a target joint, but multiple bleeding sites are common.20 Repeated hemorrhages into joints initiate the

progressive destruction of a joint, leading to fibrosis of the joint with contractures, pain, and limitation of motion.21 The number and type of hemarthroses correlate with function and future quality of life. Thus, orthopedic complications remain a serious problem. Bleeding into muscles is the next most common manifestation, and most often affects the quadriceps, iliopsoas, and forearm muscles, and sometimes are large enough to compromise neurovascular structures and produce a compartment syndrome.22 Other sites of bleeding include the nose (epistaxis), oral mucosa, gastrointestinal tract, and hematuria. Intracranial hemorrhage occurs in older children as well as neonates. In one study of children, the prevalence of ICH was 12%.23

Genetics in Hemophilia Hemophilia A and B can be “familial” or can occur as a “sporadic” mutation. Seventy percent of mild to moderate cases of hemophilia, 57% of severe hemophilia B cases, and 45% of severe hemophilia A cases are clearly familial on pedigree analysis. Regardless, in isolated cases of hemophilia the mother of the index case carries the mutation in 85% of the cases; however, the mother’s mother carried the mutation in only 11% of isolated cases. Isolated cases, referring to only one affected individual in a family, account for approximately 25% of severe hemophilia cases and 15% of mild hemophilia cases. These “isolated cases” do not include sporadic cases in which a sibling (second child) is also diagnosed with hemophilia even if there is no family history.24 One study looked for the genetic origin of sporadic cases of hemophilia A. It found that the majority of these boys inherited their Factor VIII inversion mutations from a de novo mutation that originated in the male germ cells of the maternal grandfather.25 Neither Factor VIII nor Factor IX crosses the placenta; thus, the diagnosis of hemophilia A or B can be made at birth from a sample of cord blood drawn from a vessel on the fetal side of the placenta, after the infant is born. The blood should be collected in citrate (1:10 dilution) and sent immediately to the laboratory for a Factor VIII or IX assay. The diagnosis can also be made by cordocentesis at 18 to 20 weeks of gestation. Similarly, umbilical cord blood can be collected and tested for Factor VIII or IX activity. However, prenatal diagnosis of hemophilia is now accomplished almost exclusively through molecular biological techniques analyzing various polymorphic markers from DNA obtained from chorionic villus sampling. These modern techniques, including restriction fragment length polymorphism analysis, denaturing gradient gel electrophoresis, single-strand conformation polymorphism, DNA sequencing, and others, are accurate and can be obtained earlier in the pregnancy with less morbidity than cordocentesis.26 Although several mutations in the Factor VIII and Factor IX genes have been described,2,4-6 an inversion in the intron 22 of the Factor VIII gene accounts for roughly 45% of cases of severe (not mild or moderate) hemophilia A.27 Thus, the search for inversions of Factor VIII should be considered as the

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first DNA diagnostic option in a person or a family with severe hemophilia A.28 The inversion mutation can be detected by polymerase chain reaction amplification and other molecular techniques from a person with hemophilia A or from a possible carrier, or using fetal or chorionic villus cells. These techniques can be used to diagnose virtually 100% of the intron 22 inversions seen in severe hemophilia. In one study, however, genomic analysis techniques failed to detect the defect in approximately 4% of moderately affected hemophilia A persons and approximately 12% of mildly affected persons.29

Treatment of Hemostatic Abnormalities in Hemophilia The optimal management of these patients is very complex, and requires the provision of preventive care, the use of replacement therapy, and treatment of complications of the disease and its therapy. During the first half of the 20th century, life for these patients was at best miserable because they all developed disabling arthropathy (usually before the age of 20), often leading to permanent disability. Hemophilia also had a high mortality rate from ICH. In the 1920s and 1930s, life expectancy in developed countries such as Finland and Sweden was only 8 to 11 years. As recently as 1960, life expectancy was ⬍30 years.30-33 Improvements in hemophilia care saw its share of setbacks, the most burdensome of which was the transmission of human immunodeficiency virus (HIV) and hepatitis C virus (HCV), both of which had a devastating effect on this community.34,35 With replacement therapy came two further challenges—the development of inhibitors to Factor VIII or IX, and the rising costs of treatment. Because of the increasing costs of care, developing countries struggle to provide factor concentrates to

their patients. The challenge facing clinicians today is to maintain state-of-the-art care in an environment of rising treatment costs.36 Rapid advances within the last 30 years in the evolution of factor replacement products have been made (Table 28-1). The availability of factor concentrates, the emergence of comprehensive hemophilia treatment centers, and the widespread adoption of home-administered replacement therapy led to the early control of hemorrhages and reduced the musculoskeletal damage typical in patients with inadequately treated disease.19

Blood Component Therapy Until the mid-1960s, fresh plasma or Fresh Frozen Plasma (FFP) were the only preparations that could be used to treat Factor VIII or Factor IX deficiency. Then, in 1965, Pool et al37 described a simple method for concentrating Factor VIII in the form of a cold-insoluble precipitate (cryoprecipitate). By the late 1960s, the availability of Cryoprecipitated AHF had revolutionized the treatment of hemophilia A and made possible elective surgery and outpatient treatment of bleeding episodes. Cryoprecipitates, prepared from single units of plasma, contain approximately 50% of the Factor VIII activity, vWF, and fibrinogen, as well as Factor XIII, from the starting unit of plasma. Thus, for purposes of calculation, a single bag of cryoprecipitate contains, on average, 100 IU of Factor VIII and 0.2 g of fibrinogen in a volume of 8 to 10 mL. In the late 1970s and early 1980s, approximately half of the hemophiliacs in the United States or about 9300 people developed HIV from lyophilized concentrates and cryoprecipitates. In addition, about 80% of HIV-positive hemophiliacs were eventually found to have HCV.38 As a result, cryoprecipitates

Table 28-1. Evolution of Hemophilia A and B Therapy Year

Hemophilia A

Hemophilia B

1950 1966 1975 1983 1985 1986

Plasma infusion Cryoprecipitate infusion Lyophilized concentrates Heat treatment of lyophilized products — Introduction of solvent/detergent treatment for virus inactivation Monoclonal-antibody-purified Factor VIII concentrates, eg, Monoclate —

Plasma infusion — Lyophilized prothrombin complex concentrates — Virus attention by heat treatment of lyophilized concentrates Introduction of solvent/detergent treatment for virus inactivation

1988 1990 1992 1996 2000 2003

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Recombinant first-generation Factor VIII concentrates, eg, Recombinate — Recombinant second-generation Factor VIII, eg, Kogenate FS, Helixate FS, Refacto Recombinant third-generation Factor VIII, eg, Advate

— Chromagraphic/monoclonal antibody purified plasma-derived Factor IX, eg, Mononine, Alphanine — Recombinant Factor IX, eg, BeneFix — —

Chapter 28: Congenital Disorders of Clotting Proteins and Hypercoagulable States in Pediatrics

were largely replaced by lyophilized Factor VIII concentrates and recombinant Factor VIII (rFVIII) concentrates. Regardless, cryoprecipitates are still used in many developing countries to treat persons with severe or moderately severe hemophilia A. Disadvantages of cryoprecipitates over commercially prepared concentrates include 1) a slightly increased risk of blood-borne viral disease transmission (particularly HCV), 2) a required storage temperature of ⬍20 to ⬍30ºC, and 3) marked variation in the Factor VIII and vWF content of preparations. By 1970, methods of fractionation had been developed to produce concentrates of both intermediate-purity Factor VIII and prothrombin complex concentrate (PCC), the latter of which contained the vitamin-K-dependent clotting factors (Factors II, VII, IX, and X) as well as proteins C and S. The availability of these lyophilized concentrates quickly led to home treatment (self-infusion) programs throughout the United States and abroad. For the first time, persons with hemophilia were not totally dependent on family members, hospital personnel, and emergency rooms. Bleeding episodes could be treated more promptly, less time was lost from work or school, and the incidence of progressive chronic musculoskeletal disease was decreased. However, it soon became apparent that many persons with hemophilia had developed hepatitis and had persisting alterations of liver function.39-41 Some had progressed to cirrhosis. As many as 90% of hemophiliacs became seropositive to hepatitis B surface antigen.42 Because of this serious complication, in 1980 a Factor VIII concentrate (Hemofil T, Hyland Laboratories, Glendale, CA) was developed that was heated in the dry state for 72 hours at 60ºC. Studies in chimpanzees suggested that this method of heating destroyed hepatitis viruses; however, when Hemofil T was infused into previously untreated children with hemophilia, 84% developed non-A, non-B hepatitis (currently referred to as HCV).42 In the early 1980s, the first cases of acquired immunodeficiency syndrome (AIDS) in persons with hemophilia were noted. It quickly became apparent that this too was being transmitted by blood and blood components, including clotting factor concentrates.43 Approximately 90% of persons who received Factor VIII concentrates and 55% of those who received PCCs prepared in the United States between 1979 and 1984 became seropositive for HIV. Fortunately, HIV proved to be heat labile.44 Although the dry heating method used in production of Hemofil T did not prevent HCV, it did seem to destroy HIV. Thus, by late 1984 almost all hemophiliacs in the United States were receiving dry heat-treated concentrates. Additionally, by the spring of 1985, mandatory screening of all blood and plasmapheresis donors for HIV seropositivity was in place. These measures greatly improved the safety of plasma-derived Factor VIII and Factor IX concentrates in terms of HIV transmission. Since 1987, there has not been a new case of HIV or hepatitis in the North American hemophilia population.45 In addition to better virus-attenuated products, Factor VIII concentrates purified by using murine monoclonal antibodies and immunoaffinity chromatography became commercially available in 1987.46 Several logs of virus were eliminated in the purification process. These monoclonal antibody-purified

products were further virus-attenuated by pasteurization or by solvent/detergent treatment. Creutzfeldt-Jakob disease (CJD) was another potential threat for the hemophilia community. Although there have been no documented instances of transmission of CJD or variant CJD, the mere possibility of such transmission has been cause for further concern among hemophilic patients and their physicians.38 Thus, the use of safer alternatives to blood concentrates/derivatives was explored.

Recombinant Factor VIII Despite improved methods of donor screening and virus attenuation of plasma-derived clotting factor concentrates, the transmission of certain blood-borne viruses remained a concern. Thus, recombinant clotting factor concentrates have had great appeal. After scientists described the molecular cloning of a complementary DNA (cDNA)-encoding human Factor VIII47 and the expression of human Factor VIII from recombinant DNA clones,48 scale-up, purification, and standardization were rapidly accomplished. Prelicensure clinical trials in humans began in 1987. In patients with hemophilia A, the original, “first-generation” full-length rFVIII products have demonstrated the same pharmacokinetics and clinical effectiveness as plasma-derived Factor VIII products.49,50 Despite the cost of rFVIII, many patients (especially those who are HIV seronegative) now have been switched from plasma-derived to rFVIII, because of the added margin of safety. Still, there were concerns among some that human serum albumin was in these rFVIII products (and was, in fact, the major component in the final products) and might transmit a blood-borne disease. The “second generation” of rFVIII products that were developed included truncated molecules. Because the heavily glycosylated B domain seemed to be dispensable for the hemostatic activity of Factor VIII,51 a B-domain-deleted (BDD) form of Factor VIII was developed.31,32 This much smaller molecule is secreted more efficiently by Chinese hamster ovary (CHO) cells, is less prone to proteolytic degradation, and no serum albumin is needed for stabilization of the final product. B-domain-deleted rFVIII has proven to be effective in a wide variety of situations (acute bleeding episodes, prophylaxis, home treatment, surgery, etc), is safe, and is well tolerated.52 The only “problem” with BDD rFVIII is that usual, one-stage (aPTT-based) Factor VIII assays give roughly 50% of expected values in the recipient’s plasma.52-54 There are marked differences in the phospholipid requirement in the assay of BDD rFVIII compared to the other Factor VIII preparations. Thus, if one is monitoring Factor VIII levels in a patient with hemophilia A who is receiving BDD rFVIII, one-stage Factor VIII assays will appear to be lower than expected (calculated). On the other hand, chromogenic substrate assays, although infrequently performed by US clinical laboratories, will give expected recovery values. Also, if one uses a small test vial of BDD rFVIII as the Factor VIII standard in the one-stage assay, results will be as expected. Small

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test vials of BDD rFVIII (prepared by the (NIBSC) are available for this purpose. Another “second-generation” rFVIII product is a full-length preparation formulated with sucrose, rather than human serum albumin.55 The purification process for this product (Kogenate FS, Bayer Healthcare, West Haven, CT) eliminates the need for the addition of albumin. A solvent/detergent treatment step has also been added during purification. In 2003, a third-generation Factor VIII product (Advate, Baxter, Deerfield, IL) was released. This product has no human or animal proteins either in the initial stages of preparation or in the final formulation.

Dosage and Administration of Factor VIII Factor VIII concentrates are routinely used for treating acute bleeding episodes in persons with moderate or severe hemophilia A. Factor replacements may be given either “on demand” (ie, after a bleeding episode to treat the hemorrhage) or as “prophylaxis” (ie, scheduled infusions to prevent or decrease the frequency and intensity of hemorrhages). The most common indications for treatment are acute hemarthrosis and intramuscular bleeding. Such events should be treated promptly to prevent or reduce complications (such as chronic joint disease) and to minimize the need for additional infusion of clotting factor (Table 28-2). In addition, Factor VIII concentrates are often used for prophylaxis (to prevent bleeding), for surgical coverage, and for immune tolerance induction (ITI) in persons with Factor VIII inhibitors. In calculating the dose, it can be assumed that 1 IU of Factor VIII/kg of body weight will raise the patient’s Factor VIII level by 2% (0.02 IU/mL). Thus, if the patient’s baseline Factor VIII level is 0.01 IU/mL, a dosage of 20 IU/kg would be expected to

raise the level to 40% (0.40 IU/mL). The half-life of Factor VIII is 10 to 12 hours but may be shorter if the recipient is febrile, is bleeding extensively, has a Factor VIII inhibitor, or is a small child. Serious, life-threatening bleeding into the central nervous system or intraoperative and postoperative bleeding may be treated with a continuous infusion of Factor VIII. After an initial bolus of 40 to 50 IU/kg (which will raise the patient’s Factor VIII level to 80% to 100%), a continuous infusion could be started. An initial rate of 3 to 4 IU/kg/hour should be subsequently adjusted as indicated by the recipient’s Factor VIII level. In general, a rate of 2 IU/kg/hour will achieve a Factor VIII level of 25%; 3 IU/ kg/hour will yield a 50% level; and 4 IU/kg/hour will result in a 75% level. However, these levels should not be assumed, and daily monitoring should be performed. If continuous infusion becomes a logistical issue, then twice a day bolus infusions may be substituted to keep a trough Factor VIII level ⬎50%.

Prophylaxis Chronic hemophilic arthropathy, resulting from repeated bleeding into joints, is the major cause of morbidity in persons with severe hemophilia. In those whose Factor VIII or IX levels are less than 0.01 IU/mL, bleeding episodes occur approximately 20 to 30 times per year.1 Primary prophylaxis, which is aimed at preventing spontaneous hemarthroses, was begun in Malmö, Sweden, over 35 years ago.57 All boys with severe hemophilia currently seen at the Malmö center start prophylaxis when they are 1 to 2 years old. Most standard prophylaxis regimens aim to keep the factor level ⬎1%.58 For Factor VIII deficiency, the standard implies infusions three times per week; for Factor IX replacement, the standard implies two infusions per week because of

Table 28-2. Management of Bleeding Episodes in Hemophilia: Suggested Recommendations Type of Bleeding

Examples

Factor VIII/IX Levels Desired*

Duration of Therapy

Minor

Uncomplicated hemarthroses, superficial muscle or soft tissue bleeding

20-40%

1-3 days

Moderate

Intramuscular or soft tissue bleeding with dissection; mucous membranes; dental extractions; hematuria

30-60%

2-7 days

Major

Pharynx; retropharynx; retroperitoneum; central nervous system (CNS) surgery

60-100%

5-14 days; CNS bleeding— 6 weeks to months to indefinite

Postoperative management

60-80%

7-14 days

Prophylaxis therapy— administered 3 or 4 times/week†

Trough factor ⬎1%

??

*

The required dosage for Factor VIII is determined using the formula: Required units ⫽ Body weight (kg) ⫻ Desired Factor VIII Rise (%) × 0.5 IU/kg. The required dosage for Factor IX is determined using the formula: Required units ⫽ Body weight (kg) ⫻ Desired Factor VIII Rise (%) † One study56 showed that once-a-week prophylaxis may be feasible in some patients.

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the longer half-life of Factor IX.59 Although venous access may be a problem in younger children, central venous catheters are often used. With such devices the risk of bleeding or infection is generally low but must be considered. Another historical concern has been the propensity for increased Factor VIII inhibitor formation with the increased number of infusions given in prophylaxis. However, recent reports suggest that there may be a benefit resulting in decreased inhibitor formation with prophylaxis.58 Prophylactic treatment with recombinant Factor VIII has been shown to prevent joint damage in young boys with severe hemophilia A. A prospective randomized, multicenter study conducted in the United States enrolled 65 boys ⬍30 months of age with severe hemophilia A between August 1996 and March 2000. The boys were randomly assigned to receive either prophylactic Factor VIII dosing at 25 IU/kg every other day or episodic therapy only at the time of a joint hemorrhage with an initial dose of 40 IU/kg and 20 IU/kg 24 and 72 hours after the first dose. Parents were also encouraged to re-treat up to 4 weeks with 20 IU/kg every other day until the symptoms of joint hemorrhage resolved. Radiography, magnetic resonance imaging (MRI), and a physical examination scoring system were used to assess joint damage. The prophylaxis group had a significant decrease in structural joint damage as assessed by MRI. An 86% relative risk reduction in joint damage as assessed by the scoring systems was noted as compared to on-demand therapy.60

so-called coagulation Factor IX concentrates are considerably more expensive than PCCs; however, they are far less thrombogenic.65 These high-purity virus-attenuated Factor IX concentrates and recombinant concentrates are recommended for use in neonates and children, in persons with hemophilia B undergoing surgery (particularly orthopedic surgery), in those with crush injuries or large intramuscular hemorrhages, in those with hepatocellular dysfunction, and in anyone with a history of thrombotic problems after receiving PCCs (ie, in any high-risk situation for thrombosis or DIC)—in fact, they are the preferred products for anyone with hemophilia B.

Coagulation Concentrates Available for the Treatment of Hemophilia B Until late 1990, intermediate-purity PCCs were the mainstay of treatment for persons with hemophilia B. They contain the vitamin-K-dependent clotting Factors II, VII, IX, and X, as well as proteins C61 and S. In addition to nonactivated factors, PCCs contain some coagulation factors in activated forms as well. This occasionally results in disseminated intravascular coagulation (DIC) or thromboembolic complications in recipients of these products. The risk of DIC and thromboembolism is greatest in recipients who have sustained crush injuries or extensive softtissue bleeding, or who have undergone orthopedic surgery and are immobile. In such situations, thromboplastic materials are released into the circulation. The risk is also enhanced in persons with hepatocellular disease, because clotting intermediates are not optimally cleared from the circulation, and antithrombin levels are often low.62 Because of the potential, albeit rare, thrombotic complications of activated PCCs (aPCCs), most physicians avoid concurrent treatment with aPCCs and antifibrinolytics.63 Recent studies using PCCs as prophylaxis in the treatment of hemophiliacs with inhibitor (see below) demonstrated no prothrombotic complications at doses of 75 to 100 U/kg every other day to three times per week.64 The only aPCC (FEIBA-VH, Baxter Healthcare) currently available and licensed for use in the United States is now relegated to treatment as a bypassing agent for patients with inhibitors.64 In view of the thrombogenic potential of PCCs, there was an obvious need for high-purity Factor IX concentrates. These

Dosage and Administration of Factor IX Because Factor IX is a smaller molecule than Factor VIII and diffuses from intravascular to extravascular sites, a larger dose must be given to achieve the same concentration in the circulation. Whereas a dose of Factor VIII of 1 IU/kg will raise the serum Factor VIII level by 0.02 IU/mL (2%), the same dose of (plasmaderived) Factor IX will raise the circulating Factor IX level by only 0.01 IU/mL (1%). As in the case of Factor VIII deficiency, in hemophilia B the recommended dosage for treatment of bleeding depends on the nature and severity of the bleeding episode. For most situations, dosages should be calculated to raise the Factor IX level to 0.2 to 0.3 IU/mL (20%-30%). However, larger doses are recommended for treatment of serious, life-threatening bleeding episodes and for surgery. Recombinant Factor IX does have a decrease in recovery of the product by ⬃50% to 72% with respect to plasma-derived Factor IX.70 However, the recombinant preparation has also been shown to exhibit a longer half-life of 13.7 vs 12.9 hours for the plasmaderived product to the extent that in three of the 13 patients in this recovery study, less recombinant product was infused to maintain the same baseline level of 2%.71 The difference in recovery results from a simple difference in the posttranslational modification, or namely, the differences in sulfation of tyrosine 155 and phosphorylation of serine 158, residues that play a role in the clearance of Factor IX.70 Thus, it is recommended that one use a somewhat larger dose of BeneFix than one would of a plasma-derived Factor IX product. The product package insert recommends the following calculation of dosage: number of

Recombinant Factor IX The CHO cell line used in the manufacture of recombinant Factor IX is co-transfected with a human Factor IX cDNA expression plasmid and a cDNA expression plasmid that encodes an engineered form of the paired amino-acid-cleaving enzyme, which improves the processing efficiency of profactor IX expressed in CHO cells.66,67 No albumin is used, and no human plasma, animal plasma, or animal-derived protein is used in its manufacture or purification.68 A reformulation (BeneFix, Wyeth, Collegeville, PA) now exists that seems to exhibit equal efficacy to the original preparation with a more concentrated formulation. There were no instances of inhibitor development, allergic reactions, or thrombosis in 34 patients tested.69

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Factor IX units required ⫽ body weight (kg) ⫻ desired Factor IX increase (%)⫻ 1.2. However, one should be aware that there are wide variations in recovery among individuals. Infants and children tend to have lower recovery rates than adults.68

Surgery in Persons with Hemophilia B Through the 1980s many hemophilia centers avoided elective surgery (particularly orthopedic surgery) in persons with hemophilia B because of the risk of DIC and thromboembolic complications. As noted earlier, the risk is greatest in persons undergoing extensive orthopedic surgery on the lower extremities, because there is release of thromboplastic materials into the circulation and the patient is likely to be immobile. Now that less thrombogenic plasma-derived Factor IX preparations [Mononine (CSL Behring, King of Prussia, PA) and Alphanine SD (Grifols, Los Angeles, CA)] and recombinant Factor IX (BeneFix) are available, these should be used during and after surgery. Continuous infusion of BeneFix can be used safely and efficaciously to control bleeding in the perioperative period with little to no thrombotic side effects. The stability is maximized (90%) up to 14 days when stored at 4ºC with the addition of 4 µ/ mL of unfractionated heparin (UFH). The addition of the UFH also prevents thrombophlebitis, a known complication of continuous infusion.72,73 Other Ancillary Therapeutic Options Desmopressin Desmopressin (DDAVP) is the treatment of choice for persons with mild hemophilia A74 whenever an approximately threefold increase in Factor VIII is sufficient to control bleeding. This synthetic agent effects a three- to fivefold (average of threefold) increase in Factor VIII and vWF:Ag 30 to 60 minutes after administration.75 It shortens the bleeding time and the aPTT. However, only a fraction of mild hemophiliacs and patients with von Willebrand disease (vWD) respond to this agent so a trial of desmopressin is important to confirm a response before a hemostatic challenge. The recommended dosage is 0.3 µg/kg given intravenously.76 The levels of Factor VIII (and vWF) generally remain elevated for 8 to 12 hours and the dose can be repeated at 8- to 12-hour intervals. If repeated doses are given in rapid succession, many (but not all) persons develop tachyphylaxis (a diminishing response). This is thought to reflect a depletion of the storage sites for Factor VIII and vWF. Desmopressin is remarkably free of undesirable side effects. In most persons, side effects are limited to facial flushing and a feeling of facial warmth. The antidiuretic properties of the drug seldom cause a problem. However, if repetitive doses of desmopressin are used along with large amounts of intravenous fluids, fluid and electrolyte balance must be carefully monitored to avoid hyponatremia and water intoxication. This precaution is particularly important in infants, small children, and elderly patients. In fact, when desmopressin is given to small children, fluid intake during the next 18 hours should be limited to avoid the possibility of water intoxication. Several formulations of desmopressin are available. The highly

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concentrated intranasal spray is ideally suited for outpatient and home use, but patients must be counseled not to use the spray more than twice daily and not more than for 3 to 4 consecutive days.77 For patients weighing ⬍50 kg, one spray (in one nostril) is recommended, while for those weighing ⬎50 kg, two sprays (one in each nostril) should be given. The prescription must be specific for the metered dose pump that delivers 0.1 mL (150 ␮g of desmopressin) per actuation. Antifibrinolytic Agents ε-Aminocaproic acid and tranexamic acid are antifibrinolytic agents that inhibit plasminogen activation. These agents are useful adjuncts in certain situations, to prevent lysis of a clot that has already formed as a result of specific replacement therapy. The oral mucosa has significant fibrinolytic activity, and antifibrinolytic agents are particularly useful in the control of bleeding in the oral cavity (eg, extraction of permanent teeth, lacerations of the tongue and mouth, and oral surgery).78 The recommended dosage of ε-aminocaproic acid is 75 mg/kg every 4 to 6 hours orally, after a loading dose of 100 mg/kg; the dose of tranexamic acid is 25 mg/kg every 6 to 8 hours. For invasive dentistry, antifibrinolytic therapy should be started the evening before the procedure and be continued for 7 to 10 days. Swedish investigators79 have demonstrated that transfusion requirements and postoperative bleeding after oral surgery in patients with hemophilia can be significantly reduced by the use of a tranexamic acid mouthwash in addition to systemic antifibrinolytic treatment. The mouthwash is prepared from 10% tranexamic acid for injection, diluted with sterile water. SindetPederson et al79 recommend the use of 10 mL of 4.8% tranexamic acid solution for 2 minutes four times daily. Hepatitis B Vaccine In view of the risk, albeit very slight, of posttransfusion hepatitis, all persons with hemophilia (and without, according to the American Academy of Pediatrics) should be immunized against hepatitis B. Although all units of blood or plasma collected are tested for hepatitis B surface antigen, such testing may not be 100% effective in screening out all units that might transmit hepatitis B, and the risk is multiplied substantially in the case of plasma-derived clotting factor concentrates in which plasma from 2500 to 20,000 plasma donors is used to produce a single lot of concentrate. Vaccination against hepatitis B (with recombinant hepatitis B vaccine) should be given subcutaneously as soon as the diagnosis of hemophilia has been made. Hepatitis A Vaccine Hepatitis A vaccine is also available, and is recommended for children (or any seronegative persons) with hemophilia 2 years of age or older.74 It can be given subcutaneously,80 which is preferred over intramuscular injections because of the tendency to develop muscle bleeding with intramuscular vaccination.81 The Centers for Disease Control and Prevention did investigate the seroconversion to hepatitis A or B of people with blood disorders

Chapter 28: Congenital Disorders of Clotting Proteins and Hypercoagulable States in Pediatrics

and found no seroconversions that could be attributed to blood components.82 Avoidance of Drugs that Can Cause Platelet Dysfunction Certain drugs, aspirin in particular, induce platelet dysfunction that can aggravate the bleeding tendency in persons with hemophilia (or another underlying abnormality of hemostasis). Joint or soft-tissue bleeding is often painful. If aspirin or an aspirin-containing compound is taken to relieve that pain, the bleeding tendency may worsen, and a vicious cycle may ensue. This effect of aspirin is caused by an irreversible inhibition of platelet cyclooxygenase, which inhibits prostaglandin synthesis. Thus, aspirin and all aspirin-containing compounds should be avoided by people who have underlying coagulation disorders. Acetaminophen is a good alternative to relieve mild pain in addition to rest, cooling, and elevation. Other drugs that induce platelet dysfunction include antihistamines, phenothiazines, and nonsteroidal antiinflammatory agents such as indomethacin and ibuprofen. Recently, celecoxib and other cyclooxygenase 2 (COX- 2) inhibitors known to exhibit antiinflammatory and antiangiogenic properties have been effectively utilized for analgesia in hemophilic arthropathies. COX-2 inhibitors do not interfere with platelet function as do the more traditional nonsteroidal antiiflammatory drugs.83

Treatment of Hemophilia with Inhibitors Approximately 20% to 35% of persons with hemophilia A develop inhibitor antibodies to Factor VIII, measured in Bethesda units (BU). A lesser percentage (between 2% and 5%) of persons with hemophilia B develop Factor IX inhibitors. The development of inhibitors (antibodies, primarily IgG) currently represents one of the most serious complications of hemophilia.84 These antibodies complicate bleeding episodes because of diminished responsiveness to factor concentrates and increase the risk of uncontrollable bleeding, disability, and premature death. In addition, life-threatening symptoms of anaphylaxis can occur in about 5% of patients with Factor IX inhibitors and in some cases, development of a nephrotic syndrome has been reported.85 The latter is seen, especially, in those individuals whose severe hemophilia B results from a large deletion, frameshift mutation, or stop codon in the Factor IX gene and occurs whenever a Factor IX-containing product is infused.86-88 Inhibitors are much less common in patients with mild or moderate disease. Inhibitors typically develop in early childhood, within the first 10 to 20 exposure days to exogenous factor concentrates. Compared with hemophilia patients without inhibitors, patients with inhibitors experience bleeding that is harder to control once it starts. Not surprisingly, progressive joint disease and significant mobility impairments are far more prevalent in patients with inhibitors than in those without. Soucie et al89 analyzed joint range of motion (ROM) data on 2378 children (ages 2 to 19 years) with severe hemophilia, 186 of whom had an inhibitor titer of ⬎0.5 BU at the time the joint measurements were made. Those with a measurable inhibitor had a twofold greater loss of joint ROM

(expressed as a percentage of normal ROM) when compared with those with inhibitor levels ⬍0.5 BU. In an Italian study of older inhibitor patients (mean age 36 years, range 15 to 65), it was noted on study entry that 80% were physically disabled, ⬎60% had measurable flexion contractures, and over 70% had mobility impairments.90 These studies suggest that predictable and progressive impairment in joint function in inhibitor patients begins in childhood and worsens with the passing of time, with older adults experiencing significant orthopedic disabilities. Many persons with hemophilia and inhibitors (approximately 50%) have a high response titer with ⬎5 BU/mL upon exposure to Factor VIII. These high-responder patients are particularly difficult to treat.91 The remainder of hemophiliacs with inhibitors have a low-response inhibitor with ⬍5 BU/mL.86 Approximately one-third of inhibitors (usually low-titer inhibitors) are transient.87,88

Inhibitors and Type of Replacement Product Used The early studies of the first-generation recombinant Factor VIII products demonstrated inhibitor antibodies in previously untreated patients (PUPs). This raised some concern, but as the trials progressed it became apparent that neither product was any more antigenic than plasma-derived Factor VIII.92,93 A more recent PUP trial94 studied various plasma-derived vs first- and second-generation recombinant products over a 23-year period with 72 hemophilia A patients enrolled. Thirty-one percent of the patients developed inhibitors (43% of the severely affected patients and only 8% of the moderately affected). Fifty-one patients were treated with plasma-derived products and 21 were treated with recombinant products. There was no significant difference in high-titer (⬎5 BU) inhibitor development between the two groups.94 In another PUP study95 with BDD rFVIII, 33 of 101 patients (all severely affected) developed inhibitors to Factor VIII after a median of 12 exposure days. Seventeen of the 33 patients who developed an inhibitor had peak values of ⬍5 BU, while 16 had peak values of ⭓5 BU. Eight of the 33 inhibitors were transient, disappearing despite “on demand” treatment with BDD rFVIII. Twelve additional patients (most high-responder inhibitor patients) had a good response to immune tolerance induction (ITI) with BDD rFVIII, with their latest inhibitor titers being negative.52 These results are very similar to those seen with the two full-length rFVIII preparations.95 Inhibitor development follows a typical pattern; the highest risk is within the first 20 days of exposure to Factor VIII. The management of patients with Factor VIII inhibitors has two important aspects. The first is treatment for bleeding, and the second involves attempts to eliminate the inhibitor. The management of patients with Factor IX inhibitors involves a third important consideration: the monitoring for and management of allergic reactions or frank anaphylaxis to Factor IX concentrates and Factor IX-containing agents.96 Bleeding in patients with low inhibitor titers (⭐5 BU) often can be treated with Factor VIII concentrates in usual or somewhat increased dosages, because anamnestic responses are

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minimal and hemostatic levels of Factor VIII can be achieved. However, other approaches must be used in high responders. If the inhibitor titer is ⬎5 BU, infusion of large doses of human Factor VIII concentrates often results in a marked increase in the inhibitor titer (anamnestic response) without controlling the bleed.63 The choice of treatment depends on several variables: the patient’s inhibitor concentration, whether he is naive to plasma-derived products, the degree of cross-reactivity of the inhibitor to porcine Factor VIII, the nature and extent of bleeding, product availability, and the experience and preference of the medical personnel involved.86

Prothrombin Complex Concentrates Prothrombin complex concentrates and aPCCs have been used extensively for the treatment of bleeding in patients with inhibitors since 1972 and until recently have been the mainstay of treatment for joint and soft-tissue bleeding. They can be used in both Factor VIII and Factor IX inhibitor patients. The so-called nonactivated or standard PCCs have been largely replaced by the purposely activated products, particularly FEIBA-VH (Baxter). Although they are not always effective, there is no readily available laboratory test for monitoring the recipient, and no one has demonstrated convincingly how they work,97,98 many clinicians use aPCCs.86 The recommended dose of FEIBA is 50 to 75 IU/kg every 12 to 24 hours.64 If necessary, this dosage can be repeated for a total of 3 to 5 days. However, the use (particularly for traumatic injuries) of frequent repetitive doses should be avoided and total doses should not exceed of 200 U/kg/day, because of the thromboembolic complications referred to earlier. Multiple studies have demonstrated the efficacy of FEIBA in the control of bleeding in patients with inhibitors. FEIBA has been used in treating acute bleeding, in perioperative control of bleeding, and most recently in prophylaxis for patients with inhibitors.59,64,99 Porcine Factor VIII Concentrates Factor VIII inhibitors exhibit varying degrees of species specificity. Most human Factor VIII inhibitors destroy human Factor VIII to a greater degree than Factor VIII from another species. Polyelectrolyte porcine Factor VIII was a highly purified freezedried porcine Factor VIII concentrate that had a much lower incidence of side effects than the older porcine preparations that were largely abandoned in the 1960s. Polyelectrolyte porcine Factor VIII was licensed in the United States in 1986 for life- and limb-threatening emergencies. Porcine Factor VIII may be ineffective for hemophilia patients with inhibitors because of human antibody cross-reactivity or development of porcine Factor VIII antibodies. Allergic reactions are generally mild but can be as serious as anaphylaxis. Either allergic or pyrogenic reactions are seen in as many as 40% of cases. In addition, viral contamination, particularly with parvovirus B19, is limiting the availability of porcine Factor VIII.63 On the other hand, polyelectrolyte porcine Factor VIII, unlike PCCs and aPCCs, permits the measurement of Factor VIII levels in the recipient, and it has proved lifesaving in many serious situations. It is no longer produced because of

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parvovirus infection of the swine colony that was used to manufacture this product. A recombinant version is currently undergoing clinical trials.

Recombinant Factor VIIa The development of recombinant Factor VIIa (rFVIIa) has been an important landmark in the management of hemophilia patients with inhibitors. The drug (Novo Seven, Novo Nordisk, Copenhagen, Denmark) has been used with clinical success in hemophilic patients with inhibitors who had life-threatening bleeding episodes,98 in patients with acute bleeding that was not life- or limb-threatening, as prophylaxis for joint bleeding, and as prophylaxis in elective surgical procedures.100-102 Monroe et al103 demonstrated that concentrations of Factor VIIa much higher than those found normally in the circulating blood are able to mediate a tissue-factor-independent conversion of Factor X to Factor Xa on a phospholipid surface. rFVIIa induces hemostasis in the absence of Factor VIII and Factor IX, probably by enhancing thrombin generation on the thrombin-activated platelet surface. The rFVIIa binds to the platelet membrane. Factor VIIa, in concentrations of 50 to 100 nmol/L, then proceeds to activate Factor X on the platelet surface in the absence of Factors IX and VIII. This thrombin activation recruits more platelets and enhances the platelet-to-platelet adhesion. In addition, the thrombin activation leads to a tighter fibrin clot structure by recruiting Factor XIII.104 Since the first human patient’s successful treatment with rFVIIa, during and following open synovectomy,105,106 many clinical trials with rFVIIa have been conducted. These included pharmacokinetic studies (which documented the short halflife of the product, 2 to 3 hours),107 dose-finding studies, home treatment studies,108 and multiple surgical studies.100,109 In most of these studies, as well as in a large compassionate-use database, rFVIIa has been given by intravenous (IV) bolus injections. The recommended dose is 90 µg/kg of body weight per dose, with repeat dosing (if necessary) given every 2 to 3 hours for the first 24 hours (or more), with increasing intervals of 3 to 6 hours thereafter. However, the clearance rate varies among individuals, with some children less than 15 years of age having three times the clearance rate achieved in adults.110 Adverse effects found with the use of rFVIIa are rare with the exception of lack of efficacy. The Hemophilia and Thrombosis Research Society (HTRS) Registry reported on the use of rFVIIa in treating 719 bleeding episodes in 50 hemophilia patients. There were nine reported adverse events in five of the patients. Eight of the nine events were “decreased therapeutic response.” The ninth reported adverse event was a maculopapular rash on both arms after 5 days of rFVIIa infusions.108 One of the worries of using rFVIIa has been the risk of thromboembolic events. However, studies in hemophilia patients have shown that this risk is extremely low. There is some evidence, as well, that the initial treatment dose of rFVIIa could be increased as a one-time dose without thromboembolic complications. The bleeding episodes from the HTRS Registry

Chapter 28: Congenital Disorders of Clotting Proteins and Hypercoagulable States in Pediatrics

were treated with bolus IV doses ranging from ⬍100 µg/kg to a maximum of 346 µg/kg. None of the patients experienced a thromboembolic event. The doses used were divided into four groupings: ⬍100 µg/kg, 100 to 150 µg/kg, 150 to 200 µg/kg, and ⬎200 µg/kg. Bleeding stopped in 86%, 82%, 92%, and 93% for each of the respective groups of patients including the 23 patients who were under 18 years of age. In the adult groups, bleeding stopped in 100% of the patients treated with ⬎200 µg/kg.108 In those persons (usually infants and young children) who have severe hemophilia B and develop an inhibitor, roughly 40% to 50% will have severe allergic reactions (including anaphylactic shock) when infused with any Factor IX-containing product (plasma, plasma-derived Factor IX concentrates, or recombinant Factor IX).98 Although a few such patients have been desensitized to Factor IX, for the majority of hemophilia B patients who have had severe allergic reactions to Factor-IX-containing products, rFVIIa is regarded as the treatment of choice.96

Prophylaxis Using Recombinant Factor VIIa Despite its short half-life, two recent studies demonstrated that a daily prophylactic dose of either 90 µg/kg or 270 µg/kg was effective in patients with inhibitors in decreasing the frequency of joint bleeding.102,111 Staphylococcal Protein A Immunoabsorption of Inhibitor Antibodies Staphylococcal protein A immunoabsorption columns (to rapidly reduce a very high-titer Factor VIII or Factor IX inhibitor) are widely available but not often used. In view of the availability of “bypassing” agents such as aPCC and rFVIIa, which work as well in patients with high-titer inhibitors, there is not often a need to rapidly reduce an inhibitor titer in a bleeding patient. The columns are, however, used on occasion in order to decrease an inhibitor titer in a patient in preparation for certain immune tolerance regimens, such as the Malmö regimen. The International Workshop on Immune Tolerance Induction consensus recommendations address the potential utility for immunoabsorption in patients with severe hemophilia B before immune tolerance in order to reduce the incidence of nephrotic syndrome.112 Prevention of Inhibitor Development The CANAL (Concerted Action on Neutralizing Antibodies in Severe Hemophilia A) study group studied children with severe hemophilia A who were started on regular prophylaxis at an early age (median age of 20 months). These children, who received treatment at least twice weekly, had a 60% reduction in the risk of developing inhibitors.113 Other studies have confirmed these findings.114,115 These studies infer that the institution of early prophylaxis may be a reasonable means for the prevention of inhibitor development and must be considered, particularly in patients at high risk for inhibitor development (positive family history, at-risk mutations).

Attempts to Suppress or Eradicate Inhibitors: Immune Tolerance Regimens In the 1970s, a demanding regimen for ITI, called the “Bonn protocol,” used very large and very frequent doses of both Factor VIII and aPCC (FEIBA) during a period of many months or even years.116 In the 1980s other investigators began reporting success with modifications of the Bonn regimen that involve smaller doses of Factor VIII given at less frequent intervals.97 Ewing et al117 had considerable success with a regimen consisting of a daily infusion of Factor VIII of 50 IU/kg, and others have reported118 suppression of inhibitors in a high percentage of patients given Factor VIII at 25 IU/kg on alternate days or two or three times a week. Other groups reported an enhancement of immune tolerance with the addition of intravenous γ-globulin, immunosuppressive agents, or both.119,120 Although these modifications of the Bonn regimen are costly and require good venous access and patient compliance (and a guaranteed supply of Factor VIII), it now seems that such approaches suppress many inhibitors completely and convert other high responders to low responders. Observations to date indicate that timing is extremely important in the use of ITI. The optimal time to start a regimen is when the inhibitor titer is ⬍10 BU; this is best accomplished by avoiding exposure to Factor VIII. It is important to watch the titer closely and initiate treatment promptly when the titer falls sufficiently. Of course, ITI may need to be started sooner if a 1- to 2-year period does not yield the desired titer levels or there is a life-threatening hemorrhage. The optimal dose and frequency for ITI varies between investigators and patients. Hemophiliacs with inhibitors can be divided into “good risk” and “poor risk” patients. The “good risk” patient has an inhibitor titer ⬍10 BU before the initiation of immune tolerance, has a maximum inhibitor level that never exceeded 199 BU, and is starting immune tolerance less than 5 years since the diagnosis of an inhibitor. The “poor risk” patient does not fall into those categories. There is some evidence that “good risk” patients may require less Factor VIII, but “poor risk” patients may require as much as 200 IU/kg/day to maximize the efficacy of ITI.112 Although many physicians treating hemophiliacs now institute ITI in an attempt to suppress or eradicate Factor VIII inhibitors, the success rate has been only about 70%, and is poorer when immune tolerance is attempted in hemophilia B patients with inhibitors. The North American Immune Tolerance Registry data suggested only a 36% success rate in hemophilia B patients; however, patients with allergic reactions were overrepresented.121 The success rate is hindered by the development of nephrotic syndrome, which has been reported in 13 patients internationally. This generally occurs 8 to 9 months after the start of ITI, and its etiology remains unclear. Although the nephrotic syndrome generally resolves with the avoidance of Factor IX products, this complication often mitigates further ITI.122

Gene Therapy Gene therapy replaces the missing factor or protein with a nucleic acid rather than the protein itself. The goal would be to transfect certain targeted tissues with the nucleic acid to replace

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the mutated area of the genome. Hence, the targeted tissue would produce normal protein either fully or partially correcting the factor deficiency. Hemophilia has a favorable combination of features for a positive response to gene replacement therapy. A reduced-intensity stem cell transplant in mice with stem cells transduced with the BDD porcine Factor VIII has been effective in decreasing clot times and increasing Factor VIII levels.123 A few dog models have been used successfully in transgene expression. The targeted tissues were skeletal muscle and liver/hepatocytes. The problem of immune reaction to the transgene product exists and may necessitate approximately 6 months of immune suppression. Vector safety is another important consideration in transporting the transgene product to the recipient.124 A few gene therapy trials in humans have now been completed and the preliminary data are moderately encouraging; however, the results are far from satisfactory with regard to safety and efficacy.125 In terms of efficacy, the plasma levels of Factor VIII or Factor IX reached so far are insufficient to free patients from the need for infusion of exogenous factors. No inhibitor developed in gene replacement trials, but this risk is still of concern especially in previously untreated patients. Additionally, small amounts of the viral genome have been detected in the semen of a few patients, suggesting that the risk of germline integration and passage from the vector to descendants cannot be ruled out.19,125

von Willebrand Disease von Willebrand disease is characterized by mucous membrane bleeding, excessive bruising, and excessive bleeding during and after surgery or invasive dental procedures. It is the most common of the hereditary coagulation disorders. The incidence of vWD in the United States is estimated to be between 0.82% and 1.6% of the population. Clinical severity varies greatly, and many affected individuals have minimal symptoms unless challenged by surgery. A number of subtypes of vWD have been described; the more common of these are discussed below (Types 1, 2, and 3). The basic defect is in vWF, a large multimeric plasma glycoprotein (GP) that supports the adhesion of platelets to the vascular subendothelium. It is normally present in plasma in multimers of up to 20 million daltons. The highest-molecular-weight multimers are the most important hemostatically for platelet adhesion and connective tissue binding. vWF circulates as a complex with Factor VIII and protects Factor VIII from rapid proteolytic degradation, as well as transporting it to sites of active hemostasis. vWF is synthesized in endothelial cells and megakaryocytes and is stored in organelles (in Weibel-Palade bodies of endothelial cells and in the alpha granules of platelets). The gene for vWF is located on the short arm of chromosome 12.126 vWF levels are influenced by ABO blood group,127 with the lowest levels being found in persons of blood group O and the highest in those with blood group AB. The A and B alleles code for A and B glycosyltransferase, which converts the H antigen into A and B blood groups. Blood group O individuals lack this enzyme and

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continue to express the H antigen. H-antigen-rich blood groups have significantly lower vWF and Factor VIII.128 Hormonal changes accompanying pregnancy increase the levels of vWF, Factor VIII, and fibrinogen. von Willebrand factor is a plasma glycoprotein composed of multimeric subunits. It is the high-molecular-weight (HMW) subunits that mediate platelet adhesion during vessel injury. vWF also binds to Factor VIII in the circulation and stabilizes its presence there. In the absence of vWF (Type 3 vWD), Factor VIII levels are also very low. Once released from the Weibel-Palade bodies or alpha granules, the multimers can interact with platelets, connective tissue, or other cells, but the larger subunits may also be cleaved by the ADAMTS13 metalloprotease into smaller multimers. These cleavage products are seen on an agarose gel when a von Willebrand multimer analysis is ordered. A mutation involved in multimer assembly and function results in Type 2 vWD.126 von Willebrand disease results from a quantitative or qualitative deficiency of vWF. If vWD is suspected, a battery of tests should be performed. Screening tests such as the aPTT and bleeding time may or may not be abnormal, especially in persons with mild vWD. More specific tests include quantitation of vWF: Ag by an immunoelectrophoretic assay (the Laurell technique) or by an enzyme-linked immunosorbent assay, a ristocetin cofactor (RCoF) assay as a measure of vWF activity, a Factor VIII assay, and multimeric analysis of vWF using sodium dodecyl sulfate agarose gel electrophoresis.

Classification of von Willebrand Disease A revised classification from an international subcommittee, intended to reflect the research on the function and fate of vWF and differences in pathophysiologic mechanisms that lead to particular vWD phenotypes, was recently published.126 Type 1 vWD is by far the most common of the types of vWD, accounting for at least 80% of cases. The Type 2 variants account for 10% to 15%, whereas Type3 is relatively rare (Table 28-3). Type 1 vWD is caused by autosomal dominant heterozygous mutations. The diagnosis of vWD Type 1 is difficult because of significant variations in vWF levels as discussed above. Bleeding symptoms also vary widely with vWF:Ag ranges of 30 to 50 IU/ dL⫺1. Linkage to a vWF gene mutation has been shown to be only 0.4 to 0.5 in patients with vWF:Ag levels ⬎30 IU/dL⫺1.126 The diagnosis of Type 1 vWD is characterized by a prolonged closure time on the platelet function analyzer and proportionately low levels of vWF:Ag, vWF activity, and Factor VIII. Therefore, a vWF:RCoF/vWF:Ag ratio should be proportionate (0.7-1.2) for Type 1 vWD.126,129 Multimeric analysis of the patient’s vWF reveals that all sizes of vWF multimers are present. In Type 1 vWD, vWF levels are subnormal, but the vWF produced is structurally and functionally normal. Type 2 variants are characterized by qualitative defects in vWF.126 Type 2A results either from decreased production or from accelerated clearance of the hemostatically important

Chapter 28: Congenital Disorders of Clotting Proteins and Hypercoagulable States in Pediatrics

Table 28-3. Laboratory Diagnosis of von Willebrand Disease and Suggested Treatment Options* Condition

vWF:RCoF (IU/dL)

vWF:Ag (IU/dL)

Factor VIII

vWF:RCoF/ vWF:Ag

Therapeutic Options

Type 1

⬍30†

⬍30†

↓ or Normal

⬎0.5-0.7

DDAVP/Stimate‡

Type 2A

⬍30†

⬍30-50†§

↓ or Normal

⬍0.5-0.7

DDAVP; vWF-Factor VIII concentrates

Type 2B

⬍30

⬍30-50

†§

↓ or Normal

Usually ⬍0.5-0.7

vWF-Factor VIII

Type 2M

⬍30†

⬍30-50†§

↓ or Normal

⬍0.5-0.7

vWF-Factor VIII

†

Type 2N

30-200

30-50

↓↓

⬎0.5-0.7

vWF-Factor VIII

Type 3

⬍3

⬍3

↓↓↓ (⬍10 IU/dL)

Not applicable

vWF-Factor VIII

“Low vWF”

30-50

30-50

Normal

⬎0.5-0.7

Case-by-case need

Normal

50-200

50-200

Normal

⬎0.5-0.7

*

Adapted with permission from Sadler et al.126 ⬍30 IU/dL is designated as the level for a definitive diagnosis of vWD; there are some patients with Type 1 or Type 2 vWD who have levels of vWF:RCoF and/or vWF:Ag of 30-50 IU/dL. ‡ In nonresponsive patients, use a vWF-Factor VIII concentrate. § The vWF:Ag in the majority of individuals with Type 2A, 2B, or 2M vWD is ⬍50 IU/dL. ↓ Refers to a decrease in the test result compared to the laboratory reference range. vWF:RCoF ⫽ von Willebrand factor:ristocetin cofactor; vWF:Ag ⫽ von Willebrand factor antigen; DDAVP ⫽ desmopressin.

†

HMW vWF multimers. The loss of HMW multimers causes a decrease in platelet-vWF interactions as expressed in a decreased vWF:RCoF assay result. Loss of HMW multimers can also cause a decrease in connective tissue-vWF interactions as measured in decreased vWF:collagen binding (vWF:CB) assay results. Patients may have normal or only slightly reduced vWF and Factor VIII levels but may have a loss in the intermediate and HMW multimers of vWF in plasma. Affected individuals usually have a mild-to-moderate bleeding tendency and generally have a poor response to DDAVP.126,129 In vWD Type 2B, mutations in the A1 domain results in a heightened affinity of vWF binding to platelet glycoprotein 1b (GP1b). Affected individuals often have mild thrombocytopenia caused by in-vivo platelet aggregation, and on ex-vivo testing, their platelets aggregate with very low concentrations of ristocetin (enhanced ristocetin-induced platelet aggregation). Upon secretion, the HMW multimers in vWD Type 2B bind spontaneously to platelets and are then cleaved by ADAMTS13 for excretion. The small multimers do not enhance platelet adhesion or connective tissue binding, resulting in decreases in vWF:RCoF and vWF:CB. Another disorder caused by a mutation on the platelet GP1b causes a similar phenotype with increased binding affinity of the platelet for vWF. This is called pseudo-vWD. von Willebrand disease Type 2M variants have decreased affinity of vWF for platelets. This results in an increase in HMW multimers on the agarose gel testing. These multimers have a decreased activity in platelet adhesion and decreased cleavage by ADAMTS13. Similar to Type 2B, most patients with Type 2M have a mutation in the vWF A1 domain responsible for binding the GP1b. von Willebrand disease Type 2N is an uncommon but interesting variant. It is characterized by a mutation in vWF that prevents the binding of Factor VIII but does not interfere with

platelet adhesion. Thus, uncomplexed Factor VIII is rapidly degraded in the circulation, resulting in low levels of Factor VIII, mimicking hemophilia A.126 In vWD Type 3, patients have a severe bleeding tendency and very low levels of vWF and Factor VIII. Type 3 is inherited in a autosomal recessive pattern and has levels of vWF:Ag ⬍1 to 5 U/dL. Although the vWF:Ag levels of Type 3 can approach those of severe Type 1, the two types of vWD are almost always clinically distinguishable. In addition to severe mucous membrane bleeding, Type 3 patients may bleed into joints and soft tissues. Type 3 patients are not responsive to desmopressin, whereas type 1 patients generally are responsive (see below). Most cases of vWD Type 3 are caused by deletions, nonsense, missense, and frameshift mutations in the vWF gene.126,129

Treatment of von Willebrand Disease Treatment is determined by the type of vWD (Table 28-3). The goals of therapy are to increase both vWF and Factor VIII levels, which predict clinical response. Generally, raising the vWF and Factor VIII levels into the normal range (above 50%) is sufficient to alleviate the bleeding manifestations of vWD. However, a qualitative defect (Type 2 vWD) may also require completely replacing the patient’s vWF with normal vWF to give a clinical response. For persons with Type 1 vWD, the treatment of choice is DDAVP.74,76,129,130 When given intravenously in a dosage of 0.3 µg/kg diluted in 50 mL saline over 30 minutes, the drug will effect a rapid three- to fivefold increase in vWF activity, vWF:Ag, and Factor VIII levels in most patients with Type 1 vWD and some with Type 2A. Generally, the increase in vWF and Factor VIII levels will last for 6 to 8 hours. It is important to give a patient with vWD a test dose of DDAVP to confirm responsiveness and to determine the magnitude of the response.

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Although the magnitude of response varies among individuals, the response in a given individual is generally consistent over time (ie, threefold increases in vWF activity and vWF:Ag on one occasion are likely to be duplicated during retreatment at a later date). The dose can generally be repeated every 12 to 24 hours with efficacy. Most patients treated repeatedly over a short period do become less responsive, a phenomenon called tachyphylaxis.76 Because the effectiveness of DDAVP is attributable to the rapid release of vWF from storage sites, DDAVP is ineffective in patients with Type 3 vWD, who have nothing in storage to be released. Most clinicians consider DDAVP to be contraindicated in Type 2B, because the release of the functionally abnormal vWF causes a worsening of thrombocytopenia and is hemostatically ineffective.76 Although desmopressin is generally given intravenously to hospitalized patients, the highly concentrated intranasal spray formulation is ideal for home use. It provides excellent bioavailability of the drug and effects an increase of vWF similar to that obtained with an intravenous dose of 0.3 µg/kg DDAVP. The compression metered spray pump delivers 0.1 mL (150 µg) of solution per spray. The dose for children and adolescents is one spray; for adults a spray is given in each nostril. This highly concentrated intranasal spray is particularly useful for prophylaxis before invasive dentistry or other minor surgery.131 The side effects of DDAVP are generally well tolerated. Facial flushing, mild tachycardia, and headaches are often transient and mild. DDAVP does have an antidiuretic effect and rarely can cause hyponatremia, leading to seizures. This effect has been seen most often in the use of DDAVP in small children. All patients using the drug at home must be counseled to not give repeat doses within 24 hours without consulting a physician. Thrombosis has been reported in DDAVP treatment of diseases other than vWD, and in one patient with concurrent vWD and thrombophilia.76 Replacement therapy with a plasma-derived intermediatepurity Factor VIII concentrate rich in vWF is still needed in certain situations. For patients with vWD Type 1 or Type 2A whose responses to DDAVP are not sufficient (eg, for major surgery), a clotting factor concentrate containing the HMW multimers of vWF should be given.74,130 For persons with vWD Types 2B and 3, concentrates rich in the hemostatically important HMW multimers of vWF should be given for the treatment of serious bleeding episodes or for surgical prophylaxis. The optimal replacement therapy for vWD corrects both the vWF defect (quantitatively and qualitatively) and the Factor VIII concentration. Because most commercial Factor VIII concentrates do not contain the hemostatically important HMW multimers of vWF, several investigators have conducted comparative studies of various products in patients with severe vWD. Humate-P (CSL Behring) was found to contain the highest amount of HMW multimers and therefore to be most effective for patients with vWD.74,130,132 This pasteurized concentrate is thought to be safer than cryoprecipitate, which is not virus-inactivated. For the treatment of severe bleeding or for major surgeries in patients with Type 3 vWD, some studies recommend a bolus dose of 60 to 80 IU vWF:RCoF/kg⫺1 followed by 40 to 60

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vWF:RCoF/kg⫺1 every 8 to 12 hours for a few days then spaced out to daily dosing as needed for control of bleeding. A multicenter, retrospective study132 showed an average daily use of 80 IU vWF:RCoF/kg⫺1 per day for surgery coverage. The efficacy was 97% in these patients. The on-demand treatment of bleeding averaged 72 IU vWF:RCoF/kg⫺1/day of Humate P with an efficacy of 95% in these patients. A prospective, multicenter open-label cohort study133 was conducted in patients with hereditary vWD (acquired vWD patients excluded) and a history of abnormal bleeding. The median vWF:RCoF dose after individual pharmacokinetic testing was 62.4 IU/kg⫺1 with full control (analogous to expected bleeding in a normal patient) of intraoperative bleeding in 96.3% of the patients.133 An interesting use of DDAVP and Humate P occurred in a hemophilia treatment center in Germany. Two patients had severe hemophilia A with Type 1 vWD, one patient had severe hemophilia B with Type 1 vWD, and two patients had severe hemophilia B with Type 2 vWD. All five patients experienced more mucosal bleeding than would be expected with a diagnosis of hemophilia alone. The two of these patients with severe hemophilia B and Type 2 vWD had very difficult-to-treat bleeding with recombinant replacement of Factor IX alone. Even with prophylaxis, breakthrough bleeding was a problem. These patients responded to concurrent infusions of BeneFix and Humate P or DDAVP.134 Other agents that may be useful include the antifibrinolytic drugs, ε-aminocaproic acid and tranexamic acid. For the treatment of minor mucosal bleeding, particularly in patients with mild vWD, an antifibrinolytic drug may suffice. Antifibrinolytic agents also should be used in conjunction with desmopressin or Humate-P for invasive dental procedures, tonsillectomy, or other bleeding in the oral cavity. As is true for all coagulopathies, persons with vWD should avoid aspirin, all aspirin-containing compounds, and other drugs that interfere with platelet function.

Other Inherited Disorders of Coagulation Rare hereditary deficiencies of all the other known coagulation factors have been described, and are heterogeneous. Thus, not all affected families with a particular factor deficiency have the same bleeding tendencies. Because these disorders are inherited in an autosomal recessive pattern, patients who are homozygotes or compound heterozygotes tend to bleed more. Because no specific clotting factor concentrates are licensed and available in the United States for use in these relatively rare deficiency states, the treatment of choice for bleeding in most of them is still PCCs, plasma, or cryoprecipitate. In general, the level required to achieve hemostasis is relatively low and thus can be attained by plasma in a dosage of 10 mL/kg. Once hemostasis has been achieved, continued treatment may not be necessary.135 However, plasma (solvent/detergent-treated FFP or donor-retested FFP) and cryoprecipitates have the disadvantages of potential viral contamination, volume overload, and allergic reactions.

Chapter 28: Congenital Disorders of Clotting Proteins and Hypercoagulable States in Pediatrics

Bleeding in persons with congenital afibrinogenemia or hypofibrinogenemia should be treated with cryoprecipitate in a dosage of four bags/10 kg of body weight. Cryoprecipitate contains approximately 200 mg of fibrinogen per bag, or “unit.” (Because of the long half-life of fibrinogen, replacement therapy is generally given every 3 to 4 days.) Although seldom necessary, additional doses of two bags/10 kg can be given every 3 to 4 days. In Europe a specific fibrinogen concentrate (treated to be virusfree) is available. In rare hereditary factor deficiencies (ie, Factor II, V, or X deficiencies), plasma or a PCC can be used. For serious, lifethreatening bleeding, PCCs should be used to achieve higher levels of the missing factor. Recombinant Factor VIIa is the treatment of choice for congenital Factor VII deficiency, for ondemand treatment and prophylaxis. The current recommended dose for surgical or invasive procedures is 15 to 30 µg/kg every 4 to 6 hours.100 Deficiencies of the “contact factors,” Factor XII, prekallikrein, and HMW kininogen, are rarely associated with clinical bleeding and thus do not require treatment. The hemorrhagic tendency in Factor XI deficiency is usually mild and independent of the Factor XI levels. On occasion excessive bruising, epistaxis, and postoperative bleeding have been observed. One study136 found that bleeding tendency in tissues with fibrinolytic activity (ie, oral mucosa, tonsils, nose) was increased 49% to 67%, while the tendency to bleed in other tissues was reduced to 1.5% to 40% irrespective of Factor XI deficiency genotype. Therefore, in patients with Factor XI undergoing a procedure in areas of fibrinolytic activity, an antifibrinolytic agent with on-demand replacement may be sufficient. Patients undergoing other surgeries or procedures such as circumcision or appendectomy may require on-demand therapy only for intra- or postoperative bleeding.135 FFP infused at the rate of 10 to 20 mL/kg/day usually provides sufficient Factor XI to maintain hemostasis. The aPTT, the specific assay for Factor XI, or both should be used to monitor treatment. Congenital deficiency of Factor XIII (fibrin-stabilizing factor) is a rare disorder characterized by delayed umbilical bleeding in neonates, subcutaneous bleeding, muscle hematomas, bleeding after surgery, hemarthroses, and ICH.136 It is also characterized by a lifelong bleeding tendency and impaired wound healing. Only homozygous individuals with no detectable Factor XIII activity have bleeding manifestations. Factor XIII circulates in the plasma with the A and B subunit in an A2B2 tetramer. The patients with most severe disease have a deficiency in the A subunit. The prothrombin time, aPTT, and thrombin time are normal in Factor XIII deficiency. The diagnosis used to be made exclusively by testing for clot solubility in 5 mol urea, but currently both Factor XIII assays and assays for the A- and B-subunit antigens are available. Unfortunately, the clot solubility testing is useful only for patients with zero Factor XIII activity, but bleeding may still be a problem at higher levels. Additionally, it is difficult to interpret the Factor XIII levels at low levels; the accuracy of this test at levels ⬍10% is questionable. There is no correlation between bleeding and Factor XIII levels.137 Fibrogammin

P (CSL Behring), although not licensed in the United States, is available on a clinical trial basis for patients with severe Factor XIII deficiency.74 Because the half-life of Factor XIII is long, prophylactic therapy with Factor XIII concentrates can be given with a monthly dosage of 250 IU in children and 500 IU in adults.138 If it is not readily available for some reason, a single infusion of plasma of 5 to 10 mL/kg usually will provide effective therapy for bleeding episodes.

Hypercoagulable States Congenital and Acquired Thrombophilia Thrombophilia refers to a blood condition, whether acquired or inherited, with an increased propensity to develop thrombosis. Acquired thrombophilia is seen in autoimmune and infectious or postinfectious states. Inherited thrombophilia indicates a congenital deficiency of an antifibrinolytic agent or a genetic mutation in one of the clotting factors leading to an increase risk of thrombosis. An example of acquired thrombophilia is antithrombin deficiency. Antithrombin deficiency can be seen in nephrotic syndrome, protein-losing enteropathy, and as a side effect of the commonly used chemotherapeutic agent, L-asparaginase. Additionally, protein C and S deficiencies can be seen as a consumptive process in patients with severe sepsis and DIC.139 Protein C deficiency, in particular, has been associated with an increased risk for mortality in sepsis and protein C replacement is being investigated in the treatment of meningococcal-induced purpura fulminans.140 Antiphospholipid antibodies including lupus anticoagulant often occur after infections and are self-limited. Detection of antiphospholipid antibodies in the serum for 6 weeks or more connotes antiphospholipid antibody syndrome (APAS) and is more commonly seen in adolescents. APAS may be a result of lupus or other autoimmune disorders (Table 28-4).139 Genetic mutations cause hypercoagulability by decreasing the amount or function of various proteins responsible for the regulation of coagulation. These proteins include antithrombin, protein C, protein S, plaminogen, and fibrinogen. Point mutations in genes encoding prothrombin and Factor V, such as prothrombin 20210 and Factor V Leiden, represent a genetic risk factor for thrombophilia. The prothrombin 20210 mutation Table 28-4. Risk Factors for Thrombosis in Children141 Temporary Risk Factors ● Indwelling catheters ● Surgery ● Infection Ongoing Risk Factors ● Thrombophilia—genetic or acquired ● Malignancy and chemotherapy ● Inflammatory diseases ● Prosthetic heart valves ● Sickle cell anemia

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causes a 15% to 30% increase in the production of the procoagulant, prothrombin. Factor V Leiden is a mutation that causes resistance of Factor V to physiologic downregulation by activated protein C. Other genetic hypercoagulable states include hyperhomocysteinemia and platelet membrane glycoprotein defects. In a recent study142 involving children with stroke, laboratory testing was performed to look for a prothrombotic condition in 338 of the study participants. Of these 338 children, 64% percent had laboratory results consistent with a prothrombotic condition, 24% had a Factor V Leiden mutation, and another 12% had antiphospholipid antibodies detected.142

Venous Thromboembolism in Infants and Children Incidence and Risk Factors Venous thromboembolism (VTE) is relatively rare in the pediatric population. A multicenter Canadian VTE Registry trial143 reported an incidence in the general pediatric population of 7 per million children or 5.3 per 10,000 hospital admissions in children. The risk for VTE is greatest in the neonatal and adolescent age groups with indwelling catheters representing the most common risk factor found in 33% of children with VTE. It is for this reason that VTEs in children are more commonly seen in the upper venous system, contrary to the location of VTEs in adults.144 A study145 that investigated the incidence of VTE in children with acute lymphoblastic leukemia treated with L-asparaginase, also studied risk factors for VTE in children with indwelling catheters. Left-sided catheters, particularly in the subclavian vein, inserted by percutaneous rather than venous cut-down technique were at greatest risk of catheter-associated VTE.145 The current recommendations from the American College of Chest Physicians do not support primary anticoagulation prophylaxis in children with indwelling catheters.146 Only 3.6% of children on the Canadian Registry with VTE had thrombosis with no predisposing condition.143 All others had at least one predisposing factor including catheters, cancer, congenital heart disease, infection, trauma, obesity, nephrotic syndrome, surgery, lupus, sickle cell disease, and liver failure. Twelve children had a prothrombotic condition—six had protein C deficiency and six had protein S deficiency. The contribution of a prothrombotic condition is difficult to assess from these data, because only 33% (45 of 137) of the children with VTE underwent testing for a prothrombotic condition. Umbilical venous catheters can also be a source for clot formation. These clots often go undetected and occur in the inferior vena cava and portal vein. Clots from umbilical venous catheters can cause lobar atrophy or portal hypertension that may be discovered later in life. An incidence of 3.6 per 1000 neonates admitted to the neonatal intensive care unit at Toronto’s Hospital for Sick Children has been reported.147 Anticoagulation did not appear to affect the development of portal hypertension or lobar atrophy.

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In a pediatric stroke trial,142 36% of the 1065 children studied had cerebral sinovenous thrombosis (CSVT) and 54% had arterial ischemic strokes (AIS). Of the children with CSVT, 39% had identifiable risk factors for VTE. The older infants and children had a statistically higher number of identifiable risk factors than the neonates. Systemic disease was an identifiable risk factor for children with CSVT. Systemic diseases included genetic syndromes, malignancy, chronic infection, hypertension, and chronic prenatal conditions. Of the children with genetic syndromes, 38.5% had Down syndrome. Other genetic syndromes were not specified but were associated with congenital heart disease or other chronic medical conditions. Acute infections were also reported more commonly in children with CSVT than those with AIS.

Diagnosis and Treatment The diagnostic evaluation for VTE is dependent upon the location of the suspected clot and the clinical index of suspicion. Clots related to umbilical venous catheters are best diagnosed with ultrasound and/or venography. Clots related to indwelling central venous catheters often develop in the upper venous system. The initial evaluation would include a color Doppler ultrasound, but often venography and an echocardiogram are necessary to identify the suspected clot. Doppler ultrasound carried a sensitivity of only 37% in one study,145 but it has the advantage of a noninvasive evaluation. The sensitivity of bilateral venography was found to be 79%, but venography is a more invasive method of detection. Venography was the most sensitive for the central upper venous system, whereas Doppler appeared superior in the diagnosis of jugular venous clots. The diagnosis of VTE in the upper venous system should encompass Doppler sonography, echocardiography, and the more invasive alternative, venography, as needed.148 Data are limited on the optimal treatment of VTE in children. Evidence-based evaluation and management of pediatric thrombosis and thrombophilia is in its infancy. Most of the information available today is based largely on small case series or extrapolated from adult recommendations.141 The agents currently available for antithrombotic therapy in children include UFH, low-molecular-weight (LMW) heparin, warfarin, and tissue plasminogen activator (tPA). UFH is an effective drug, but has a short half-life of 25 minutes in neonates (to 1 hour in adults).149 LMW heparins are being used increasingly for initial therapy for acute thrombosis in children. In one study,150 53% of 190 children treated with enoxaparin for thromboembolism had complete resolution of the thrombus. Children with arterial clots and clots that were not occlusive at diagnosis were more likely to have complete resolution of their clots after taking enoxaparin. Forty-eight percent of the children with VTE had complete resolution of the thrombus; this is not appreciably different from the adult data of VTEs treated with LMW heparins. Age at diagnosis, location of the thromboembolus (excluding arterial vs venous), and initial treatment with fractionated vs UFH did not appreciably affect the complete

Chapter 28: Congenital Disorders of Clotting Proteins and Hypercoagulable States in Pediatrics

resolution of the clots. The mean dose of enoxaparin also did not affect the complete resolution of the clot.150 LMW heparins have many advantages over UFH and other anticoagulants in children, and they are rapidly becoming the treatment of choice for children with hypercoagulability and clots. The advantages of treatment with LMW heparin include: effectiveness equal to UFH, a lower frequency of bleeding complications, decreased incidence of heparin-induced thrombocytopenia, minimal monitoring required (the pharmacokinetics of LMW heparin are more predictable than those of UFH), decreased amount of drug-to-drug interactions, and possibly a decreased risk of osteoporosis with long-term treatment relative to UFH. The current recommended dose of LMW heparin depends on the anti-Factor Xa level in a sample drawn 4 to 6 hours after a subcutaneous dose of LMW heparin. The targeted therapeutic anti-Factor Xa level is generally 0.5 to 1 U/mL. Neonates and infants less than 2 to 3 months of age or 5 kg of weight generally require a higher dose per kilogram to maintain therapeutic anti-Factor Xa levels. Because of increased clearance in children, LMW heparins (especially enoxaparin) are administered subcutaneously twice a day.146 Schobess et al151 recently evaluated 80 children over 3 months of age with DVT in an open-label pilot randomized safety study, using once-a-day enoxaparin administration, with a target 4-hour anti-Factor Xa activity between 0.5 and 0.8 IU/mL. The children were stratified to receive either once daily or twice daily doses. The mean duration of treatment was 5 months, with a median follow-up of 24 months. The authors found no significant differences between the two groups with regard to the study endpoints, postthrombotic syndrome, rethrombosis, bleeding, or therapy-related death.151 Systemic or local thrombolysis should be strongly considered in children with “high-risk” clots that present within 2 weeks of symptomatic onset. Low-dose (0.03-0.06 mg/kg/hour) systemic infusions of tPA for 12 to 96 hours have been shown to be effective in children.152 Warfarin, despite being an oral agent, is difficult to administer in children and requires frequent monitoring with dose adjustments.153 Children with thrombophilia do not always require lifelong anticoagulation even if a genetic cause for hypercoagulability is isolated. A single thrombophilic gene mutation can certainly cause an increased risk of thrombosis in childhood; however, it is not necessarily implicated in a worse outcome or greater risk of recurrence.154 For instance, no increase in DVT-related pulmonary embolus was seen in otherwise-healthy children diagnosed with thrombophilia.155 An elevated D-dimer in the absence of infection or chronic inflammation can be indicative of excessive thrombin generation and suggest a need for prolonged anticoagulation. In addition, a recurrence of thrombus could be a strong indication for prolonged or possibly lifelong anticoagulation with or without an isolated genetic thrombophilia.139 Because prospective randomized studies in children are lacking, one strategy to select optimal antithrombotic therapy is to tailor treatment based upon assessed risk for an unfavorable clot outcome. Manco-Johnson,

in a review of antithrombotic therapy, suggested using patientspecific and thrombus-specific characteristics to stratify the likelihood of a poor outcome.141 Thus, children without underlying thrombophilia in whom a transient triggering event has resolved are classified as “low risk” while those with at least three thrombophilic traits, or those with persistence of antiphospholipid antibodies who at the time of presentation have an occlusive DVT, a Factor VIII level ⬎150 U/dL, and a D-dimer level ⬎500 ng/mL are considered “high risk” (Table 28-5).

Complications A Canadian study reports a recurrence rate of approximately 8%.144 The recurrence rate is higher with increasing age of the child at diagnosis. Mortality is uncommon but occurs in 2.2% of children with deep vein thrombosis (DVT)/pulmonary embolus.156 Postthrombotic syndrome (PTS), also referred to as postphlebitic syndrome, is an important consequence of DVT. PTS describes limb pain, swelling, development of visible collateral circulation, and chronic venous insufficiency. PTS can often be severe enough to limit the child’s activity. The incidence of PTS following childhood DVT is difficult to assess and estimated between 10% and 63% depending on the study.157,158 It is felt to be underreported and underdiagnosed in childhood. The wide variation in reporting has been due in part to a lack of standardization among assessment tools used to evaluate this problem in children. A pediatric scale for PTS has recently been validated in children.159 PTS has been reported to increase in patients with elevated Factor VIII and D-dimer levels. A thrombolytic treatment study in high-risk pediatric DVT was shown to significantly

Table 28-5. Risk Assessment for Poor Outcome from a Thrombotic Event in Children141 Patient Characteristics ● Low-risk features – Transient triggering event – No thrombophilic trait ● Standard risk features – Less than three thrombophilic traits – Factor VIII ⬍150 U/dL and D-dimer ⬍500 ng/mL at the time of presentation ● High-risk features – At least three thrombophilic traits – Persistent antiphospholipid antibody ⬎6 weeks – Factor VIII ⬎150 U/dL and D-dimer ⬎500 ng/mL at the time of presentation Thrombus Characteristics ● Low-risk features – Thrombus resolved within 6 weeks – Nonocclusive thrombus ● High-risk features – Occlusive deep venous thrombosis – Persistence of thrombus at 6 weeks

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decrease the incidence of PTS in children with “high-risk DVT.”160 High risk was defined as acute lower extremity DVT and measured Factor VIII level ⭓150 U/dL and/or D-dimer ⭓500 ng/ mL. The patients were stratified to receive thrombolytic therapy with an initial low-dose infusion of tPA. The tPA was continued until radiographic studies showed complete resolution of the thrombus. All of the patients received anticoagulation with either UFH to maintain a plasma anti-Factor Xa level of 0.3 to 0.7 U/mL or enoxaparin to maintain a level of 0.5 to 1.2 U/mL for at least 7 days. The long-term anticoagulation was accomplished with enoxaparin or warfarin for at least 3 months from diagnosis. Pulmonary embolus is a well-known complication of DVT in adults, but it is likely that many cases in children go unrecognized. Pulmonary embolus occurred in 16% or 39 children with central venous catheters in the Canadian Pediatric Thrombophilia Registry.161 Eleven of these children had isolated episodes, and the remaining 28 developed pulmonary embolus after catheter-related DVTs. Thirteen of the DVTs were located in the lower venous system, and 15 DVTs were located in the upper venous system. There is no single “best” imaging technique for pulmonary embolus. A positive helical computed tomography scan confirms the diagnosis, whereas a normal ventilationperfusion scan rules this out.141

is helpful for peripheral catheter-related arterial thromboembolism. The diagnosis of AIS generally is confirmed with an MRI (79% of the cases in the stroke study). Magnetic resonance angiography can be helpful for small or recurrent clots that are not well visualized on a standard MRI. Recurrent embolic AIS could occur from a cardiac defect or cardiac vegetations, which should be ruled out with contrast echocardiography. Children who develop Kawasaki disease should be treated with aspirin therapy and IV gammaglobulin replacement within 10 days of onset. The aspirin doses should begin at 80 to 100 mg/ kg/day during the initial or acute phase, up to 14 days from the onset of symptoms. Aspirin should be continued in lower doses, 3 to 5 mg/kg/day for at least 7 weeks more.146 As discussed previously, children with arterial thrombosis have been found to be more responsive to treatment with LMW heparin than children with venous thrombosis, particularly in a nonocclusive clot. LMW heparin has also been used acutely to treat children with AIS, particularly in children found to have vasculopathies, thrombophilia, or antiphospholipid antibodies.142 Neonatal strokes are generally not treated and generally do not recur. Randomized clinical trials are necessary to identify the thromboembolic states that mandate treatment and determine the optimal treatments when treatment is needed.

Arterial Thromboembolism in Infants and Children Incidence and Risk Factors Arterial thromboembolism occurs most often in the setting of catheter-related endothelial trauma.139 In the neonatal period, umbilical artery catheterization with vascular injury precipitates the thrombus. After the neonatal period, peripheral arterial catheterization and cardiac catheterization are the most common precipitating events.144 Arterial thromboembolism in childhood is rare without a catheter-related event, but some predisposing risk factors have been identified. Familial hyperlipidemia, hyperhomocysteinemia, Takayasu arteritis, Kawasaki disease, congenital arterial malformations, and congenital heart disease all serve as predisposing risk factors for arterial thromboembolism. In addition, children who test positive for lupus anticoagulant are at risk for venous and arterial thrombosis. Arterial ischemic stroke is rare in neonates, with an estimated annual incidence of 12 per 100,000 in neonates and 0.5 to 6 per 100,000 in children older than 1 year.144 In the pediatric stroke study,142 675 children had AIS; of these children, 46% had identifiable risk factors for arterial thromboembolism, and 60% had vasculopathy. Cardiac disorders were found in 11% and other chronic disease occurred in 24%. Ten of the 115 children with cardiac disease and AIS were older children with a patent foramen ovale. Approximately 65% of the children, equal numbers for CSVT and AIS, tested positive for a prothrombotic disorder. Diagnosis and Treatment The diagnostic workup for arterial thromboembolism depends on the suspected location of the clot or ischemia. Angiography

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Summary Hemophilia A (Factor VIII deficiency) and hemophilia B (Factor IX deficiency) are clinically indistinguishable, sex-linked coagulopathies that affect approximately 20,000 persons in the United States. Hemophilia A is the more common of the two and accounts for 80% of the cases. In the past, the treatment of hemophilia with plasma-derived products resulted in extremely high rates of hepatitis and HIV seroconversion. However, improved donor screening tests and newer virus attenuation and purification methods have resulted in much safer plasma-derived Factor VIII and Factor IX concentrates. Recombinant Factors VIII and IX concentrates are also available. Although the currently available plasma-derived Factor VIII concentrates seem to be safe in terms of both hepatitis and HIV, one cannot be absolutely sure of the safety of these products because of the risk of emerging infections. The majority of Factor VIII and Factor IX used in the United States is now recombinant (DNA-derived). Desmopressin is considered the treatment of choice for bleeding in patients with vWD Type 1, with plasma-derived Factor VIII concentrates rich in the HMW multimers of vWF being used in those with Type 2 variants and Type 3 vWD. FFP (either solvent/detergent-treated or donor-retested) and cryoprecipitates are still used to treat many of the rare coagulation factor deficiencies, but aPCCs and rFVIIa are used more and more in rare bleeding disorders with good efficacy and limited side effects. Venous and arterial thromboembolism in infancy and childhood have been described with increasing frequency over the past decade. The thrombus location, quality, and characteristics

Chapter 28: Congenital Disorders of Clotting Proteins and Hypercoagulable States in Pediatrics

of the patients (including age, genetic thrombophilia, immobility, etc) all affect the type and duration of treatment. LMW heparin or enoxaparin has been used to treat many pediatric patients with thromboembolism with good efficacy and minimal toxicities. The optimal type and duration of treatment for pediatric thromboembolism remains to be determined using prospective, randomized, controlled studies.

Disclaimer The authors have disclosed no conflicts of interest.

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Management of Congenital Hemolytic Anemias Bruce I. Sharon Associate Professor, Department of Pediatrics, University of Illinois College of Medicine and University of Illinois Hospital, Chicago, Illinois, USA

The congenital hemolytic anemias comprise a heterogeneous group of intrinsic red cell abnormalities that may be conveniently classified as disorders of hemoglobin (hemoglobinopathies and thalassemia syndromes), red cell enzyme-deficiency disorders, and abnormalities of the red cell membrane and cytoskeleton. In patients affected with any of these conditions, Red Blood Cell (RBC) transfusions may be indicated to compensate for the decreased oxygen-carrying capacity associated with the underlying anemia. In addition, pathophysiologic consequences unique to each of these disorders may lead to other indications for transfusion. The hemoglobin-related abnormalities have special significance in clinical transfusion medicine, and particular emphasis is devoted to this group of disorders.

individuals carrying this gene is localized in central West Africa, where the gene frequency for sickle hemoglobin (HbS) may be as high as 0.14. Appreciable numbers of affected individuals have also been identified in other parts of equatorial Africa, the Mediterranean, the Saudi Arabian peninsula, and India. This distribution substantially coincides with regions that historically have had endemic Plasmodium falciparum malaria. Ample epidemiologic evidence suggests that the βS gene was subject to positive selection pressure because of the survival advantage that it confers in heterozygous individuals who are infected by the malarial parasite. In the New World, the βS gene arrived as a result of the importation of slaves from Africa; in North America its gene frequency is approximately 0.04 to 0.05 among those of African ancestry. (Thus, approximately 10% of African Americans are sickle heterozygotes.)

Hemoglobinopathies and Thalassemias The hemoglobinopathies are characterized by structural changes affecting the protein globin portion of the hemoglobin molecule, resulting from DNA mutations in the corresponding globin genes. Most forms of thalassemia are characterized by the production of diminished quantities of globin chains that are structurally normal. In certain globin-gene disorders, the molecular defect produces both a quantitative and a qualitative abnormality; examples include hemoglobin (Hb) E (a β-globin abnormality) and Hb Constant Spring (an α-globin abnormality). This discussion on hemoglobin disorders focuses on sickle cell disease (SCD) and the α- and β-thalassemias.

Sickle Cell Disease Epidemiology The geographic distribution of the sickle globin (βS) gene in the Old World is shown in Fig 29-1. The greatest concentration of

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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Molecular and Cellular Pathophysiology The fundamental defect in SCD is the substitution of thymine for adenine in the sixth codon of the gene for the β-globin chain, leading to a replacement of glutamic acid by valine at this site. In contrast to normal hemoglobin tetramers, HbS has an altered surface charge that promotes the formation of lengthy polymeric chains (gelation) when in the deoxygenated state. The oxygen affinity of dilute, unpolymerized HbS is similar to that of normal hemoglobin. However, the oxygen affinity of concentrated HbS solutions is decreased,2 thereby representing a further stimulus for molecular polymerization. Once a critical nucleation step has been achieved, the polymerization process may quickly progress to form rapidly lengthening fibers, which can further organize into filaments and even thicker strands. At the cellular level, this is reflected by the conversion of normal, deformable, biconcave red cells to rigid, highly viscous cells with the characteristic sickle shape. For the most part, the process of HbS gelation and erythrocyte sickling is a reversible one, but after repeated cycles these cells may become irreversibly sickled cells (ISCs). These ISCs maintain an abnormal sickled shape even in the absence of HbS polymerization and are probably a consequence of cumulative red

Chapter 29: Management of Congenital Hemolytic Anemias

(A)

Hb S Hb E Hb C Malaria (B)

b Thalassemia Malaria Figure 29-1. The geographic distribution of (A) hemoglobins S, C, and E and (B) β-thalassemia. The regions where P. falciparum malaria was formerly endemic are also indicated.1(p238)

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cell membrane damage sustained during repeated sickle-unsickle cycles. Irreversibly sickled cells are much more rigid than biconcave red cells and have a much shorter life span, thus appreciably contributing to the hemolytic anemia that is characteristic of this disorder. Curiously, there is no close correlation between the percentage of circulating ISCs in an individual and the clinical severity of the disease. As the process of sickling progresses and an increasing number of cells assume the sickle shape and become rigid, blood viscosity rises sharply. This change causes a marked delay in the transit of blood through the microvasculature, which in turn leads to increased oxygen extraction at the local tissue level. The resultant decrease in hemoglobin oxygen saturation further increases the propensity toward sickling, thus completing a selfpropagating cycle. A critical determinant for the initiation of the sickling process is the intraerythrocytic concentration of deoxygenated HbS (deoxy-HbS). As the concentration of deoxy-HbS rises, the delay time (ie, the time required to achieve a critical minimum polymer nucleus) diminishes exponentially, approximately on the order of the 15th to 30th magnitude.3 Several genetic and cellular (erythrocyte) modulators of disease severity have been proposed and are reviewed below.4,5 Some act by altering the intraerythrocytic concentration of HbS, whereas others exert their effect through unrelated and, in some cases, undetermined mechanisms. Genetic factors that contribute most importantly to the severity of SCD include the concurrence of α-thalassemia, β0- or β⫹-thalassemia, or any of several other hemoglobin abnormalities including HbC, HbD, HbO, and syndromes of hereditary persistence of fetal hemoglobin (HPFH) (see Sickle Cell Syndromes). Other genetic determinants of the clinical expression of SCD appear to be related to the DNA “background” on which the sickle mutation is found. The β-globin gene cluster is located on the short arm of chromosome 11 and contains the β-, δ-, and γ-globin genes, whose globin chain products, in combination with α chains, form hemoglobins A, A2, and F, respectively. The DNA “haplotype” of a given region is defined by the local array of restriction endonuclease fragment-length polymorphisms. Three major DNA haplotypes have been described for the African βS-globin region,6 and it appears that at least certain measures of SCD severity may correlate with the different DNA haplotypes, probably in large part because of the differences they mediate in fetal hemoglobin (HbF) expression. Despite the influence that these genetic factors may have on the expression of SCD, they clearly are not the sole determinants of disease severity. Individuals with apparently identical globin genotypes can vary markedly in their clinical courses. Several erythrocyte-related factors have also been implicated as possible modulators of disease severity.4,5 They include the erythrocyte levels of 2,3-diphosphoglycerate (2,3-DPG; also known as 2,3-bisphosphoglycerate) or adenosine triphosphate, glucose6-phosphate dehydrogenase (G6PD) deficiency, calcium or zinc deficiency, the ISC count, the degree of intraerythrocytic polymerization of deoxy-HbS, and the degree of cellular dehydration.4,5

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Increasingly, the role of extraerythrocytic factors in the vasoocclusive process is being appreciated. Sickle erythrocytes have been shown to have abnormal interactions with coagulation and other plasma proteins, neutrophils, platelets, and vascular endothelium.7,8 The consequence is a red cell environment characterized by abnormally adherent sickle erythrocytes, vascular inflammation, oxidant damage, and excessive stimulation of the coagulation system. Chronic intravascular hemolysis further contributes to these pathologic processes.9 These abnormalities may lead to increased transit time through the microcirculation, or cause altered vascular tone; either consequence has the potential to enhance the likelihood of vasoocclusion. Certain interactions, such as those between neutrophils and the endothelium, result in inflammatory or oxidative injury to the vessel wall. Subsequently, damaged vascular endothelium contributes to increased adhesion of sickle erythrocytes. Thus, the vasoocclusive process is propagated by a cycle of events: sickle cell adhesion damages endothelium and vessel walls, and the damaged microvasculature in turn becomes a more potent nidus for adhesion, and ultimately, vasoocclusion. The interplay between sickle red cells and other factors in their environment that contribute to sickle vasoocclusion is illustrated in Fig 29-2. Sickle red cells have increased adherence to vascular endothelium,11 and this property appears to have a strong correlation with the severity of vasoocclusive disease.12 Red cell adhesion occurs through direct interaction between erythrocyte and endothelial proteins, or through plasma proteins such as thrombospondin and von Willebrand factor, which act as bridges between red cell and endothelial binding sites. Elevated levels of red cell and endothelial binding sites, and plasma proteins contributing to adhesion, have been demonstrated in SCD. Nitric oxide (NO) is an important vasodilator that is synthesized by the vascular endothelium,13 and low levels in SCD contribute to the pathophysiology of complications such as acute chest syndrome (ACS) and stroke.14,15 In addition, levels of the vasoconstrictor endothelin (ET-1) are elevated in SCD, and further contribute to increased vascular tone. Increased plasma homocysteine levels, probably related to folic acid deficiency, appear to be associated with an increased risk of stroke.16

Mechanisms of Anemia The anemia of sickle cell (HbSS) disease is characterized by increased peripheral destruction of the HbS-containing red cells. Progressive membrane damage to these spiny, brittle, poorly deformable erythrocytes is further intensified by secondary changes, including alterations in membrane lipid composition and oxidant damage. Erythrocyte production is substantially increased, in partial compensation for the rapid peripheral destruction. However, compensation is incomplete because of the rightward shift of the oxygen-hemoglobin binding curve that results from deoxy-HbS polymerization. The red cell life span in patients with SCD is approximately 5 to 20 days. Sickle cell complications, including acute splenic sequestration crisis (ASSC) and aplastic crisis, intensify the anemia (see Clinical Features).

Chapter 29: Management of Congenital Hemolytic Anemias

Endothelium

ET-1

NO

3 Cytokines

Hb Sickle Erythrocyte Excess Oxidants

6

LW

Leukocyte K⫹

4

2

H2O

BCAM/LU

C

CD36

Platelet ?

α4 β1

5 TSP

vWF

Laminin VCAM-1

1

GPIb

αv β3

Representative examples in each area are provided. Thick lines indicate binding. Please see the text for additional details. 1. Adhesion. Binding between membrane proteins on erythrocytes and endothelial cells is responsible for increased adhesion between the two. Circulating plasma factors, eg, thrombospondin (TSP) or von Willebrand factor (vWF), serve as bridges between these membrane proteins. Alternatively, direct binding may occur, eg, between α4β1 integrin of the erythrocyte and VCAM-1 of the endothelial cell. In addition, erythrocytes may bind to laminin, an endothelial extracellular matrix protein. (VCAM-1 ⫽ vascular cell adhesion molecule 1; GPIb ⫽ glycoprotein Ib; BCAM/LU ⫽ basal cell adhesion molecule/Lutheran blood group; LW ⫽ LW blood group protein) 2. Erythrocyte membrane pump dysfunction. Loss of K⫹ and water result in intraerythrocytic dehydration, increasing the propensity for sickle polymerization. 3. Vasoconstriction/Vasodilation.The endothelium elaborates endothelin-1 (ET-1) and nitric oxide (NO). ET-1 is a potent vasoconstrictor, and may be produced in excess in sickle cell disease (SCD). NO is a potent vasodilator, and diminished production in SCD can lead to abnormal vasoconstriction. On the other hand, NO is normally inactivated by oxyhemoglobin. In a severe anemic state such as SCD, decreased hemoglobin concentration causes diminished inactivation, or relative excess, of NO. This may produce inappropriate localized vasodilation, and contribute to ventilation/perfusion mismatching seen in acute chest syndrome. NO may also inhibit endothelial adhesion of erythrocytes and platelet aggregation. 4. Leukocytes. Leukocytes in SCD are often increased in number, and demonstrate increased adhesion to both endothelium and erythrocytes. They are a source of cytokines and other mediators of inflammation. 5. Platelets. Increased platelet adhesion and aggregation, and a thrombogenic state of coagulation factors (C) may impede flow. 6. Oxidant damage. Increased generation of oxidants by sickle erythrocytes leads to denaturation of hemoglobin (Hb) and membrane lipid peroxidation. Figure 29-2. Schematic representation of factors potentially influencing the vasoocclusive process.10

In addition, other conditions common in sickle cell patients contribute to anemia, including fever or infection or both, immune hemolysis, G6PD deficiency, and folic acid deficiency.

Sickle Cell Syndromes Because of the prevalence of other α- and β-globin mutations within the African sickle cell population, SCD is a heterogeneous

group of disorders. In each individual syndrome, the primary determinants of sickling are the intracellular concentration of HbS and the propensity of other nonsickle globins present within the cell to participate in the polymerization process. Fetal hemoglobin is virtually totally resistant to incorporation into sickle globin polymers, whereas both adult hemoglobin (HbA) and HbC participate to some degree in this process.

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Table 29-1. Hematologic Values Associated with Common Sickle Cell Syndromes in Adults Hemoglobin Genotype

Hemoglobin Concentration (g/dL)

Mean Cell Volume (fL)

HbF (%)

HbAA (normal)* HbAS* HbSS HbS/β 0 thalassemia HbS/β⫹ thalassemia HbSC HbS/HPFH† HbSS/α-thal trait

12-18 12-16 6.5-9.5 6.0-12.5 6.5-14.0 8.5-12 7-18 7.5-10

80-100 82-92 80-98 63-88 62-84 82-92 75-89 60-80

1 1 2-20 1.4-20 1-15 1-8 15-85 5-20

*No associated clinical disease; included for comparison. Hereditary persistence of fetal hemoglobin.

†

However, patients with HbSC disease have a higher percentage of HbS in their erythrocytes than do individuals with sickle cell trait (HbAS). In addition, HbSC erythrocytes have a membrane defect that leads to intracellular dehydration, causing a further increase in the intraerythrocytic hemoglobin concentration.17 Because of both these factors, HbSC erythrocytes are far more likely to undergo sickling than are HbAS cells. As would be predicted from these differences, the relative expected disease severity is: HbSS, HbS/β0 thalassemia ⬎ HbSC, HbS/β⫹ thalassemia ⬎ HbS/HPFH ⬎ HbAS. Concomitant α-thalassemia in individuals with HbSS disease does not appear to impose a significant change in the frequency of pain crises. However, some forms of chronic organ damage are increased in this disorder because of higher hemoglobin levels, which could lead to increased blood viscosity.18 Characteristic hematologic indices of some of the important sickle syndromes are shown in Table 29-1.

Transfusion Therapy in Sickle Cell Disease The chronic anemia of SCD is usually well tolerated, but during intercurrent illnesses RBC transfusions may be required. Transfusions may also be indicated in order to limit or prevent vasoocclusive complications, either acute or chronic. As indicated in Table 29-2, some of the complications of SCD are characterized by both anemia and vasoocclusive manifestations. In general, for complications in which anemia is the predominant concern, traditional transfusion goals such as adequate hemoglobin concentration are appropriate. In contrast, for complications in which sickle cell vasoocclusion is the overriding concern, the primary objective of RBC transfusion is to achieve a favorable balance between the relative concentrations of normal and sickle red cells. Transfusion in Patients with Sickle Cell Vasoocclusion The purpose of transfusion in the management of sickle cell vasoocclusion is to diminish the likelihood of intravascular sickling through the dilution and replacement of the recipient’s sickle cells by transfused, nonsicklable cells. Clinical experience has shown that vasoocclusion is unlikely to occur with mixtures of AA (normal) and SS (sickle) cells if the relative concentration

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Table 29-2. Acute and Chronic Complications of Sickle Cell Disease that May Require Transfusion Because of Anemia or Vasoocclusion Anemia

Anemia and Vasoocclusion

Vasoocclusion

Acute

Aplastic crisis

Acute chest syndrome Acute splenic sequestration Acute papillary necrosis Acute multiorgan failure syndrome Sepsis Surgery

Stroke Priapism Intractable pain crisis

Chronic

Pregnancy Pregnancy, high risk Hematuria Pulmonary artery Chronic renal hypertension failure Cardiac failure

Skin ulcer Prophylaxis for: Stroke (primary, secondary prophylaxis) Splenic sequestration Intractable pain

of HbS-containing cells is less than 30% to 40%. This finding is supported by in-vitro data from Lessin et al,19 who showed that the relative resistance to flow of mixtures of AA and SS red cells rose sharply when the proportion of SS cells exceeded 40%. Schmalzer et al20 showed that at a fixed concentration of sickle cells (HS/HT or HS alone, where HT is the total hematocrit and HS is the hematocrit of sickle cells), the viscosity (η) rose with the hematocrit (HT), because of the addition of AA cells (Figs 29-3 and 29-4). These authors also showed that deoxygenation does not change the viscosity of AA cells but causes appreciable increases in the viscosity of SS cells, especially at higher hematocrits (Fig 29-3). The rise in viscosity was steepest with higher HS values, and at fixed absolute (HS) or relative (HS/HT) concentrations of sickle cells the effective oxygen delivery (HT/η)20 declined when HT rose through the physiologic range (20% to 40%) (Fig 29-5). This effect, too, was more pronounced with higher HS (or HS/HT). These results indicate that in suspensions of normal cells (HS or HS/HT ⫽ O), as HT rises, the benefit provided by increased oxygen-carrying capacity is nearly equally

Chapter 29: Management of Congenital Hemolytic Anemias

(A)

(A)

8

15

pO2 ⫽ 150 mmHg

6

18

HS/HT

HS/HT 1.0 0.5

␩ 4

0

0

12 HT/␩

0.2 0.3 0.4

9 6

2

3

1.0

pO2 ⫽ 37 mmHg

0

(B)

HS/HT 8

(B)

0

1.0

pO2 ⫽ 37 mmHg

15

6

0.2

0

12

0.5

␩

HS%

HT/␩

E

5 10 15 20 25

S

9

T

4 0

6

2

3

0

0 0

0

10

20

30

40

50

Total hematocrit (HT), % Figure 29-3. The rise of viscosity (η) with HT at given proportions of sickle cells to total cells (HS/HT) in the suspension. (See text for definition of terms.) (A) Oxygenated cell suspensions. (B) Deoxygenated suspensions.20

8 HS% 6

pO2 ⫽ 37 mmHg

10

20

30

40

50

Total hematocrit (HT), % Figure 29-5. The HT/η ratio for suspensions containing mixtures of AA and SS cells. (A) Increasing HS/HT ratios cause a lowering of the HT/η curve. (B) The same data as in A is plotted with constant levels of HS. The relative merits of simple vs exchange transfusion can be analyzed by using the curves for HT/ η (B) to illustrate a hypothetical patient. Point S represents a patient with sickle cell disease with HT ⫽ HS ⫽ 20% before transfusion treatment. After simple transfusion to raise the HT to 35%, Point S moves along the same HT/η curve (HS ⫽ 20%) to Point T, and there is a drop of about 11% in oxygen delivery (HT/η). However, if an exchange transfusion is carried out to lower the HS level of the patient to 5%, although the HT is raised to 35% with AA cells, the result would be at Point E, where the HT/ η is 11% more than at Point S and about 23% more than at Point T. 20

25

pO2 ⫽ 37 mmHg

15 ␩(cP) 4

5 0

2

0 0

10

20

30

40

50

Total hematocrit (HT), % Figure 29-4. The data from Fig 29-3 (B) replotted to show the rise in viscosity (η) with HT at various hematocrit levels of sickle cells (HS).20

offset by the adverse effect of increased viscosity, so that the effective oxygen delivery remains nearly constant (Fig 29-5). Conversely, in the presence of sickle cells (higher HS or HS/HT), the effect upon viscosity predominates and a net decrease in the effective oxygen delivery results. These data strongly emphasize the rheologic advantages of exchange transfusion over simple transfusion when used in the management of patients with sickle cell vasoocclusion. Either type of transfusion produces relative enrichment of normal over sickle cells, but the exchange method has the added benefits of rapidly diminishing HS and limiting the rise in HT. Both effects are helpful in reducing viscosity and improving oxygen delivery to tissues (Fig 29-5).

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The distribution of HbS among the erythrocytes significantly influences the overall likelihood of sickling. For example, in mixtures of AA and SS erythrocytes, the SS cells fully retain the capacity to sickle, whereas AA cells, of course, lack any potential to sickle. Similarly, in HbSC disease or other clinically significant compound heterozygous condition, each HbS-containing cell retains the potential to sickle under suitable physiologic conditions. When planning transfusions for managing sickle vasoocclusion in individuals with compound heterozygous conditions, it is therefore most prudent not to focus on the percentage of the HbS, but rather to consider the percentage of cells that are sicklable. For example, if the recommendation for a particular indication in HbSS disease is to achieve a HbS concentration of 30%, it should be reinterpreted in HbSC disease to mean that transfusion should achieve 30% sicklable cells (yielding approximately 15% HbS) or an HbA concentration of 70%. Clinical Features At birth, infants with homozygous (HbSS) SCD are clinically and hematologically normal because of the predominance of HbF during this period. During the ensuing several months, as the percentage of HbF naturally declines, anemia and other hematologic abnormalities become increasingly apparent. Susceptibility to clinical sequelae of SCD typically begins at 3 to 6 months of age. Various clinical findings in young children with SCD, including early onset of dactylitis (“hand-foot syndrome”), severity of anemia, and presence of leukocytosis, may be predictive for disease severity and mortality,21,22 but some of these relationships (eg, early onset dactylitis) have not been found consistently.23 Acute Sickle Cell Crises The hallmark clinical expressions of SCD are acute sickle cell “crises.” Acute Pain Crisis. These episodes, which are believed to result from vasoocclusion, account for the majority of hospitalizations of sickle cell patients in the United States. Pain crises may involve practically any area of the body but most often are musculoskeletal or soft tissue in origin. These episodes may be brought on by a variety of initiating conditions, including fever, infection, acidosis, and hypoxia, but frequently there is no identifiable precipitant. Occasionally, pain crises are so severe as to be unresponsive to high-dose narcotic analgesia, and they often occur with great enough frequency to be debilitating and severely disruptive of school or work. Acute dactylitis involves the distal extremities and characteristically occurs in infancy. Frequently, this complication represents the initial identifiable clinical event in children with SCD. RBC transfusions are rarely indicated for the treatment of severe or protracted episodes of acute pain crisis or for the prevention of frequent recurrences. When severe acute pain crisis is unresponsive to the standard therapy of intravenous hydration and analgesia, exchange transfusion designed to lower the HbS to less than 40% to 50% may produce relief. Debilitating cycles of frequent pain crises may be arrested by regular courses of

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transfusion that maintain the HbS at less than 40% to 50%. However, this approach is associated with a high risk of recurrence when the transfusions are stopped. Acute Splenic Sequestration Crisis. In patients with SCD, repetitive microocclusive events occur in the spleen throughout early childhood, so that by adulthood the spleen is often “autoinfarcted” and lacks any appreciable blood circulation. Before reaching this state, and primarily in early childhood, the spleen is subject to acute obstruction by sickle cells in the efferent circulation. In about one-half of reported cases, there is a history of infectious illness. The period of risk for acute splenic sequestration is typically prolonged until the early adult years in patients with the milder sickle syndromes (eg, HbSC and HbS/β⫹-thalassemia). Splenic sequestration is a rapidly progressive and potentially fatal process. The clinical course may progress from a state of relative well-being to circulatory shock within just a few hours. Upon presentation, the hemoglobin concentration may be as low as 2 g/dL. Volume resuscitation can be initiated with crystalloid solution followed by prompt transfusion of RBC units. The volume of RBC transfusions should be sufficient to restore baseline hemoglobin levels. One or 2 days following transfusion, splenomegaly will typically diminish and the hemoglobin will spontaneously rise, indicating resolution and liberation of formerly sequestered blood cells into the circulation. In contrast to the typical, rapidly progressive course of acute splenic sequestration crisis (ASSC), subacute, minor episodes may be seen, especially in children with chronic hypersplenism. If splenectomy is being considered to avert recurrence of ASSC or hypersplenism, a limited transfusion program of 1 year may resolve the disease process and avoid surgery. At the least, transfusion can stave off surgery until the patient reaches an age when splenectomy would pose less risk of sepsis. As in other long-term transfusion programs intended to prevent sickle vasoocclusion, the HbS concentration should be maintained at less than 30% to 40%. However, repeated ASSC episodes have even been reported with HbS concentrations as low as 14% to 16%.24 Aplastic Crisis. This complication typically occurs in children of an age and history similar to those of children who experience splenic sequestration. Aplastic crisis is a transient, self-limited cessation of erythrocyte production caused in most, if not all, cases by infection with human parvovirus.25 Because the life span of sickle red cells is only 5 to 20 days, any temporary cessation of erythrocyte production can lead to severe anemia within a short time. Aplastic crisis is characteristically self-limited, and spontaneous recovery usually occurs within a few days of onset. Decisions regarding transfusion must take into account the patient’s clinical status as well as the hemoglobin concentration and any evidence of recovery, as reflected by the reticulocyte count. When possible, an early decision about transfusion helps to diminish the period of mandatory close observation and shorten the hospital stay. Transfusion to or near the baseline hemoglobin level is sufficient, and under such circumstances there need not be concern that transfusion will delay the recovery from marrow aplasia.

Chapter 29: Management of Congenital Hemolytic Anemias

Stroke In addition to the pain episodes that apparently result from vasoocclusion, patients with SCD may also experience major, in some cases catastrophic, ischemic events. Sickle vasoocclusion in the cerebral circulation may cause cerebrovascular accidents, producing hemiplegia, seizures, coma, or death. In children, strokes usually are caused by cerebral infarction, whereas subarachnoid or cerebral hemorrhages become more prevalent with advancing age. Over two-thirds of strokes in patients with SCD occur during childhood, and approximately 11% of children with SCD suffer from stroke.26 In the absence of further intervention, approximately two-thirds of sickle cell patients with stroke will have a recurrent episode, usually within 3 years.26 Functional recovery after a first stroke is variable, and subsequent episodes may produce additional and frequently more devastating sequelae. The acute mortality in adult patients is especially high and may approach 50%. Acute cerebral events that produce focal neurologic deficits lasting less than 24 to 48 hours—transient ischemic attacks (TIAs)—may be a harbinger of stroke in these patients. In sickle cell patients, chronic neurologic damage, manifest as silent cerebral infarcts (SCIs) on magnetic resonance imaging, is twice as common as overt stroke, and is associated with impaired cognitive function.27 SCI is a risk factor for the development of stroke. The primary acute therapy for stroke in patients with SCD is exchange transfusion to limit further intracerebral sickling. Exchange transfusion is preferred rather than simple transfusion because it allows a greater reduction in the fraction of circulating sickle cells, and it more rapidly improves the rheologic properties of circulating blood. In addition, the risk of stroke recurrence appears to be lower with exchange rather than simple transfusion.28 At a minimum, a single-volume exchange should be performed, with the aim of achieving a HbS concentration of approximately 30%. In more severe cases, a greater level of protection may be desirable during the acute phase; a doublevolume exchange would be expected to reduce the level of HbS to approximately 10%. The recurrence rate of stroke in sickle cell patients is high. Chronic transfusion to maintain the HbS concentration below 30% to 40% has been effective in reducing the incidence and severity of recurrent neurologic events.29 However, this beneficial effect may be temporary, lasting only for the duration of transfusion therapy. For example, the incidence of recurrence is reduced to approximately 10% during transfusion therapy but increases again within 1 year after the cessation of transfusions.30 When transfusions are stopped after 5 to 12 years, the risk of stroke recurrence still remains elevated.31 The appropriate duration of therapy, therefore, remains a vexing clinical question. Common practice is to continue transfusion until 18 years of age, if not longer.32 The following management of stroke is advised. Acute exchange transfusion aims to achieve rapid reduction of circulating sickle cells to less than 30%. If cerebral angiography is planned, a further decrease of HbS to 20% should be achieved.

Chronic transfusion therapy to maintain the HbS concentration at less than 30% is advised for at least 5 years. In order to diminish the transfusion load, it may be possible to relax the transfusion criteria after 5 years and maintain the level of HbS at less than 50%.33 Beyond that period, as noted above, transfusions should be continued at least until the patient is approximately 18 years of age, and possibly indefinitely. Transfusion therapy should be monitored by hemoglobin fractionation for determination of the percentage of HbA and HbS, as well as hemoglobin and reticulocyte determinations. If frequent fractionation measurements are impractical, it may be possible to estimate an individual’s transfusion requirement with reasonable accuracy by measuring the reticulocyte count and hemoglobin level after the patient’s individual pattern has been established.34 Complications resulting from transfusions, such as hepatitis, alloimmunization, and iron overload may necessitate early cessation of chronic transfusion. Improvements in cerebral radiographic findings should not be misinterpreted as evidence that risk of stroke recurrence has abated. All these treatment considerations unfortunately underscore a great deficiency in the attempt to prevent stroke in sickle cell patients—they are designed to prevent a second stroke, but ignore the considerable morbidity that occurred as a result of the first event. Is there a reliable means to identify prospectively those patients who are at high risk for developing a first stroke, so that they can be offered prophylactic therapy? Children with elevated cerebral blood flow velocity, as measured by transcranial Doppler (TCD) ultrasonography, have an increased risk for developing stroke during the several years following abnormal studies. On the basis of this screening method, the Stroke Prevention Trial in Sickle Cell Anemia (“STOP”) tested the hypothesis that regular prophylactic transfusions in patients with abnormal TCD findings could prevent a first stroke.35 A total of 1934 children ages 2 to 16 years were screened using TCD, and 206 were found to have abnormal results. Of 130 children who were deemed suitable for the study, 63 patients were randomly assigned to receive regular transfusions designed to maintain the concentration of HbS at less than 30%, and 67 patients were assigned to receive standard care (no transfusions). During the course of the trial, 10 patients dropped out of the transfusion group, and two patients assigned to standard care transferred to the transfusion group. After a follow-up period of up to 30 months, there were 10 cerebral infarctions and one intracerebral hematoma in the standard care group, compared with one infarction in the transfusion group. Because of the obvious benefit afforded by prophylactic transfusion, this study was terminated early in order to allow patients in the standard care group to elect to receive regular transfusions. Patients who received such primary stroke prophylaxis were more likely to revert to normal TCD findings,36 suggesting that chronic transfusion can actually aid in the healing of abnormal vasculature. Is it safe to stop prophylactic transfusion after TCD velocities have normalized? A follow-up study (“STOP 2”) addressed that question, and found that once preventive transfusions were stopped, many patients again developed abnormal TCD

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results, or even stroke, within just a few months.37 A recommended duration for primary stroke prevention has not been established. In patients with abnormal TCD results and evidence of SCI, chronic transfusion has also been demonstrated to lower the risk of new SCI.38 Whether transfusion of patients with a history of SCI but normal TCD results could lower the incidence of new SCI is currently under study (“Silent Infarct Transfusion or SIT Trial”).39 Despite these promising results, many questions remain unanswered. That regular transfusions are highly effective in preventing stroke is hardly surprising. The crucial question is, given the limitations of TCD methodology, do the benefits of primary stroke prevention via chronic transfusion outweigh the risks? The positive predictive value of high-risk TCD results is only about 35% to 40%,40,41 implying that a majority of patients deemed as high risk for stroke by TCD testing would be subjected to the risks of chronic transfusion unnecessarily. A recent analysis employed computer modeling to hypothetically compare the projected benefits and risks of several common primary stroke prevention strategies.42 The key parameter assessed in this study was life expectancy. Several assumptions were employed in this study, including an 80% participation rate of families for whom preventive transfusions were recommended, use of the oral medication deferasirox for iron chelation, and an 80% compliance rate with that treatment. This study found that all the commonly recommended primary stroke prevention strategies employing transfusion resulted in a shorter life expectancy than no transfusion at all. In all cases, but to varying extents, the life expectancy gain produced by averting strokes was outweighed by increased mortality from iron overload. The decision model highlighted the major adverse impact that iron accumulation has in any long-term transfusion regimen. Indeed, in the analysis, if 100% compliance with oral iron chelation was assumed instead of 80%, then the adverse impact of iron accumulation was averted in virtually all the analyzed regimens. Some limitations of this study should be recognized. Some of the assumed benefits of primary stroke prevention may have been overstated because the data employed were derived from controlled clinical trials, in which rates of adherence, monitoring, and follow-up are undoubtedly better than those that would be achieved in general clinical settings. Conversely, some benefits of chronic transfusion may have been understated, because this study focused on life expectancy as an outcome, and did not address any potential improvements in quality of life. Ultimately, it is perhaps understandable that primary stroke prevention with chronic transfusion has met considerable resistance from physicians and sickle cell families.41 Alternative, effective medical strategies certainly would be highly welcome. Hydroxyurea has been found capable of lowering TCD flow velocities,43 but it may not be sufficiently reliable to be clinically useful.44 Acute Chest Syndrome The clinical picture of ACS includes new pulmonary infiltrates on chest radiograph, often accompanied by a combination

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of fever, cough, chest pain, and hypoxia. Infection is the most common cause in young children, whereas vasoocclusion is probably the most likely etiology in adults.45 Especially in children, one complication can precipitate the other. Regardless of the underlying cause, the clinical course can be extremely variable; the most severe cases can progress to an acute respiratory distress syndrome (ARDS)-like picture within just a few hours. Among those patients with an infectious etiology of ACS, the most common pathogens are Chlamydia pneumoniae, Mycoplasma pneumoniae, and respiratory syncytial virus. Pulmonary fat embolism, presumably caused by infarction of bone during a vasoocclusive event, is another important cause of ACS.46 Serum levels of secretory phospholipase A2 (sPLA2), an enzyme that hydrolyzes phospholipids into free fatty acids and lysophopholipids, has been described as a useful biochemical marker that is markedly elevated during episodes of ACS, and correlates with its clinical severity.47 sPLA2 levels rise 24 to 48 hours before clinical presentation of ACS, and thus it is a useful tool to predict impending disease48; serum levels of C-reactive protein, a much more readily available test, appear to parallel those of sPLA2.49 RBC transfusions are often necessary for the treatment of this complication if it is associated with hypoxemia. Prompt treatment helps to avert clinical deterioration. In critical situations, a single-volume exchange transfusion is recommended, because this approach poses no risk of volume overload and achieves a rapid replacement of sickle cells with normal red cells. In less urgent situations, simple transfusions are usually satisfactory. Hepatic Crisis Sickling in the liver produces sharp right upper quadrant abdominal pain, hyperbilirubinemia, and other abnormal liver function values. The clinical picture often resembles that seen in acute cholelithiasis. Because asymptomatic gallstones are coincidentally seen in approximately one-half of all young adult patients with SCD, it is important to distinguish between the two conditions. Treatment with supportive care generally suffices. Acute hepatic sequestration has been rarely described, and is characterized by a rapidly enlarging liver accompanied by a significant decrease in the hemoglobin concentration. RBC transfusion may be required; in order to prevent hyperviscosity stemming from release of sequestered cells, exchange transfusion is preferred. Priapism Prolonged, painful, and unwanted penile erection occurs in up to 89% of male patients with SCD50 and appears to be most prevalent in sickle cell patients who have unusually high rates of hemolysis.51 Common clinical pictures include prolonged erection (lasting more than 4 hours), which often results in severe pain, difficulty in urination, and even impotence, or brief episodes lasting only a few hours, which may be only moderately painful with no long-term sequelae, and which may occur in clusters (“stuttering priapism”).

Chapter 29: Management of Congenital Hemolytic Anemias

Two types of priapism have been described based on blood flow patterns in the penile vasculature. High-flow priapism is caused by dysregulated arterial inflow stemming from cavernosal arterial injury, and is uncommonly associated with ischemia or pain. High-flow priapism rarely requires urgent treatment. In contrast, low-flow priapism is thought to be caused by venous sickle vasoocclusion within the corpora cavernosa, and is often associated with localized ischemia and pain. This type is much more common in SCD, and requires prompt attention to avert long-term sequelae.52 In milder priapism of very recent onset, intravenous hydration and analgesia are the mainstays of therapy, but this approach is often insufficient. If priapism has lasted longer than 4 hours, then aspiration of the copora cavernosa under local anesthesia with instillation of epinephrine, a mixed α- and β-agonist, has proven to be effective.53 Instillation of phenylephrine, which primarily has α-agonist activity, can be used instead of epinephrine, and might be preferable.52 Simple and exchange transfusion have been employed in the management of acute priapism, but their role is unclear. Seeler54 obtained satisfactory results by transfusing sufficient RBC units to double the hemoglobin concentration. In more severe or prolonged episodes (those lasting longer than 6 to 12 hours), a single-volume exchange transfusion may be considered. If this is successful, relief of pain occurs relatively soon, but complete detumescence may take several days or even weeks. If this approach is unsuccessful, a surgical shunt between the corpora cavernosa and corpus spongiosum may relieve the circulatory obstruction. If surgery is required, an exchange transfusion will have prepared the patient for anesthesia and surgery (see Special Indications for Transfusion). However, acute neurologic events, including seizure and stroke, have been reported in children who have undergone partial exchange transfusion for priapism, and the association between the two has been termed the ASPEN syndrome (association of sickle cell disease, priapism, exchange transfusion, and neurologic events).55 This complication frequently begins with acute onset of severe headache. The etiology of these neurologic complications is not well understood, but it has been speculated that release of vasoactive substances is involved.52 If RBC transfusions are used in the management of acute priapism, it is important to ensure that they not delay the use of more definitive therapies, such as intracavernosal injection, or shunt placement. Hematuria Significant urinary blood loss from acute papillary necrosis in patients with SCD may require transfusions for correction of anemia. It is unclear whether transfusion accelerates healing of the renal papillae in these patients. Acute papillary necrosis has also been described in patients with sickle cell trait. Infection Patients with SCD have impaired host defense function and increased susceptibility to infections, particularly to Streptococcus pneumoniae and Haemophilus influenzae b. In addition, bouts of infection are considerably more severe and protracted in patients

with SCD, compared to normal individuals. Sepsis may hinder erythropoiesis, as well as accelerate hemolysis. In severe infections, the red cell T cryptantigen may be exposed, leading to red cell polyagglutination.56 Transfusions may be indicated in sickle cell patients with sepsis in order to improve oxygen-carrying capacity and to prevent widespread sickling that might result from associated hypoxia or acidosis. Acute Multiorgan Failure Syndrome This relatively uncommon but life-threatening complication typically occurs after an episode of unusually severe pain crisis, and is probably caused by widespread vasoocclusion. Acute failure develops in at least two of three organs: lungs, liver, or kidney. The onset of organ failure is often accompanied by fever, rapid decrease in hemoglobin concentration and platelet count, nonfocal encephalopathy, and rhabdomyolysis.57 It is curious that this catastrophic complication seems to preferentially affect patients who previously had relatively mild disease, relatively high steady-state hemoglobin levels, and no prior chronic organ damage. Aggressive transfusion therapy, either through the use of repeated simple transfusions, and/or exchange transfusions, is associated with improved survival in this severe complication.57,58 Chronic Effects of Sickle Cell Disease Chronic organ damage from SCD produces an additional spectrum of disease manifestations. RBC transfusions play a role in the management of these complications. Cardiac Effects. Cardiomegaly, especially left ventricular hypertrophy, most often reflects chronic anemia, but may also result from sickle cardiomyopathy. Even though the coronary circulation is a site of high oxygen extraction, the incidence of myocardial infarction is diminished in sickle cell patients. Apparently, the short transit time through the coronary circulation is less than the delay time for sickling. Pulmonary Effects. Pulmonary arterial hypertension (PAH) affects about a third of adult patients with SCD, and is associated with a significantly increased risk of death.59 PAH is diagnosed by Doppler echocardiography, with a finding of tricuspid regurgitant jet velocity of at least 2.5 m/second. This condition is strongly associated with increased rates of hemolysis, a finding which supports the pathophysiologic process alluded to previously (see Molecular and Cellular Pathophysiology). Hemolysis is injurious to endothelium, and contributes to increased vascular tone by lowering levels of NO.9 Chronic lung disease with ventilation-perfusion mismatch or reduction in lung volume may result from repeated or severe episodes of pneumonia or pulmonary infarction. Sickle cell patients with the most severe PAH are treated with a variety of medical therapies, including oral selective pulmonary artery vasodilators, prostacyclin analogues, hydroxyurea, and inhaled NO.60 In addition, they are often treated with exchange transfusions.59 Preliminary data indicate that early stages of PAH may be reversed by transfusion therapy.61

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Hepatobiliary Effects. Cholelithiasis occurs in approximately 40% to 50% of patients with SCD by the time they reach young adulthood, but in the majority of cases it is asymptomatic. Cholecystectomy is required for those patients who are symptomatic, but many have managed asymptomatic patients expectantly; some however, do advocate elective cholecystectomy for those with cholelithiasis who are asymptomatic.62 Renal Effects. The renal medulla is a hypertonic, acidotic environment that readily encourages intravascular sickling. Beginning in early childhood, intrarenal ischemia leads to an impaired ability to concentrate urine maximally (hyposthenuria). Infrequently, patients with SCD also develop nephrotic syndrome and overt renal failure. Their anemia may also worsen in the face of frank renal failure. Skeletal Effects. Avascular necrosis of the femoral head produces Legg-Perthes-like radiographic changes, and is a common cause of chronic pain and debilitation in patients with SCD. This complication is associated with higher hemoglobin levels and concomitant presence of α-thalassemia. Sickle-cell-induced infarction of the vertebral bodies is a cause of severe back pain, and is seen as a “fishmouth” appearance of the vertebrae on radiographic examination. Dermal Effects. Adolescent and adult sickle cell patients are prone to develop leg ulcers, usually in the anterior tibial region or adjacent to the medial malleolus. Infection is common, and healing occurs slowly; the recurrence rate is high. When severe, leg ulcers adversely affect mobility, employability, and overall quality of life. When local measures are unsuccessful in the management of severe ulcers, a transfusion program lasting several months may achieve satisfactory results.63 However, the role of transfusion in the management of skin ulcers is debatable.58 Ophthalmic Effects. Patients with SCD are at increased risk for ophthalmic disease, which occurs in numerous forms.64 Nonproliferative lesions, which frequently can be identified by direct ophthalmoscopic examination, affect a variety of structures, including the retina, macula, choroid, and optic disc. Proliferative retinopathy occurs primarily in adolescent or older patients with HbSC or HbS/β⫹-thalassemia, and indirect ophthalmoscopy and fluorescein angiography are used to detect the characteristic retinal vascular changes. In severe cases, there can be progression to vitreous hemorrhage and retinal detachment, and even blindness. However, spontaneous resolution of milder disease occurs quite commonly,65 and so optimal management of proliferative sickle retinopathy is not always clear. Early stages of sickle eye disease may not produce obvious vision changes; therefore, routine examinations are advised.

and death. The fetus is at increased risk for growth retardation, premature birth, stillbirth, and spontaneous abortion. In mothers with HbSS disease, maternal complications and fetal death occur most frequently in the third trimester, whereas fetal death is more frequent in the first trimester in mothers with HbSC disease.66 In 1971, Fort et al67 reported an overall maternal death rate of more than 6% and an infant perinatal mortality rate of approximately 45% in mothers with SCD. These authors concluded that measures should be taken to prevent pregnancies in these women, including sterilization or abortion. Since 1972, the maternal death rate has been less than 2% and the perinatal death rate has been less than 5%.68 Recommendations for the prenatal care of the mother with SCD have included prophylactic transfusion at the onset of pregnancy or at the beginning of the third trimester, prophylactic transfusion only in those pregnancies considered high risk, or obstetric management without transfusion. Many of the earlier studies of transfusion in pregnant women with SCD are difficult to evaluate because of differences in design and the use of historical controls. The most informative study to date is that of Koshy et al.69 In this prospective study, 72 pregnant women with SCD were randomly assigned to receive either prophylactic RBC transfusions or only therapeutically indicated transfusions for defined medical or obstetric emergencies. Those patients assigned to the prophylactic transfusion study arm received an average of 12 RBC units during the course of pregnancy, while nearly one-half of the controls received an average of only 6.5 RBC units. When patients with high-risk factors (ie, multiple gestation or a history of previous perinatal mortality) were excluded, there were no significant differences in maternal and fetal outcome between the prophylactic transfusion and control groups, except (as might be expected) that the number of pain crises in the prophylactic transfusion group was significantly reduced. These findings suggest that prophylactic transfusion therapy may be unnecessary, and better avoided, in pregnant women with SCD who lack underlying high-risk factors. Prophylactic transfusions have been advised for pregnancies in women with SCD who have high-risk features such as multiple gestatation or history of perinatal loss.70 Other indications for transfusion include toxemia, septicemia, acute renal failure, hypoxemia, and ACS.71 It is important to recognize that even when highrisk features are absent, and prophylactic transfusions are avoided, between one-quarter and one-half of pregnant women with SCD will require RBC transfusion on an emergency basis because of major complications.70,72 This should serve as a sobering reminder that all pregnant women with SCD require meticulous care.

Special Indications for Transfusion in Sickle Cell Patients Pregnancy Pregnancy poses special risks to the mother with SCD and to her fetus. Potential maternal complications include an increased frequency of painful vasoocclusive crises, worsening of anemia, increased infection (especially urinary tract infections), toxemia,

Surgery The patient with SCD is at increased risk for perioperative complications including sudden death, pulmonary infarction, infection, and pain crisis. Changes in regional perfusion, accidents of anesthesia (eg, aspiration, difficulty in intubation, etc), or a variety of surgical circumstances may alter oxygenation, hydration,

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Chapter 29: Management of Congenital Hemolytic Anemias

temperature, or acid-base balance and lead to localized or generalized sickle cell vasoocclusion. Some studies have reported favorable surgical results without the use of prophylactic transfusion. Homi et al73 avoided transfusion throughout the entire perioperative period in 137 of 200 sickle cell patients who underwent surgery with general anesthesia. In the remaining patients transfusions were given either to restore the hemoglobin to baseline levels or to combat anticipated or actual blood loss. Six patients died, all during the postoperative period. Detailed transfusion information related to these fatalities was not provided, but five of the six patients had undergone emergency surgery and were in poor physical condition preoperatively. In a nonrandomized study of 66 patients with sickle syndromes (50 HbSS, 13 HbSC, 3 HbS/β thalassemia) who underwent 82 surgical procedures, no major differences in perioperative morbidity and mortality were seen, regardless of whether transfusions were given preoperatively, intraoperatively, postoperatively, or not at all.74 Other groups have routinely employed elective preoperative transfusion with good results. Janik and Seeler75 described 32 patients with HbSS disease who underwent 46 operations following preparative RBC transfusions of 15 to 20 mL/kg, designed to increase the hematocrit to a minimum of 36%. The posttransfusion level of HbS was between 32% and 55%. In this series, there was no reported morbidity or mortality. Prophylactic exchange transfusions designed to achieve hematocrit values of more than 35% and HbA concentrations of more than 40% were administered in another study of 42 patients with SCD.76 There were no reported deaths, and postoperative complications were infrequent and unremarkable; there were no apparent cases of vasoocclusive crisis or pulmonary embolus. Preoperative exchange transfusions have also been given successfully to patients with SCD before vitreoretinal surgery in order to diminish the risk of anterior segment ischemia.77 The Preoperative Transfusion in Sickle Cell Disease Study Group has provided the most comprehensive comparison of conservative and aggressive transfusion regimens. In 1995, this group reported results from a prospective study of 551 patients (a total of 604 operations), some of whom were randomly assigned to one of two transfusion regimens.78 The aggressive transfusion regimen was intended to achieve a HbS concentration of 30% or less, while the conservative transfusion regimen sought to attain a minimum hemoglobin concentration of 10 g/dL. Although some overlap existed in the hematologic features of the two groups, data were analyzed on an intent-to-treat basis. Cholecystectomies, and otolaryngologic and orthopedic procedures accounted for more than three-fourths of the operations performed. A serious or life-threatening complication, most commonly ACS, was experienced by about 20% of the patients in each group. The aggressively transfused group used twice as many RBC units and had twice the alloimmunization rate, in comparison with the conservatively managed group. The study group reported a subset of 364 patients who underwent cholecystectomy, using either traditional (58% of patients)

or laparoscopic techniques (42% of patients).79 In this study, 110 patients were randomly assigned to receive aggressive preoperative transfusion, 120 patients were randomly assigned to receive conservative transfusion, 37 patients were nonrandomly assigned to receive no transfusion, and 97 patients were nonrandomly assigned to receive transfusion. Among all groups combined, the total complication rate was 39%, and sickle cell events occurred in 19%. However, patients who were nonrandomly assigned to receive no transfusion had a sickle cell complication rate of 32%. Patients who underwent laparoscopic procedures had longer anesthesia time but shorter duration of hospitalization, compared with those who had an open abdominal procedure. The incidence of complications did not differ between these two groups. In a review of 118 patients who underwent elective tonsillectomy and adenoidectomy or myringotomy, and who were randomly assigned to either conservative or aggressive preoperative transfusion protocols, the study group found no major differences in complication rates.80 About one-third of all patients experienced serious, nontransfusion-associated complications. In all these reports, the study group concluded that a conservative preoperative transfusion regimen had equivalent efficacy to an aggressive transfusion regimen in preventing perioperative complications. Furthermore, the conservative transfusion regimen was associated with one-half the transfusion-associated complications. Therefore, a conservative preoperative transfusion approach was advocated. However, other interpretations of the data should be considered as well. For example, because of the overlap in percentage of HbS achieved between the aggressively and conservatively transfused groups, the two groups may not have differed from each other sufficiently to produce clinically distinct outcomes. Also, the aggressively transfused group may not have been transfused intensely enough to provide adequate physiologic protection from sickle cell events. The experience from Duke University supports the latter hypothesis: a very low complication rate was experienced by sickle cell patients there who were preoperatively transfused to mean hemoglobin values of 11.2 g/dL and an average of 21% HbS.81 Based upon all these results, the following guidelines would seem to be appropriate for most sickle cell patients who are undergoing surgery: 1. The most critical aspect of perioperative management is meticulous anesthetic and surgical care, avoiding hypoxemia, dehydration, acidosis, and hypothermia. 2. Preoperative RBC transfusions are warranted when prolonged general anesthesia is used or anticipated (ie, when there is reasonable likelihood that local anesthesia will prove to be inadequate). For uncomplicated minor procedures in which brief inhalation anesthetic is used (eg, some dental procedures), transfusion is seldom necessary. 3. The specific method of transfusion—regular or partial exchange—is not as important as the hematologic endpoint. A preoperative HbS of 30% to 40% and hemoglobin concentration in an intermediate range (10 to 12 g/dL) are desirable,

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based upon empirical clinical experience and data cited previously. Special surgical situations characterized by low regional blood flow, especially if in a critical anatomic site (eg, major cardiac, orthopedic, or neurosurgery), may warrant even more aggressive preoperative transfusion. The choice of the method of transfusion largely depends upon the clinical circumstance. When surgery is urgent, a single-volume exchange transfusion (60 to 70 mL/kg) will rapidly achieve the hematologic goal, but when sufficient time is available before elective surgery, repeated regular transfusions over 2 to 4 weeks can be employed, with the attendant benefits of greater technical ease, lower procedural risk, and possibly a lower overall transfusion requirement. 4. In the various compound heterozygous sickle syndromes (with the exception of HbS/β0 thalassemia, which has a hemoglobin pattern similar to that of HbSS disease) it is safest not to monitor the percentage of HbS per se, but to consider the percentage of sicklable cells. 5. In most circumstances no special measures need to be taken in the preoperative management of patients with sickle cell trait. Patients with sickle cell trait probably do not even require transfusion before cardiopulmonary bypass.82 However, prophylactic transfusion may well be appropriate for patients with sickle cell trait who will be undergoing orthopedic surgery requiring prolonged tourniquet application, although this view is not universally held.83 Radiologic Imaging Cerebral or coronary angiography in patients with SCD should be performed only after prophylactic transfusion has achieved a HbS concentration of less than 20% and with adequate hydration.84 Magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA) have largely supplanted catheter angiography for neuroimaging in SCD, and can be safely performed using gadolinium contrast without concern for inducing vasoocclusion.85 Intravenous urography may be performed without preparative transfusion, but vigorous hydration should be maintained both before and after the procedure.

Donor Selection and Transfusion-Related Complications in Patients with Sickle Cell Disease Alloimmunization Sensitization to transfused red cell antigens (alloimmunization) may lead to an inconvenient and costly delay or even a life-threatening inability to find compatible blood. In addition, significant alloimmunization in patients with SCD may result in delayed hemolytic transfusion reactions (DHTRs) that are often serious and occasionally even lethal. Whenever there is a prolonged or repeated requirement for transfusion, it is prudent to obtain a full red cell antigen profile (including, for example, C, E, K, Fya, and Jkb phenotypes) of the recipient before transfusion therapy is initiated. This information will be very useful later if alloimmunization is suspected. Studies of adult and pediatric patients who have been transfused suggest that the overall risk of alloimmunization in SCD is

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about 20% to 30%. Many of these studies, however, were retrospective and did not examine the patients’ transfusion histories before the study. Patients who are frequently transfused appear to be somewhat more likely to develop alloantibodies than those who receive infrequent transfusions. Chronically transfused patients who develop alloantibodies often appear to do so early in their transfusion courses86 and are also more likely to develop multiple alloantibodies. In this regard, patients with SCD appear to be similar to the population at large: there is a subpopulation of “hyperresponders” who readily become alloimmunized, but the majority are nonresponders in spite of numerous transfusions.87 Approximately one-half to two-thirds of alloantibodies identified in patients with HbSS disease have been directed at antigens of the Rh blood group system, and the majority of these are antiRh(E). Anti-Kell (K) and anti-Kidd (Jka and Jkb) account for approximately 20% of alloantibodies in transfused patients with SCD.87 Lewis antibodies account for up to one-third of alloantibodies, but as in the general population these are generally regarded as “naturally occurring” and do not appear to be clinically significant. The impact of racial differences between donors and recipients on the rates of alloimmunization of patients with HbSS disease has been examined in several studies. Overall, approximately 20% to 30% of transfused patients with SCD become alloimmunized, in comparison with 5% to 10% of thalassemia patients who are transfused in the United States.88 The increased incidence of alloimmunization in patients with SCD has been attributed to the greater disparity in race and red cell phenotypic profile that exists between typical blood donors and sickle cell recipients, in comparison with other recipients.88,89 In many urban centers, often 90% to 95% of blood donors are of European ancestry88,89 and often possess a variety of Rh, Duffy, Kell, and Kidd antigens that are less frequently found in recipients of African ancestry.90 Orlina et al89 calculated that a Black recipient has a 33% chance of compatibility for these antigens from a Black donor pool, in comparison with an only 3% chance of compatibility with the typical urban donor pool of 90% White and 10% Black donors. However, 70% to 80% of Black populations are Fy(a–b–), whereas more than 99% of White populations are positive for one or both antigens, yet the frequency of Duffy alloimmunization in transfused sickle cell patients is only 15% or less.87 When the blood donor and recipient belong to different racial groups, the risk of alloimmunization depends not only on whether the two populations have different frequencies of blood group antigens but also on the intrinsic antigenicity of each blood group antigen. Previous recommendations have included selecting donors 1) according to an extended red cell antigen profile, so that compatibility is achieved for Kell and secondary Rh group antigens (ie, C and E), 2) according to race, and 3) according to no special criteria other than the standard ABO group and Rh(D) type. If any special preselection of donors is performed, it should be maintained on a regular basis, because hyperresponders often

Chapter 29: Management of Congenital Hemolytic Anemias

become immunized after only 10 to 20 transfusions. Under most circumstances, it is not cost effective or feasible to preselect donors for sickle cell patients. Routine donors are usually acceptable. If a patient with SCD has clinically significant alloantibodies, then specific compatibility testing must, of course, be performed. Because of the existence of hyperresponders, it is advisable to match as closely as possible all individuals who have developed one or more alloantibodies. In such cases, the search for compatible blood is hastened by screening units obtained from same-race donors.88 Delayed Hemolytic Transfusion Reaction This complication is caused by acute destruction of transfused red cells beginning several days after transfusion. It is typically caused by an alloantibody to a blood group antigen produced in anamnestic fashion in a previously sensitized patient. Clinically, delayed hemolytic transfusion reaction (DHTR) is characterized by fever and a precipitous decrease in hemoglobin, often to levels lower than those that existed before transfusion. Hemoglobinuria is often seen, resulting from intravascular hemolysis. DHTR has distinctive features when present in patients with SCD, and thus it may even constitute a unique syndrome in this setting.91 It occurs in about 5% to 20% of regularly transfused patients with SCD, an incidence considerably higher than that seen in other multitransfused populations.92 Pain is commonly experienced by sickle cell patients with DHTR and it may be readily confused with that from vasoocclusive crisis. Marked reticulocytopenia is occasionally seen, which contributes to the anemia. When DHTR is suspected, serial hemoglobin fractionation may be a useful means to document alloimmune hemolysis, by showing a rapid fall in donor-cell-derived HbA. However, although hemolysis of donor red cells is typically the principal concern, destruction of autologous cells can occur as well through a phenomenon that has been referred to as “bystander hemolysis.”93 In this process, antigen-negative red cells are destroyed by autoantibodies with an indeterminate specificity, or by deposition of antigen-antibody complexes on red cell membranes, causing complement activation and lysis of red cells. These reactions are an especially serious threat, because this potentially life-threatening complication is not always prevented by the use of phenotypically matched blood. Corticosteroids, intravenous immune globulin, and erythropoietin have been used to treat this complication. In the face of a DHTR, additional transfusion should be avoided whenever possible in order to prevent exacerbation of the hemolytic process. Blood from Sickle Cell Trait Donors for Patients without Sickle Cell Disease The oxygen-carrying capacity of HbAS blood is comparable to that of normal (HbAA) blood, and in the great majority of general, elective transfusion settings, HbAS blood may be transfused without any untoward consequence. However, this blood should be avoided in neonates (especially ventilator-dependent premature infants), in patients with severe cardiopulmonary illness or shock, and in those who require high-volume

blood transfusions (eg, in cases of severe acute hemorrhage). All of these conditions are sometimes associated with hypoxemia and acidosis severe enough to cause sickling of sickle trait erythrocytes. Leukocyte reduction filters often are unsuccessful, and HbAS erythrocytes sometimes hemolyze during the process of deglycerolization.94,95 Accordingly, RBC units destined for leukocyte reduction or frozen storage should be screened to detect, and possibly eliminate, units containing sickle cell trait. Because HbAS blood is acceptable for routine transfusion to individuals without sickle cell disease, there should be no objection to using autologous transfusion in patients with sickle cell trait, as long as none of the risk factors cited above is present.96 Blood from Sickle Cell Trait Donors for Patients with Sickle Cell Disease Blood obtained from donors with sickle cell trait is undesirable for use in patients with SCD, primarily because the resultant mixture of HbSS and HbAS cells will obfuscate hemoglobin fractionation results that are generally used to monitor the progress of transfusion therapy. In addition, in nearly all sickle cell conditions requiring transfusion therapy, there is a danger of progressive sickling. As noted above, even HbAS blood can sickle under severely unfavorable physiologic conditions and thus should be avoided.

Thalassemias Epidemiology The frequency of thalassemia genes in world populations is surprisingly high, with the thalassemias in aggregate representing the most prevalent of all known genetic diseases.97 Occasional individuals with thalassemia have been observed in native populations in virtually all areas of the world where they have been sought, but most cases are concentrated within a broad subtropical “thalassemia belt” extending from Western Europe to Southeast Asia (Fig 29-1). Within this region there are areas with especially high frequencies of thalassemia genes, with as many as 50% of some populations having one or more forms of thalassemia. The characterization in recent years of the underlying mutations of the thalassemia syndromes has added considerably to our understanding of the population genetics of these disorders. In general, each of the geographic areas with a high prevalence of thalassemia, particularly the β form, has a characteristic and often unique group of thalassemia mutations. In most of these populations, a small number of mutations account for most of the observed cases. These mutations probably arose independently in each population, and they achieved high frequency because of strong selective pressure. There is considerable information suggesting that resistance to P. falciparum malaria is the selective force responsible for the high frequency of thalassemia. One line of evidence in support of this hypothesis is the geographic concurrence of the “thalassemia belt” and the areas where malaria was formerly endemic (Fig 29-1).

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Molecular Basis for the Thalassemia Syndromes More than 200 distinct thalassemia mutations are now known. A summary of the major categories of these molecular abnormalities is shown in Table 29-3; a detailed description is given elsewhere.1(pp194-195) In spite of the considerable diversity of forms and sites of mutations in the globin genes, as well as the variety of mechanisms by which they lead to inhibition of globin-chain synthesis, the clinical phenotypes of most forms of thalassemia are similar (see below). For most clinical purposes, therefore, the identification of the specific mutations of thalassemia patients is of little importance. However, for prenatal diagnosis, and because of the potential for ameliorating the effects of thalassemia genes, this information may eventually assume fundamental significance. Pathophysiology In most of the symptomatic forms of thalassemia, the pathophysiology follows the same mechanism. The pathogenesis of homozygous β-thalassemia (“Cooley anemia”) serves as a representative example. Marrow erythroid cells of affected individuals exhibit a diminution or absence of hemoglobin β-chain synthesis. Although this defect is partially compensated for by an increase in the synthesis of HbF γ chains, a major disparity nevertheless exists between α-chain and non-α-chain synthesis (Fig 29-6), with αchain synthesis occurring in considerable excess. The deficiency of β and γ chains results in severe underhemoglobinization of the red cells because of insufficient quantities of HbA (α2 β2) or HbF (α2γ2). The excess of uncombined α chains that accumulate in the red cells of these patients is also responsible for other deleterious effects. Uncombined α subunits are unstable and precipitate within erythrocytes. This process, which is accompanied by oxidation of heme iron, releases superoxide ions, which in turn inflict oxidative damage on intracellular, and particularly membrane, components of the red cell.98,99 In severe forms of β-thalassemia, the latter process results in destruction of a major fraction of erythroid cells, even before they are released from the marrow, producing what has been termed “ineffective erythropoiesis.” In compensation for this process, these patients

develop an enormous expansion of their marrow, a change that produces characteristic bony deformities most apparent in the skull and face. The resulting anemia in the most severe forms of homozygous β-thalassemia (β0) is incompatible with long-term survival unless periodic transfusions are given.

Compound Heterozygous Forms In geographic areas with high frequencies of thalassemia, both α- and β-thalassemia alleles may coexist in the same population, and both types of abnormalities may therefore occur in the same individual. The combination of homozygous β0-thalassemia with concomitant α-thalassemia produces a clinically mild form of

␥

␤

␣ ␣ non-␣ ⫽ 1.0

Normal

␥

␤

␣ ␣ non-␣ ⫽ 0.3

␣-thalassemia (Hb H disease)

␥ ␤-thalassemia (Homozygous ␤⬚)

␣ ␣ non-␣ ⫽ 3.5

Globin protein Radioactive amino acid incorporation Figure 29-6. Synthesis of globin chains by erythroid cells from a normal individual (top), a patient with α-thalassemia (middle), and a patient with severe β0-thalassemia (bottom).1(p192)

Table 29-3. Examples of Molecular Abnormalities of Thalassemia Genes Abnormality

Consequence

Globin-gene deletions, partial or complete Nucleotide substitutions in the promoter regions Nucleotide substitutions or deletions at splice-junction sites Nucleotide substitutions in the coding segments or introns Nucleotide substitutions in the transcription-termination signal region Nucleotide substitutions in translation-initiation codons Nucleotide deletions or insertions Nucleotide substitutions in the translation-termination codons Mutations that produce highly unstable globin chains

Absence of globin synthesis; formation of fusion genes; elevated expression of HbF Inhibition of globin-gene transcription Prevention of the formation of normal globin messenger RNA (mRNA) Abnormally spliced globin mRNA Formation of abnormal globin mRNA Inhibition of mRNA translation Formation of premature termination codons Production of extended-length globin chains Rapid degradation of globin chains within the erythrocytes

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Cooley anemia,100 undoubtedly because a more nearly normal balance is achieved between α-chain and β-chain synthesis, so that the harmful series of events described above is attenuated. Hemoglobin E, an abnormal hemoglobin prevalent in Southeast Asia, produces thalassemia-like hematologic effects, possibly by two separate mechanisms. First, HbE is an unstable variant and may undergo β-chain loss by intracellular precipitation or degradation or both. Second, the HbE mutation produces an aberrant splicing site, leading to a decreased production of βE messenger RNA (mRNA).101 Large series of patients with the compound heterozygous combination of HbE and β0 thalassemia have been reported.102 This syndrome has variable expression, with more severely affected individuals requiring periodic transfusions to maintain adequate hemoglobin levels.

Homozygous β-Thalassemia (Cooley Anemia) Clinical Features and Natural History The clinical and hematologic features of the major forms of βthalassemia are summarized in Table 29-4. Because the molecular abnormalities that cause these disorders are within, or in close proximity to, the β-globin genes, the onset of expression of β-thalassemia occurs some months after birth when HbF is replaced by HbA. Affected infants are therefore normal at birth, and evidence of the disease first appears by the middle or end of the first year. Anemia gradually develops, accompanied by progressive enlargement of the liver and spleen and cardiovascular changes related to congestive heart failure. A large fraction of the cardiac output is shunted through the hypertrophied marrow of untransfused thalassemia patients and this change contributes to an expansion of the plasma volume, resulting in increasing

hemodilution and worsening cardiac failure. Before the introduction of modern transfusion methods, the size of the spleen and the liver in these patients progressed to a massive degree, primarily because of reticuloendothelial cell proliferation. Hyperexpanded marrow also results in a thinning of cortical bone, predisposing these children to fractures. Craniofacial bone deformities are variable in degree but often severe, frequently resulting in considerable malocclusion caused by maxillary hyperplasia. In the absence of transfusion support, children with severe β-thalassemia succumb to the effects of anemia and heart failure within the first years of life. Milder forms of β-thalassemia are accompanied by a slower rate of progression and correspondingly longer survival. Transfusion Therapy for Patients with β-Thalassemia The earliest transfusion regimens for patients with β-thalassemia consisted of administering mainly whole blood as infrequently as possible and only when severe anemia developed.102 This form of management protected these children from early death from progressive heart failure but did not mitigate the full expression of the disease. A critical turning point in the approach to the transfusion management of β-thalassemia patients came in 1964, when reports by Schorr and Radel103 and Wolman104 compared the clinical status of patients whose hemoglobin levels had been maintained at different levels. The principal finding was that children with thalassemia fared better if their hemoglobin levels were maintained above 8 g/dL. Compared with other β-thalassemia patients, the group with a high hemoglobin level exhibited more normal growth, a lesser extent of liver and spleen enlargement, less bony deformity and orthodontic abnormalities,

Table 29-4. Clinical and Hematologic Features of the Major Forms of β Thalassemia Type

Hemoglobin Findings

Hematologic Changes

Clinical Features

Heterozygous β0 (severe) (high A2)

A2, 3.5-7.5% F, 1-6%

Possible splenomegaly and mild icterus

β⫹ (mild) (high A2)

A2, 3.5-7.5%

β silent carrier

δβ (high F)

A2 and F, normal (F-containing cells sometimes detectable by slide elution test) A2, normal or low F, 5-20%

Erythrocyte microcytosis and hypochromia; mild to moderate anemia Erythrocyte microcytosis and hypochromia; mild or absent anemia Hematologically normal

Erythrocyte microcytosis and hypochromia; mild or absent anemia

Usually none

Homozygous β⫹

F, 30-95%

β⫺

F, 40-80%

δβ (high F)

F, 100%

Markedly abnormal red cell morphology with microcytosis and hypochromia, nucleated red cells; severe anemia Poikilocytosis, anisocytosis, target cells; moderate anemia Poikilocytosis, anisocytosis, hypochromia,microcytosis; microcytosis; mild to moderate anemia

Pallor, jaundice, bone deformities with abnormal facies, hepatosplenomegaly, usually transfusion-dependent Pallor, hepatosplenomegaly, jaundice; transfusions not usually required Mild jaundice, hepatosplenomegaly usually present

Usually none None

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and a considerably lower frequency of cardiac enlargement. Although these patients had received a larger number of transfusions and had correspondingly higher levels of iron stores, their considerably improved clinical status demonstrated that, at least in younger children, the hemoglobin level was more important than iron overload as a determinant of clinical status.104 The findings from these studies led rapidly to the introduction of transfusion regimens that sought to maintain higher hemoglobin levels, usually higher than 9.5 or 10 g/dL. This approach, frequently referred to as “hypertransfusion,” usually consists of RBC transfusions of about 20 mL/kg, given whenever the patient’s hemoglobin falls below the targeted level. Once these patients are fully transfused, the requirement to maintain them on the hypertransfusion regimen is no greater than that needed to maintain patients at a lower hemoglobin level.105 Immediate posttransfusion hemoglobin levels typically average 14 g/dL, returning to baseline after 3 to 6 weeks. Hypertransfusion is also accompanied by a reduction in gastrointestinal absorbance of iron.106 The rate of iron accumulation can therefore be lessened while maintaining a more desirable hemoglobin level. In addition to improved clinical status, the correction of anemia by hypertransfusion also exerts a suppressive effect on the erythropoietic drive that causes marrow hypertrophy. The bony abnormalities, which in the past were so characteristic of this disease, are now entirely preventable,107 and these patients now experience no increased risk of fractures. In addition, the marrow suppressive effect of hypertransfusion prevents the release of the deformed, short-lived Cooley anemia erythrocytes and delays the onset of hypersplenic changes, permitting splenectomy to be postponed safely until an older age. Appropriately transfused patients with Cooley anemia gradually experience increasing splenomegaly caused by reticuloendothelial proliferation. Although this process is considerably delayed by hypertransfusion, it cannot be totally prevented. The development of hypersplenism shortens the transfusion interval and increases transfusion requirements. Piomelli and Loew108 have suggested that RBC transfusion of more than 200 mL/kg per year in these patients is usually an indication for splenectomy. Propper et al109 have proposed a transfusion regimen for Cooley anemia that maintains the hematocrit above 35%, but this “supertransfusion” approach is no longer recommended.97 Indeed, moderate transfusion regimens that target a baseline hemoglobin level of 9 to 10 g/dL appear to offer the best combination of preventing the long-term adverse consequences of anemia and excessive erythropoiesis, while minimizing iron accumulation.110 Individuals with heterozygous β-thalassemia (β-thalassemia trait) ordinarily have no special need for transfusions. An exception is during pregnancy, when women with thalassemia trait often develop more severe anemia111 and occasionally require transfusion support.112 Those who have inherited a combination of both mild and more severe β-thalassemia mutations, termed thalassemia intermedia, exhibit a variable clinical course; some experience a mild clinical course that rarely requires intervention,

464

while others may require regular RBC transfusions, at least for some period.113

α-Thalassemia Wasi et al,102 in summarizing their experience in Thailand with a large number of patients with HbH disease (Table 29-5), showed that this syndrome often produced Cooley anemia-like changes, with anemia, bony deformities, and splenomegaly. However, these changes appear to reflect, in part, the effects of infections and other environmental factors superimposed upon the genetic abnormality. HbH disease in the United States generally exhibits a relatively mild thalassemia-intermedia-like syndrome and seldom requires blood transfusion except during surgery. The hydrops fetalis form of α-thalassemia, in which α-chain synthesis is totally lacking, has until recent years been invariably fatal, the infants surviving no longer than a few hours after birth. A few recent examples have been reported of such infants who received vigorous resuscitative measures, including transfusions at birth, with survival beyond the neonatal period.114 This syndrome is characterized by the production almost exclusively of homotetramers of γ and β chains (Table 29-5). These abnormal hemoglobins have virtually no physiologically useful oxygen-transporting capacity because of their very high oxygen affinity. Infants who survive are therefore at great risk of developing hypoxic encephalopathy during the neonatal period and are totally dependent on transfused RBC units throughout their lives.

Effects and Management of Chronic Transfusion Therapy Hemosiderosis Each RBC unit contains approximately 250 mg of iron. Unfortunately, physiologic mechanisms for the elimination of excess iron are lacking. Signs of clinical toxicity often become apparent when total body iron reaches 400 to 1000 mg/kg of body weight, and levels in excess of this are potentially lethal.115 Once reticuloendothelial sites of iron storage become saturated, parenchymal deposition increases and tissue damage ensues. The primary targets of iron toxicity are the heart, liver, pancreas, and other endocrine organs. Hepatotoxicity, expressed initially by fibrosis and subsequently cirrhosis, is the most common early manifestation of transfusion-related iron overload. Cardiac toxicity, causing cardiomyopathy and arrhythmia, has been the most frequent cause of death in chronically transfused patients. The most practical measurement of body iron stores is the serum ferritin concentration, which is in equilibrium with storage iron up to a level of approximately 4000 µg/L.115 However, in the face of a high total body iron burden, and in a variety of disease states (eg, acute inflammatory conditions, hepatocellular damage), the reliability of serum ferritin as a marker for the total body iron burden diminishes. Serum ferritin is probably even less reliable as a marker of total body iron in patients with SCD.116 The hepatic iron concentration determined from liver biopsy specimens is an excellent indicator of total body iron stores in

Chapter 29: Management of Congenital Hemolytic Anemias Table 29-5. Clinical and Hematologic Features of α Thalassemia Form of α Thalassemia

Genotype

Silent carrier

Hemoglobin Findings

Hematologic Changes

Clinical Features

Clinically normal

Newborn

Adults

αα/α–

1-2% Hb γ4 (Bart’s)

No abnormality

Usually no abnormality; mild microcytosis may be present

α-Thalassemia trait

αα/–– or α–/–α–

2-10% Hb γ4

No abnormality

Red cell microcytosis and hypochromia Usually normal with mildly abnormal morphology; anemia mild or absent

HbH disease

α–/––

20-30% Hb γ4

5-25% Hb H (β4); may be traces of Hb γ4

Moderately severe anemia with Pallor, jaundice marked anisopoikilocytosis and hepatosplenomegaly; microcytosis; red cell inclusion bodies cholelithiasis occurs demonstrable by supravital staining commonly in adults

Hydrops fetalis

––/––

80% Hb γ4, with the remainder Hb β4 and Hb Portland (ζ2γ2)

—

Severe anemia, markedly abnormal erythrocyte morphology with anisopoikilocytosis, hypochromia, and pronounced erythroblastemia

patients with thalassemia major.116 Despite the relative difficulty in obtaining such specimens, this method is recommended for the reliable measurement of the total amount iron stored in the body.117 A superconducting quantum interference device susceptometer measures body iron noninvasively and produces results equivalent to those obtained by liver biopsy, but this method is available in just a few centers worldwide.116 MRI has been used to assess levels of iron in the liver, has recently been standardized, and may soon become the optimal method for the indirect measurement of iron in the liver.118 MRI measurement of iron in the heart has also been studied, but this technique has not been fully standardized.116,117 When faithfully administered, treatment with daily subcutaneous deferoxamine has been shown to arrest or even reverse hepatic119 and cardiac120 iron toxicity in thalassemia patients, especially when started early in the course of transfusion therapy. This drug has also been effective in patients with sickle cell anemia.121 Deferoxamine is usually prescribed in a daily dose of 50 to 75 mg/kg of body weight, administered subcutaneously over 8 to 12 hours (usually overnight) via a portable infusion pump. Because of the parenteral route of administration and the special measures required to use the pump, this agent is cumbersome to administer. The infusion may also be painful, especially for children. Not surprisingly, compliance is often poor, thus limiting the effectiveness of this therapy. Oral iron chelators have been developed that offer the promise of far greater ease of administration, and hopefully, improved compliance. Deferiprone has been studied for over 20 years, and is currently available in Europe. Concerns were initially raised that deferiprone could be linked to an increased incidence of hepatic fibrosis, but further studies appear to have relieved that concern. Its main toxicity appears to be agranulocytosis, and so patients

Massive hepatosplenomegaly, generalized edema with ascites and pleural and pericardial effusion; nearly all are stillborn or die shortly after birth

receiving this medication should undergo frequent blood counts. Deferiprone may have only partial efficacy in reducing liver iron concentrations, but it actually might be superior to deferoxamine in lowering cardiac iron.116 Deferasirox is the first oral iron chelator licensed in the United States, and is almost exclusively excreted in the bile, in contrast to deferoxamine and deferasirox, which are primarily excreted in the urine. Deferasirox is generally well tolerated, but it may cause nephrotoxicity. Studies to date have demonstrated that deferasirox is effective in reducing liver iron content, while only preliminary data show that it can lower cardiac iron. Both oral iron chelators, deferiprone and deferasirox, appear to be more effective than deferoxamine in binding to intracellular iron, while deferoxamine may have an advantage in chelating extracellular iron. Attempts have been made to combine therapy with both deferoxamine and either deferiprone or deferasirox. Early data seem to suggest that this type of combination indeed offers some additive benefit. The transfusion regimen employed for the long-term management of chronically transfused patients can significantly affect the rate of iron accumulation. In sickle cell patients who require protracted courses of transfusion, exchange procedures can considerably decrease the rate of accumulation.122

Erythrocyte Enzyme Abnormalities Enzyme-mediated metabolic pathways in the red cell function to maintain the hemoglobin and the cell membrane in a reduced state. These objectives are accomplished primarily through the reactions linked to the pentose phosphate shunt and, for metabolic energy requirements, through the glycolytic pathway. Many enzyme deficiencies in these metabolic pathways have

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been described. The majority are inherited as autosomal recessive abnormalities. Two exceptions—glucose-6-phosphate dehydrogenase (G6PD) deficiency and phosphoglycerate kinase deficiency— are transmitted in an X-linked recessive mode. G6PD deficiency is prevalent in the same geographic areas in which there is a high frequency of hemoglobin disorders. In common with many of the hemoglobinopathies and thalassemia syndromes, hemizygous carriers of G6PD abnormalities are more resistant to P. falciparum malaria. Two common clinical forms of G6PD deficiency are the African variant, designated GdA-, and the Mediterranean variant, GdMediterranean. Both are characterized by an accelerated age-dependent decrease in red cell G6PD activity. In the cells of affected males or homozygous females with the African GdA- variant, there is a comparatively slow decline in enzyme activity with age, so that only a minority of cells, the most senescent, are substantially lacking in enzyme activity. The GdMediterranean variant has much less enzyme activity, and in affected males even newly released reticulocytes have a substantial deficiency in enzyme activity; the mature circulating erythrocytes are essentially devoid of measurable G6PD activity. G6PD catalyzes the initial step in the pentose phosphate shunt pathway, which is essential in the mature red cell for the regeneration of reduced nicotinamide adenine dinucleotide phosphate (NADPH) from nicotinamide adenine dinucleotide phosphate (NADP). The reduced form is a required cofactor for the enzyme glutathione reductase, which together with glutathione peroxidase protects the hemoglobin, the supporting structures, and the enzymes of the red cell against oxidative damage. In the face of G6PD deficiency, erythrocytes may be extremely vulnerable to oxidant stress, resulting from exposure to certain drugs, chemicals, and infectious agents. During periods of oxidant stress, the hemoglobin may undergo intracellular precipitation to form Heinz bodies. These precipitates, which become associated with the cell membrane, are normally removed in the spleen, but in the process the cell may become damaged and undergo rapid destruction. Both the African and the Mediterranean types of G6PD deficiency are associated with little or no hematologic abnormality under normal circumstances. In individuals with the African variant, only a comparatively small fraction of circulating red cells are significantly deficient in G6PD at any given time, and even though severe hemolysis and anemia may result from an episode of oxidative stress, the process is typically self-limited. The younger population of red cells that remains after hemolysis has occurred retains a higher level of enzyme activity, and these cells are thus relatively protected from further oxidative challenge. In individuals with the Mediterranean variant, all of the circulating erythrocytes are severely G6PD deficient and hence susceptible to oxidant challenge. Patients with this variant are at risk for acute, life-threatening hemolysis if exposed to a significant oxidant challenge. Transfusions are often lifesaving during episodes of acute hemolysis. Pyruvate kinase (PK) deficiency occurs most often in northern Europeans and is the most prevalent enzyme deficiency

466

disorder affecting the glycolytic pathway. Impaired synthesis of adenosine triphosphate in PK-deficient red cells results in potassium depletion, intracellular dehydration, and impaired passage of the erythrocytes through the spleen. Premature destruction ensues in the spleen, liver, or marrow, resulting in variable degrees of anemia. In severe forms of the disease, splenectomy may ameliorate the degree of hemolysis, but ongoing transfusion support may nevertheless be required to maintain an adequate hemoglobin level in these patients.

Disorders of the Red Cell Membrane The primary functions of the red cell membrane are to maintain the structural integrity of the cell and to regulate cation transport and permeability. The most prevalent of the red cell membrane disorders include hereditary spherocytosis (HS) and hereditary elliptocytosis (HE). Rarer membrane-related disorders include a group of hereditary stomatocytosis syndromes and hereditary pyropoikilocytosis. Hereditary spherocytosis predominates in individuals of northern European extraction, whereas HE has been found in numerous population groups. There are numerous variants of each of these disorders, and in most cases inheritance follows an autosomal dominant pattern. The extent of hemolysis in HS and HE varies considerably among the different variants. In some cases hemolysis is fully compensated and barely perceptible, whereas in more severe forms anemia may be pronounced. The underlying defects in these disorders are structural abnormalities of the red cell membrane cytoskeleton, but this alone is not the direct cause of hemolysis. Rather, the misshapen cells have impaired deformability and difficulty in traversing the splenic circulation, and undergo destruction within the spleen. Accordingly, splenectomy is frequently effective in ameliorating the hemolysis and the anemia, especially in HS. Before splenectomy, patients with these hemolytic disorders are also at risk of developing acute episodes of aplasia accompanied by reticulocytopenia and a rapid decrease in hemoglobin levels. These episodes typically follow a trivial viral illness, and they have been linked to the same parvovirus agent that causes aplastic episodes in patients with SCD.25 During these aplastic crises, as in sickle cell disease, RBC transfusions may be required to sustain life.

Acknowledgment The author gratefully acknowledges the contributions of Dr. George R. Honig, who co-authored previous editions of this chapter.

Disclaimer The author has disclosed no conflicts of interest.

Chapter 29: Management of Congenital Hemolytic Anemias

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transcranial Doppler testing and chronic transfusion. Pediatrics 2007;120:e1107-16. Zimmerman SA, Schultz WH, Burgett S, et al. Hydroxyurea lowers transcranial Doppler flow velocities in children with sickle cell anemia. Blood 2007;110:1043-7. Bernaudin F, Verlhac S, Coic L, et al. Long-term follow-up of pediatric sickle cell disease patients with abnormal high velocities on transcranial Doppler. Pediatr Radiol 2005;35:242-8. Johnson CS. The acute chest syndrome. Hematol Oncol Clin North Am 2005;19:857-79. Vichinsky EP, Neumayr LD, Earles AN, et al. Causes and outcomes of the acute chest syndrome in sickle cell disease. N Engl J Med 2000;342:1855-65. Styles LA, Schalkwijk CG, Aarsman AJ, et al. Phospholipase A2 levels in acute chest syndrome of sickle cell disease. Blood 1996;87:2573-8. Styles LA, Aarsman AJ, Vichinsky EP, et al. Secretory phospholipase A2 predicts impending acute chest syndrome in sickle cell disease. Blood 2000;96:3276-8. Bargoma EM, Mitsuyoshi JK, Larkin SK, et al. Serum C-reactive protein parallels secretory phospholipase A2 in sickle cell disease patients with vasoocclusive crisis or acute chest syndrome (letter). Blood 2005;105:3384-5. Mantadakis E, Don Cavender J, Rogers Z, et al. Prevalence of priapism in children and adolescents with sickle cell anemia. J Pediatr Hematol Oncol 1999;21:518-22. Nolan VG, Wyszynski DF, Farrer LA, et al. Hemolysis-associated priapism in sickle cell disease. Blood 2005;106:3264-7. Rogers ZR. Priapism in sickle cell disease. Hematol Oncol Clin North Am 2005;19:917-28. Mantadakis E, Ewalt DH, Don Cavender J, et al. Outpatient penile aspiration and epinephrine irrigation for young patients with sickle cell anemia and prolonged priapism. Blood 2000;95:78-82. Seeler RA. Intensive transfusion therapy for priapism in boys with sickle cell anemia. J Urol 1973;110:360-1. Siegel JF, Rich MA, Brock WA. Association of sickle cell disease, priapism, exchange transfusion and neurological events: ASPEN syndrome. J Urol 1993;150:1480-2. Weisz-Carrington P. Principles of clinical immunohematology. Chicago: Year Book, 1986:183. Hassel KL, Eckman JR, Lane PA. Acute multiorgan failure syndrome: A potentially catastrophic complication of severe sickle cell pain episodes. Am J Med 1994;96:155-62. Josephson CD, Su LL, Hillyer KL, et al. Transfusion in the patient with sickle cell disease: A critical review of the literature and transfusion guidelines. Transfus Med Rev 2007;21:118-33. Gladwin MT, Sachdev V, Jison ML, et al. Pulmonary hypertension as a risk factor for death in patients with sickle cell disease. N Engl J Med 2004;350:886-95. Castro O, Gladwin T. Pulmonary hypertension in sickle cell disease: Mechanisms, diagnosis, and management. Hematol Oncol Clin North Am 2005;19:881-96. Vichinsky EP. Pulmonary hypertension in sickle cell disease. N Engl J Med 2004;350:857-9. Curro G, Meo A, Ippolito D, et al. Asymptomatic cholelithiasis in children with sickle cell disease. Ann Surg 2007;245:126-9. Charache S, Lubin B, Reid CD. Management and therapy of sickle cell disease. NIH publication 84-2117. Bethesda: US Department of Health and Human Services, 1984.

64. Emerson GG, Lutty GA. Effects of sickle cell disease on the eye: Clinical features and treatment. Hematol Oncol Clin North Am 2005;19:957-73. 65. Downes SM, Hambleton IR, Chuang E, et al. Incidence and natural history of proliferative sickle retinopathy: Observations from a cohort study. Ophthalmology 2005;112:1869-75. 66. Powars DR, Sandhu M, Niland-Weiss J, et al. Pregnancy in sickle cell disease. Obstet Gynecol 1986;67:217-28. 67. Fort AT, Morrison JC, Berreras L, et al. Counseling the patient with sickle cell disease about reproduction: Pregnancy outcome does not justify the maternal risk! Am J Obstet Gynecol 1971;111:324-7. 68. Hassell K. Pregnancy and sickle cell disease. Hematol Oncol Clin North Am 2005;19:903-16. 69. Koshy M, Burd L, Wallace D, et al. Prophylactic red-cell transfusions in pregnant patients with sickle cell disease. N Engl J Med 1988;319:1447-52. 70. Seoud MAF, Cantwell C, Nobles G, et al. Outcome of pregnancies complicated by sickle cell and sickle-C hemoglobinopathies. Am J Perinatol 1994;11:187-91. 71. Koshy M, Burd L. Management of pregnancy in sickle cell syndromes. Hematol Oncol Clin North Am 1991;5:585-96. 72. Howard RJ, Tuck SM, Pearson TC. Pregnancy in sickle cell disease in the UK: Results of a multicentre survey of the effect of prophylactic blood transfusion on maternal and fetal outcome. Br J Obstet Gynecol 1995;102:947-51. 73. Homi J, Reynolds J, Skinner A, et al. General anesthesia in sickle cell disease. Br Med J 1979;1:1599-601. 74. Bischoff RJ, Williamson A, Dalali MJ, et al. Assessment of the use of transfusion therapy perioperatively in patients with sickle cell hemoglobinopathies. Ann Surg 1988;207:434-8. 75. Janik J, Seeler RA. Perioperative management of children with sickle hemoglobinopathy. J Pediatr Surg 1980;15:117-20. 76. Morrison JC, Whybrew WD, Bucovaz ET. Use of partial exchange transfusion preoperatively in patients with sickle cell hemoglobinopathies. Am J Obstet Gynecol 1978;132:59-63. 77. Jampol LM, Green JL, Goldberg MF, et al. An update on vitrectomy surgery and retinal detachment repair in sickle cell disease. Arch Ophthalmol 1982;100:591-3. 78. Vichinsky EP, Haberkern CM, Neumayr L, et al. A comparison of conservative and aggressive transfusion regimens in the perioperative management of sickle cell disease. Preoperative Transfusion in Sickle Cell Disease Study Group. N Engl J Med 1995;333:206-13. 79. Haberkern CM, Neumayr LD, Orringer EP, et al. Cholecystectomy in sickle cell anemia patients: Perioperative outcome of 364 cases from the National Preoperative Transfusion Study. Preoperative Transfusion in Sickle Cell Disease Study Group. Blood 1997;89:1533-42. 80. Waldon P, Pegelow C, Neumayr L, et al. Tonsillectomy, adenoidectomy, and myringotomy in sickle cell disease: Perioperative morbidity. Preoperative Transfusion in Sickle Cell Disease Study Group. J Pediatr Hematol Oncol 1999;21:129-35. 81. Adams DM, Ware RE, Schultz WH, et al. Successful surgical outcome in children with sickle hemoglobinopathies: The Duke University experience. J Pediatr Surg 1998;33:428-32. 82. Warltier DC. Sickle cell disease and anesthesia. Anesthesiology 2004;101:766-85. 83. Eichhorn JH. Preoperative screening for sickle cell trait (editorial). JAMA 1988;259:907.

Chapter 29: Management of Congenital Hemolytic Anemias

84. Stockman JA, Nigro MA, Mishkin MM, et al. Occlusion of large cerebral vessels in sickle-cell anemia. N Engl J Med 1972;287:846-9. 85. Zimmerman RA. MRI/MRA evaluation of sickle cell disease of the brain. Pediatr Radiol 2005;35:249-57. 86. Blumberg N, Ross K, Avila E, et al. Should chronic transfusions be matched for antigens other than ABO and Rh0(D)? Vox Sang 1984;47:205-8. 87. Alarif L, Castro O, Ofosu M, et al. HLA-B35 is associated with red cell alloimmunization in sickle cell disease. Clin Immunol Immunopathol 1986;38:178-83. 88. Vichinsky EP, Earles A, Johnson RA, et al. Alloimmunization in sickle cell anemia and transfusion of racially unmatched blood. N Engl J Med 1990;322:1617-21. 89. Orlina AR, Sosler SD, Koshy M. Problems of chronic transfusion in sickle cell disease. J Clin Apheresis 1991;6:234-40. 90. Sosler SD, Jilly BJ, Saporito C, et al. A simple, practical model for reducing alloimmunization in patients with sickle cell disease. Am J Hematol 1993;43:103-6. 91. Petz LD, Calhoun L, Shulman IA, et al. The sickle cell hemolytic transfusion reaction syndrome. Transfusion 1997;37:382-92. 92. Garratty G. Severe reactions associated with transfusion of patients with sickle cell disease (Editorial). Transfusion 1997;37:357-61. 93. King KE, Shirey RS, Lankiewicz MW, et al. Delayed hemolytic transfusion reactions in sickle cell disease: Simultaneous destruction of recipients’ red cells. Transfusion 1997;37:376-81. 94. Brecher ME, ed. Technical manual. 15th edition. Bethesda, MD: AABB, 2005:192. 95. Ould amar AK. Red blood cells from donors with sickle cell trait: A safety issue for transfusion? Transfus Med 2006;16:248-53. 96. Romanoff, ME, Woodward DG, Bullard WG. Autologous blood transfusion in patients with sickle cell trait. Anesthesiology 1988;68:820-1. 97. Olivieri N. Medical progress: The β-thalassemias. N Engl J Med 1999;341:99-109. 98. Rachmilewitz EA. Denaturation of the normal and abnormal hemoglobin molecule. Semin Hematol 1974;11:441-62. 99. Rund D, Rachmilewitz E. β-Thalassemia. N Engl J Med 2005;353: 1135-46. 100. Furbetta M, Tuveri T, Rosatelli C, et al. Molecular mechanism accounting for milder types of thalassemia major. J Pediatr 1983;103:35-9. 101. Orkin SH, Kazazian HH Jr, Antonarakis SE, et al. Abnormal RNA processing due to the exon mutation of βE-globin gene. Nature 1982;300:768-9. 102. Wasi P, Na-Nakorn S, Pootrakul S, et al. α- and β-Thalassemia in Thailand. Ann N Y Acad Sci 1969;165:60-82. 103. Schorr JB, Radel E. Transfusion therapy and its complications in patients with Cooley’s anemia. Ann N Y Acad Sci 1964;119:703-8.

104. Wolman IJ. Transfusion therapy in Cooley’s anemia: Growth and health as related to long-range hemoglobin levels. Ann N Y Acad Sci 1964;119:736-47. 105. Gabutti V, Piga A, Nicola P, et al. Haemoglobin levels and blood requirements in thalassaemia. Arch Dis Child 1982;57:156-8. 106. Cavill I, Worwood M, Jacobs A. Internal regulation of iron absorption. Nature 1975;256:328-9. 107. Piomelli S, Danoff SJ, Becker MH, et al. Prevention of bone malformations and cardiomegaly in Cooley’s anemia by early hypertransfusion regimen. Ann N Y Acad Sci 1969;165:427-36. 108. Piomelli S, Loew T. Management of thalassemia major (Cooley’s anemia). Hematol Oncol Clin North Am 1991;5:557-69. 109. Propper RD, Button LN, Nathan DG. New approaches to the transfusion management of thalassemia. Blood 1980;55:55-60. 110. Cazzola M, Borgna-Pignatti C, Locatelli F, et al. A moderate transfusion regimen may reduce iron loading in β-thalassemia major without producing excessive expansion of erythropoiesis. Transfusion 1997;37:135-40. 111. Schuman JE, Tanser CL, Peloquin R, et al. The erythropoietic response to pregnancy in β-thalassaemia minor. Br J Haematol 1973;25:249-60. 112. Hocking IW, Ibbotson RN. The effect of the β thalassaemia trait on pregnancy with particular reference to its complications and outcome. Med J Aust 1966;2:397-400. 113. Borgna-Pignatti C. Modern treatment of thalassaemia intermedia. Br J Haematol 2007;138:291-304. 114. Singer ST, Styles L, Bojanowski J, et al. Changing outcome of homozygous α-thalassemia: Cautious optimism. J Pediatr Hematol Oncol 2000;22:539-42. 115. Gordeuk VR, Bacon BR, Brittenham GM. Iron overload: Causes and consequences. Annu Rev Nutr 1987;7:485-508. 116. Kwiatkowski JL, Cohen AR. Iron chelation therapy in sickle-cell disease and other transfusion-dependent anemias. Hematol Oncol Clin North Am 2004;18:1355-77. 117. Jensen PD. Evaluation of iron overload. Br J Haematol 2004; 124:697-711. 118. Hershko C. Oral iron chelators: New opportunities and new dilemmas. Haematologica 2006;91:1307-12. 119. Maurer HS, Lloyd-Still JD, Ingrisano C, et al. A prospective evaluation of iron chelation therapy in children with severe β-thalassemia. Am J Dis Child 1988;142:287-92. 120. Wolfe L, Olivieri N, Sallan D, et al. Prevention of cardiac disease by subcutaneous deferoxamine in patients with thalassemia major. N Engl J Med 1985;312:1600-3. 121. Cohen AR, Schwartz E. Excretion of iron in response to deferoxamine in sickle cell anemia. J Pediatr 1978;92:659-62. 122. Porter JB, Huehns ER. Transfusion and exchange transfusion in sickle cell anemias, with particular reference to iron metabolism. Acta Haematol 1987;78:198-205.

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30

Blood Component Transfusions for Infants Ronald G. Strauss Professor Emeritus, Pathology and Pediatrics, University of Iowa College of Medicine, Iowa City, Iowa, USA

Many aspects of hematopoiesis are either incompletely developed in preterm infants or are adapted to serve the fetus (ie, the intrauterine counterpart to a live-born preterm neonate). This lack of development and/or adaptation to extrauterine life diminishes the capacity of the neonate to produce red cells, platelets, and neutrophils—particularly during the stress of lifethreatening illnesses encountered after preterm birth such as sepsis, severe pulmonary dysfunction, necrotizing enterocolitis, and immune cytopenias. Similarly, hepatic function is immature, and the result is low levels of plasma clotting proteins. The serious medical and surgical problems of preterm birth can be further complicated by phlebotomy blood losses, bleeding, hemolysis, and consumptive coagulopathy. Thus, preterm infants begin life with quantities of blood cells and clotting proteins that are barely adequate. Furthermore, these infants have a diminished ability to increase production adequately to compensate for the hematologic problems they experience. These circumstances lead to the need for blood component transfusions. Preterm infants, especially those with a birthweight less than 1.0 kg and with respiratory distress, are given numerous red cell transfusions early in life because of several interacting factors. Neonates delivered before 28 weeks of gestation (birthweight
1.0 kg) are born before the bulk of iron transport has occurred from mother to fetus via the placenta and before the onset of marked erythropoietic activity of fetal marrow during the third trimester. Hence, preterm infants of very low birthweight enter extrauterine life with low iron stores and a small circulating volume mass of red cells. Soon after preterm birth, severe respiratory disease can lead to repeated blood sampling for laboratory studies and, consequently, to replacement red cell transfusions. As a final factor, preterm infants are unable to mount an effective erythropoietin (EPO) response to decreasing numbers of red cells, and this factor contributes to the diminished ability to compensate for anemia and enhances the need for transfusions. Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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The physiology of hematopoiesis in the fetus and newborn is discussed in detail in Chapter 25, hemolytic disease of the fetus and newborn in Chapters 26 and 27, congenital hemolytic anemias in Chapter 29, and the physiology and disorders of plasma clotting and anticoagulant proteins in Chapter 28. Accordingly, only the aspects of physiology and pathophysiology that pertain to neonatal transfusion medicine, in general, are included here. The emphasis is on transfusion management during the first several weeks after birth.

Red Cell Transfusion Pathophysiology of Anemia of Prematurity During the first weeks of life, all infants experience a decline in the number of circulating red cells caused by physiologic factors. In sick preterm infants, phlebotomy blood losses also contribute to the decline. In healthy term infants, the nadir blood hemoglobin value rarely decreases to less than 9 g/dL (mean  11 to 12 g/dL) at an age of approximately 10 to 12 weeks.1 Because this postnatal decrease in hemoglobin level is universal and is well tolerated by term infants, it is commonly called the physiologic anemia of infancy. Among preterm infants, this decline occurs at an earlier age and is more pronounced in severity. Mean hemoglobin concentration decreases to approximately 8 g/dL in infants of 1.0 to 1.5 kg birthweight and to 7 g/dL in infants weighing less than 1.0 kg.2 In preterm infants, this marked decline in hemoglobin frequently is exacerbated by phlebotomy blood losses and may be associated with symptomatic anemia that necessitates red cell transfusions, making the anemia of prematurity unacceptable as a “purely physiologic” event. Physiologic factors that influence erythropoiesis and the biologic characteristics of EPO are critical in the pathogenesis of the anemia of prematurity. Growth is extremely rapid during the first weeks of life, and red cell production by neonatal marrow must increase commensurately to avoid a decreasing hematocrit caused by an insufficient number of circulating red cells being diluted within the expanding blood volume. It is widely accepted that the

Chapter 30: Blood Component Transfusions for Infants

circulating life span of neonatal red cells in the bloodstream is shorter than that of adult red cells. This shorter survival of neonatal red cells, quite possibly, is an artifact, in part, because studies of transfused autologous red cells labeled with biotin or radioactive chromium may underestimate red cell survival in the infant’s bloodstream for technical reasons. In healthy adults—when body size is stable so that blood and red cell volumes are constant (ie, not increasing with growing body size and commensurate increase in erythropoiesis), when no transfusions are given, and when large volumes of blood are not being taken for laboratory studies—the gradual disappearance of transfused labeled red cells, caused by dilution with red cells produced endogenously by the marrow, accurately reflects red cell survival in the bloodstream. In contrast, one or more of these confounding factors (ie, growth, red cell transfusions, phlebotomy) exists in infants— particularly, sick preterms—thus introducing error into the calculations performed when red cell survival is determined, based on disappearance of labeled red cells. In addition, a key clinical factor is the need for repeated blood sampling to monitor the condition of critically ill neonates. Small preterm infants are the most critically ill, require the most frequent blood sampling, and suffer the greatest proportional loss of red cells because their circulating cell volumes are smallest. In the past, the mean volume of blood removed for sampling has been reported to range from 0.8 to 3.1 mL/kg/day during the first few weeks of life for preterm infants requiring intensive care. Promising “in-line” devices that withdraw blood, measure multiple analytes, and then reinfuse the sampled blood have been reported.3,4 They have decreased the need for red cell transfusions. However, until these devices are proven more extensively to be practical, effective, and safe, replacement of blood losses from phlebotomy will remain a critical factor responsible for transfusions given to critically ill neonates—particularly, transfusions given during the first 4 weeks of life. A key reason that the nadir hemoglobin values of preterm infants are lower than those of term infants is that preterm infants have a relatively diminished EPO plasma level in response to anemia.5 Although anemia provokes EPO production in premature infants, the plasma levels achieved in anemic infants, at any given hematocrit, are lower than those observed in comparably anemic older persons.6 Erythroid progenitor cells of preterm infants are quite responsive to EPO in vitro—a finding suggesting that inadequate production of EPO (not marrow unresponsiveness) is the major cause of physiologic anemia.7 The mechanisms responsible for the diminished EPO output by preterm neonates are only partially defined and, likely, are multiple. One mechanism is that the primary site of EPO production in preterm infants is in the liver, rather than kidneys.8 This dependency on hepatic EPO is important because the liver is less sensitive to anemia and tissue hypoxia—resulting in a relatively sluggish EPO response to the decreasing hematocrit. The timing of the switch from the liver to kidneys is set at conception and is not accelerated by preterm birth. Viewed from a teleologic perspective, decreased hepatic production of EPO under in-utero

conditions of tissue hypoxia may be an advantage for the fetus. If this were not the case, normal levels of fetal hypoxia in utero could trigger high levels of EPO and produce erythrocytosis and consequent hyperviscosity. Following birth, however, diminished EPO responsiveness to tissue hypoxia is disadvantageous and leads to anemia because it impairs compensation for low hematocrit levels caused by rapid growth and red cell losses caused by phlebotomy, clinical bleeding, hemolysis, etc. Diminished EPO production cannot entirely explain low plasma EPO levels in preterm infants, because extraordinarily high plasma levels of EPO have been reported in some fetuses and infants.9,10 Moreover, macrophages from human cord blood produce normal quantities of EPO messenger RNA and protein.11 Thus, additional mechanisms contribute to diminished EPO plasma levels. For example, plasma levels of EPO are influenced by metabolism (clearance) as well as by production. Data in human infants12 have demonstrated low plasma EPO levels resulting from increased plasma clearance, increased volume of distribution, more rapid fractional elimination, and shorter mean plasma residence times than comparative values in adults. Thus, accelerated catabolism accentuates the problem of diminished EPO production, so that the low plasma EPO levels are a combined effect of decreased synthesis plus increased metabolism.

Red Cell Transfusion Practices Red cell transfusions are given to maintain the hematocrit at a level judged best for the clinical condition of the infant.13 General guidelines acceptable to most neonatologists are listed in Table 30-1. However, many aspects of neonatal red cell transfusion therapy are controversial and vary from center to center. This lack of consistency stems from incomplete knowledge of the cellular and molecular biology of erythropoiesis during the perinatal period, of the pathophysiologic effects of neonatal anemia, and of the infant’s response to red cell transfusions. In some instances the value of red cell transfusions is clear (eg, to manage anemia that has caused congestive heart failure), but in others it is not (eg, to correct irregular patterns of heart or respiratory rates), and practices are based largely on logical assumptions. Although well-designed clinical trials are being reported,14,15 they are not completely mutually supportive and questions Table 30-1. Guidelines for Small-Volume Red Cell Transfusions ● ● ● ●

Maintain 40% to 45% hematocrit for severe cardiopulmonary disease Maintain 30% to 35% hematocrit for moderate cardiopulmonary disease Maintain 30% to 35% hematocrit for major surgery Maintain 20% to 25% hematocrit for infants with stable anemia, especially with: Unexplained breathing disorders Unexplained tachycardia Unexplained poor growth

Words in italics must be defined locally. For example, “severe” pulmonary disease may be defined as requiring mechanical ventilation with 0.35 FiO2 and “moderate” as less intensive assisted ventilation.

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Section II: Part II

remain. Therefore, it is important that pediatricians critically evaluate the guidelines in Table 30-1 and apply them in light of neonatal practice at their respective institutions. An important controversy that is still unresolved is the wisdom—or lack, thereof—of prescribing Red Blood Cell (RBC) units for neonates using restrictive guidelines (ie, low pretransfusion hematocrit values) vs liberal guidelines (ie, conventional, relatively high pretransfusion hematocrit values). Two randomized controlled trials have been published14,15 and, although many of their results agree, they disagree in one extremely important way—specifically, whether preterm infants are at increased risk of brain injury when given RBCs per restrictive guidelines (ie, due to undertransfusion). In both trials, preterm infants were randomly assigned to receive small-volume RBC transfusions per either restrictive or liberal guidelines—with guidelines based on a combination of the pretransfusion hematocrit or hemoglobin level, age of the neonate, and clinical condition at the time each transfusion was given. Both studies found that neonates in the restrictive transfusion group received fewer transfusions, without an increase in mortality or in morbidity based on several clinical outcomes. However, one critical discrepancy was present. Bell et al14 found increases in apnea, intraventricular bleeding, and brain leukomalacia in infants transfused per restrictive guidelines, whereas Kirpalani et al15 found no differences between infants in the restrictive vs liberal groups. Moreover, rates of serious outcomes were fairly high in both groups of the Kirpalani study—perhaps, a result of the extreme prematurity of the infants. However, because neonates in the liberal transfusion group of Bell et al likely had substantially higher hematocrit/hemoglobin levels than neonates in the liberal group of Kirpalani et al (an average hematocrit of 6% or hemoglobin level of 2 g/dL higher), it is reasonable to speculate that the higher hematocrit levels, in some way, protected liberally transfused infants in the Bell study.14,15 Until more information is available, it seems wise to transfuse preterm neonates using conventional, relatively liberal guidelines (ie, do not place infants at possible risk of undertransfusion by strict conservative practices). The rationale for the conventional, relatively liberal guidelines (Table 30-1) follow in the paragraphs below.

Maintain Hematocrit >40% to 45% for Severe Cardiopulmonary Disease In neonates with severe respiratory disease, such as those requiring high volumes of oxygen with ventilator support, it is customary to maintain the hematocrit above 40% to 45% (hemoglobin concentration above 13-14 g/dL)—particularly when blood is being drawn frequently for testing. This practice is based on the belief that transfused donor red cells, containing adult hemoglobin with its superior interaction with 2,3-diphosphoglycerate (2.3-DPG), will provide optimal oxygen delivery throughout the period of diminished pulmonary function. Consistent with this rationale for ensuring optimal oxygen delivery in neonates with pulmonary failure, it seems logical to maintain the hematocrit

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above 40% in infants with congenital heart disease that is severe enough to cause either cyanosis or congestive heart failure.

Maintain Hematocrit 30% to 35% for Moderate Cardiopulmonary Disease Following similar logic, it seems reasonable to maintain the hematocrit above 30% to 35% for moderate cardiopulmonary disease (Table 30-1). Maintain Hematocrit >30% for Major Surgery Definitive studies are not available to establish the optimal hematocrit for neonates facing major surgery. However, it seems reasonable to maintain the hematocrit 30% because of limited ability of the neonate’s heart, lungs, and vasculature to compensate for anemia. Additional factors include the inferior off-loading of oxygen to tissues by the infant’s own red cells because of the diminished interaction between fetal hemoglobin and 2,3-DPG plus the developmental impairment of neonatal pulmonary, renal, hepatic, and neurologic function. Because this transfusion guideline is simply a recommendation—not a firm indication—it should be applied with flexibility to individual infants facing surgical procedures of varying complexity (ie, minor surgery may be judged not to require a hematocrit 30%). The amount of anticipated blood loss must be strongly considered in preoperative transfusion decisions—with a likelihood of large blood loss, some physicians might like the preoperative hematocrit to be relatively high. Maintain Hematocrit >20% to 25% for Stable Infants with Symptomatic Anemia The clinical recommendations for red cell transfusions in preterm infants who are not critically ill but, nonetheless, develop moderate anemia (hematocrit
25% or hemoglobin level
8 g/dL) are extremely variable. In general, infants who are clinically stable with modest anemia do not require red cell transfusions, unless they exhibit significant clinical problems that are ascribed to the presence of anemia or are predicted to be corrected by donor red cells.16,17 As an example, proponents of red cell transfusions to treat disturbances of cardiopulmonary rhythms believe that a low blood level of red cells contributes to tachypnea, dyspnea, apnea, and tachycardia or bradycardia because of decreased oxygen delivery to the respiratory center of the brain. Red cell transfusions might decrease the number of apneic spells by improving oxygen delivery to the central nervous system. However, results of clinical studies have been contradictory.14,16,17 Another controversial clinical indication for red cell transfusions is to maintain a reasonable hematocrit level as treatment for unexplained growth failure. Some neonatologists consider poor weight gain to be an indication for transfusion, particularly if the hematocrit is
25% and if other signs of distress are evident (eg, tachycardia, respiratory difficulty, weak suck and cry, and diminished activity).18 In this setting, growth failure has been ascribed to the increase in metabolic expenditure required to support the work of labored breathing. In the past, a

Chapter 30: Blood Component Transfusions for Infants

hematocrit below 30% was of concern and often led to transfusion. However, results of clinical studies have not supported this practice,16,17 and no apparent rationale exists to justify maintaining any predetermined hematocrit level by prophylactic, smallvolume red cell transfusions in stable, growing infants who seem to be otherwise healthy. In practice, the decision of whether to transfuse is based on the desire to maintain the hematocrit concentration at a level judged to be most beneficial for the infant’s clinical condition. Investigators who believe this “clinical” approach is too imprecise have suggested the use of “physiologic” criteria such as red cell mass,19 available oxygen,20 mixed venous oxygen saturation, and measurements of oxygen delivery and utilization21 to develop guidelines for transfusion decisions. In one study of 10 human infants with severe (oxygen-dependent) bronchopulmonary dysplasia, improvement of physiologic endpoints was shown (increased systemic oxygen transport and decreased oxygen use) as a consequence of small-volume red cell transfusions.21 However, these promising but technically demanding methods are, at present, difficult to apply in the day-to-day practice of neonatology. The application, in infants, of data obtained from studies of animals and adult humans that correlate tissue oxygenation with the clinical effects of anemia and the need for red cell transfusions is confounded by the differences between infants and adults in hemoglobin oxygen affinity, ability to increase cardiac output, and regional patterns of blood flow. Another physiologic factor considered in transfusion decisions is use of the circulating red cell volume/mass rather than the hematocrit or hemoglobin level. Although circulating red cell volume/mass is a potentially useful index of the blood’s oxygen-carrying capacity, it cannot be predicted accurately from hematocrit or hemoglobin concentration levels; hence, it must be measured directly.22 Unfortunately, circulating red cell volume/ mass measurements and other “physiologic” criteria for transfusions are not widely available for clinical use.

Red Blood Cell Products to Transfuse Most transfusions are given to preterm infants as smallvolume transfusions (10 to 20 mL/kg body weight) of RBCs in extended-storage media (additive solution AS-1, AS-3, AS-5) at a hematocrit of approximately 55% to 60% (Table 30-2). Alternatively, some neonatologists prefer RBCs in citrate-phosphate-dextrose-adenine (CPDA) solution at a hematocrit of approximately 70%—although the superiority of this last solution over extended-storage media has not been shown. Some centers prefer to centrifuge RBC aliquots before transfusion to prepare a uniformly packed RBC concentrate (hematocrit 80%).23 Most RBC transfusions are infused slowly over 2 to 4 hours. Because of the small quantity of extracellular fluid (storage media) infused very slowly with small-volume transfusions, the type of anticoagulant-preservative solution used poses no risk for most premature infants.24,25 Accordingly, the traditional use of relatively fresh RBCs (
7 days of storage) has been replaced in many centers by the practice of transfusing aliquots

Table 30-2. Formulation of Red Cell Anticoagulant-Preservative Solutions Constituent

CPDA

AS-1

AS-3

AS-5

Volume (mL) Sodium chloride (mg) Dextrose (mg) Adenine (mg) Mannitol (mg) Trisodium citrate (mg) Citric acid (mg) Sodium phosphate (monobasic) (mg)

63* None 2010 17.3 None 1660 206 140

100† 900 2200 27 750 None None None

100† 410 1100 30 None 588 42 276

100† 877 900 30 525 None None None

* Approximately 450 mL of donor blood is drawn into 63 mL of citrate-phosphatedextrose-adenine (CPDA) solution. One Red Blood Cell unit (hematocrit, ~70%) is prepared by means of centrifugation and removal of most plasma. Results of calculations will be slightly different if 500 mL of donor blood is drawn. †

When additive solution AS-1 or AS-5 is used, 450 mL of donor blood is first drawn into 63 mL of CPD, which is identical to CPDA except it contains 1610 mg dextrose per 63 mL and has no adenine. When AS-3 is used, donor blood is drawn into CP2D, which is identical to CPD except it contains double the amount of dextrose. After centrifugation and removal of nearly all plasma, the cells are resuspended in 100 mL of the additive solution (AS-1, AS-3 or AS-5) at a hematocrit of approximately 55% to 60%.

of RBCs from a dedicated unit (or part of a unit) of RBCs stored up to 42 days in efforts to diminish the high donor exposure rates among infants who undergo numerous transfusions.23,25 Neonatologists who object to prescribing stored RBCs and insist on transfusing fresh RBCs generally express the following four concerns: 1) the increase in the level of potassium in the plasma (ie, supernatant fluid); 2) the decrease in the level of 2,3-DPG; 3) the possible risks of additives such as mannitol and the relatively large amounts of glucose (dextrose) and phosphate present in extended-storage preservative solutions; and 4) the changes in red cell shape and deformability that may lead to poor flow through the microvasculature. Although these concerns often are legitimate for large-volume (25 mL/kg) transfusions, particularly when infusion is rapid, they do not apply to small-volume transfusions for the following reasons. After 42 days of storage in extended-storage media (AS-1, AS-3, AS-5) at a hematocrit of approximately 60%, extracellular (“plasma”) potassium levels in RBC units approximate 50 mEq/L (0.05 mEq/mL), a value that at first glance seems alarmingly high. Simple calculations, however, show the actual dose of ionic potassium transfused in the extracellular “plasma” fluid is small. An infant weighing 1 kg given a 15-mL/kg transfusion of RBCs stored 42 days in extended-storage media at a hematocrit of 55% to 60% receives only 0.3 mEq. The potassium concentration of RBCs stored in CPDA solution at a hematocrit of 70% increases to 70 to 80 mEq/L after the 35 days of permitted storage, and the dose of potassium infused with a 15-mL/kg transfusion to a 1-kg infant is 0.3 to 0.4 mEq. These doses are quite small when compared to the usual daily potassium requirement of 2 to 3 mEq/ kg and have been shown in several clinical studies not to cause hyperkalemia.25

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Table 30-3. Quantity (Total mg/kg) of Additives Infused During a Transfusion of 15 mL/kg AS-1, AS-3, or AS-5 RBCs at Hematocrit 60%

Table 30-4. Mean Change in Blood Chemistry Levels During RBC Transfusions27,28

Additive

AS-1

AS-3

AS-5

Toxic Dose*

Value

Sodium chloride Dextrose Adenine Citrate Phosphate Mannitol

54.0 132.0 1.6 Trace Trace 45.0

24.6 66.0 1.8 37.8 16.6 None

52.6 54.0 1.8 Trace Trace 31.5

137 mg/kg/day 240 mg/kg/hour 15 mg/kg/dose 180 mg/kg/hour 60 mg/kg/day 360 mg/kg/day

* The accuracy of toxic dose is difficult to predict because infusion rates are slow, allowing metabolism and distribution of additives from blood into extravascular sites. In addition, dextrose, adenine, and phosphate enter red cells and are somewhat sequestered and not immediately “available” in the extracellular solution. Data on potential toxic doses from Luban et al.24 Calculations for quantity of additives infused assumed the following: 1) total volume of RBC unit  300 mL, consisting of 100 mL additive solution  180 mL completely packed red cells  20 mL of primary anticoagulant trapped between the red cells; 2) 15 mL/kg transfusion  9 mL red cells  6 mL additive solution; 3) quantity of additive solution infused per single RBC transfusion  quantity in 100 mL/(100  6); 4) trace amounts of trisodium citrate, citric acid, and phosphate were carried over by primary anticoagulant trapped between red cells.

By 21 days of storage, 2,3-DPG is totally depleted from RBCs as reflected by a P50 value that decreases from approximately 27 mm Hg in fresh blood to 18 mm Hg in stored red cells at the time of outdate. Because of the effects of high fetal hemoglobin levels in neonatal red cells, the 18 mm Hg value of red cells transfused after maximum storage corresponds to the expectedly low P50 value obtained from studies of red cells produced by many healthy preterm infants at birth. Thus, both older stored red cells and endogenous red cells from neonates have a similarly reduced ability to off-load oxygen compared with fresh adult red cells. However, older adult red cells in banked RBC units provide an advantage over the infant’s own cells because 2,3-DPG and the P50 of transfused adult cells (but not endogenous infant red cells) increase rapidly after transfusion. When studied in the setting of small-volume (15 mL/kg) transfusion, 2,3-DPG levels were maintained in infants given stored RBCs.26 The quantity of additives present in RBCs in extendedstorage media is unlikely to be dangerous for neonates given small-volume transfusions (15 mL/kg).24,25 Regardless of the type of suspending solution, the quantity of additives is quite small in the clinical setting in which a neonate would receive a single, small-volume transfusion over 2 to 4 hours. With AS-1, AS-3, and AS-5 as examples (Table 30-3), the dose of extended-storage media additives transfused during a typical small-volume transfusion is estimated to be far less than levels believed to be toxic.24 This assumption was proved correct in clinical studies in which infants received one or more transfusions.24 The safety and efficacy of transfusing stored RBCs is documented by the results of several laboratory tests performed before and after small-volume RBC transfusions in preterm infants (Table 30-4). During storage in conventional preservative solutions, red cells sustain decreases in 2,3-DPG and adenosine triphosphate, and

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Hematocrit (%) Glucose (mg/dL) Lactate (mmol/L) pH Calcium (mg/dL) Sodium (mEq/L) Potassium (mEq/L)

Change* 1 to 21 Days of Storage (n = 78)

22 to 42 Days of Storage (n = 42)

12  5 12  24 0.6  1.1 0.00  0.08 0.1  0.5 0.3  4.6 0.2  0.8

12  4 16  28 0.2  0.3 0.00  0.06 0.0  0.8 0.4  4.7 0.2  0.6

* Change ( SD) is posttransfusion value minus pretransfusion value. Statistical tests used were t test for pH, sodium, potassium, and glucose (normal distribution) and Wilcoxon rank sum test for hematocrit, calcium, and lactate (abnormal distribution). No statistically significant differences were found (p values all 0.05) comparing 1 to 21 days vs 22 to 42 days of storage.

they undergo membrane and cytoskeletal changes that lead to the formation of echinocytes and microvescicles and to decreased deformability. The last changes may lead to diminished perfusion of the microvasculature and, consequently, to tissue and organ dysfunction. For the past few years, the argument has raged over whether critically ill adult patients are harmed by receiving “old” RBC units (usually defined as stored 15 or 21 days), and whether mortality, multi-organ failure, infections, need for mechanical ventilation, length of stay in intensive care units and in the hospital, etc could be diminished by transfusing only fresh RBC units. Older, largely observational, studies generally support the idea that “old” RBC units put critically ill patients at risk, particularly if they receive multiple transfusions. Several more recent studies have challenged this concept and have cautioned against insisting on transfusions of fresh RBCs.29,30 Well-designed clinical trials are needed to resolve this question. The situation is similar for neonatal RBC transfusions (ie, neonates are often critically ill and questions about morbidity caused by transfusing stored RBCs have been raised). However, well-designed trials have addressed efficacy and safety and, within the limits of the number of infants studied, fresh and stored cells have been documented to be equivalent.14,25,26 Moreover, the intravascular recovery 24 hours after transfusion and long-term survival of stored red cells is normal, when measured in human infants using biotinylated red cells.31 Because the risks of multiple donor exposure can be nearly eliminated by giving infants cells taken from dedicated, stored RBC units and because increased risks of transfusing stored vs fresh cells have not been demonstrated, it seems prudent to continue transfusing stored RBCs for small-volume transfusions.

Approach to Anemia of Prematurity For many infants with a birthweight less than 1.0 kg, anemia of prematurity is severe and necessitates therapy with red cell

Chapter 30: Blood Component Transfusions for Infants

transfusions and/or the administration of recombinant human erythropoietin (rHuEPO) plus iron. For decades, red cell transfusion has been the standard of care and continues to be so. A key principle is that allogeneic donor exposure should be minimized by transfusing cells from dedicated RBC units stored as long as permitted (eg, 42 days for extended-storage anticoagulant-preservative solutions). At the University of Iowa DeGowin Blood Center, preterm infants who need transfusions are assigned to dedicated RBC units suspended in extended-storage (42-day) solutions. At the time the first transfusion is ordered, one-half of a freshly collected unit (stored 7 days) is dedicated to a preterm infant with a birthweight of 1.0 kg or less. The rest of the unit can be assigned to another infant. Thus, one complete unit can serve two very-low-birthweight infants simultaneously. When transfusions are ordered, aliquots are removed sterilely and issued.23 Although units are used throughout 42 days of storage, if a unit has been stored 14 days without an infant being assigned to it, it has become relatively aged (14 of its 42 storage days have lapsed), and it enters the general inventory to be used for older patients as needed. This plan has been demonstrated to be cost-effective.32 Recognition of low plasma EPO levels and adequate erythropoietic activity in preterm infants provides a rational basis to consider rHuEPO as treatment for the anemia of prematurity. Because the inadequate quantity of EPO is the major cause of anemia—not a subnormal response of erythroid progenitors to EPO—it is logical to assume that rHuEPO will correct EPO deficiency and will effectively treat the anemia of prematurity. Unfortunately, rHuEPO has not been widely accepted in clinical neonatology practice because its efficacy is incomplete. On one hand, clonogenic erythroid progenitors from neonates respond well to rHuEPO in vitro and rHuEPO and iron effectively stimulate erythropoiesis in vivo as evidenced by increased blood reticulocyte and red cell counts in recipient infants (ie, efficacy successful at the marrow level). On the other hand, when the primary goal of rHuEPO therapy is to eliminate transfusions, rHuEPO often fails (ie, efficacy at the clinical level has not been consistently successful).13,33 By 2000, over 20 controlled clinical trials assessing the efficacy of rHuEPO to eliminate RBC transfusions in the anemia of prematurity were published with inconsistent results. To investigate the extent and reasons for the inconsistencies, a meta-analysis was conducted of the controlled clinical studies published between 1990 and 1999.33 To be included, a reported study had to prospectively enroll a treatment group of preterm infants under 4 months of age treated with rHuEPO and a concurrent control group not given rHuEPO. Twenty-one reports were eligible for inclusion in the meta-analysis. However, because the experimental design and conduct of the studies were extremely variable, only four reports were judged to fulfill all of the highly desired characteristics of being effectively blinded, having highquality experimental design (ie, randomized, placebo-controlled, all dropouts well-explained, etc), using conservative transfusion practices (rather than liberal transfusions, which suppress

endogenous erythropoiesis), and enrolling a majority of very preterm infants with birthweights
1.0 kg. Two major conclusions emerged from the meta-analysis.33 First, the controlled trials of rHuEPO to treat the anemia of prematurity differed from one another in multiple ways and, consequently, produced markedly variable results that could not be adequately explained. Hence, it was judged impossible to make firm recommendations regarding use of rHuEPO in clinical practice to treat the anemia of prematurity. Second, when the four studies with highly desired characteristics were analyzed separately, rHuEPO was found to be efficacious in significantly reducing transfusion needs. However, the magnitude of the effect of rHuEPO on reducing the total red cells transfusions given to infants throughout their initial hospitalization was, in fact, relatively modest and of questionable clinical importance. Although the meta-analysis was unable to recommend how to use rHuEPO in clinical practice, it was apparent that: 1) relatively large or stable preterm infants, shown to respond best to rHuEPO plus iron at the marrow level, are given relatively few red cell transfusions with today’s conservative transfusion practices and, accordingly, have little need for rHuEPO when the goal is to avoid transfusions; and 2) extremely small preterm infants, who are critically ill and unstable, have not consistently responded to rHuEPO plus iron when the outcome measure is to reduce need for transfusions. Several reports published after 2000 have provided useful information. Donato et al,34 assessing the effects of rHuEPO very early in life, randomly assigned 114 neonates with birthweights
1.25 kg to receive either rHuEPO or placebo during the first 2 weeks of life, followed by a 6- week treatment period during which all infants were given rHuEPO, iron, and folic acid. During the first 3 weeks of life, rHuEPO increased reticulocytes and hematocrit values, but there was no difference in red cell transfusions. However, at the end of all treatment (8 weeks), a subgroup of infants with birthweights
0.8 kg and phlebotomy losses 30 mL/kg who were given rHuEPO shortly after birth received fewer total red cell transfusions than infants initially given placebo (3.4  1.1 vs 5.4  3.7, p
0.05). Similarly, Yeo et al35 found a modest advantage for a subgroup of very-lowbirthweight infants given rHuEPO. Infants with birthweights 0.8 to 0.99 kg were given fewer transfusions with rHuEPO than control infants not given rHuEPO (2.1  1.9 vs 3.5  1.6, p
0.04). A randomized blinded trial by Meyer et al36 found an advantage for very-low-birthweight infants given rHuEPO. Neonates with birthweights
1.7 kg plus criteria that predicted a likely need for red cell transfusions were randomly assigned either to receive rHuEPO beginning shortly after birth or to experience a sham treatment to simulate placebo injections. Iron was given to all infants, but at a much lower dose (unfortunately) to control infants not given rHuEPO than to infants given rHuEPO—thus, creating two variables/differences (rHuEPO and iron dose, rather than just rHuEPO) being assessed in treated vs control infants. There was no overall difference in red cell transfusions, but in a subset of infants with birthweights
1.0 kg, transfusions given

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late (ie, after 30 days of age) were reduced by rHuEPO vs transfusions given to controls (0.5  0.7 vs 1.6  1.1, p  0.01). Two reports defined rHuEPO “success” as maintaining a hematocrit of 30% without need for any transfusions. Maier et al37 randomly assigned 219 neonates with birthweights 0.5 to 0.99 kg to receive early rHuEPO (from the first week of life for 9 weeks), late rHuEPO (from the fourth week of life for 6 weeks), or no rHuEPO. “Success” was modest (13% with early rHuEPO, 11% with late rHuEPO, and 4% with no rHuEPO). Only early rHuEPO administration was significantly superior to no rHuEPO (p  0.026). Avent et al38 randomly assigned 93 neonates with birthweights 0.9 to 1.5 kg to receive low-dose rHuEPO (250 units/kg), high dose rHuEPO (400 units/kg), or no rHuEPO. Treatment began within 7 days of life and continued until discharge (median 32 days and maximum 74 days). “Success” was met by 75% of low-dose rHuEPO infants, 71% of high-dose rHuEPO infants, and 40% of no rHuEPO infants (p
0.001). The number of red cell transfusions given to all infants treated with rHuEPO vs those with no rHuEPO was not significantly different. The authors concluded that rHuEPO does not significantly further decrease red cell transfusions in infants with birthweights of 0.9 to 1.5 kg, when phlebotomy losses are small and relatively few transfusions are given per stringent transfusion guidelines. The observation by Avent et al38 that the benefits of rHuEPO in reducing the number of red cell transfusions given can be equalled by stringent or conservative transfusion guidelines has been made by others. Franz and Pohlandt39 assessed both the number of red cell transfusions given and red cell transfusion guidelines in four prospective, randomized trials of rHuEPO given to preterm neonates. To be selected for analysis, the clinical trials had to include ventilated infants (ie, sick infants likely to receive red cell transfusions). The authors found that, when restrictive transfusion guidelines were followed, the number of red cell transfusions and the volume of red cells transfused were similarly low in infants either given or not given rHuEPO.36 Similarly, Amin and Alzahrani40 found no difference in the number of red cell transfusions, whether or not rHuEPO was given to preterm infants with birthweights 1.0 kg, when transfusions were given per strict transfusion guidelines. Another alternative to allogeneic transfusions is use of placental blood as a source of autologous red cells.41,42 Although collection, storage, and transfusion of placental blood after delivery deserves careful consideration, at least three obstacles must be overcome before autologous transfusions of stored placental red cells can be adopted for clinical use. One concern is that a sufficient quantity of red cells will not be obtained consistently from placentas of preterm infants to avoid a significant number of allogeneic transfusions. The second issue is the sterility of placental blood. Because bacterial contamination of placental blood is a distinct possibility,41 extensive testing will be needed to ensure absolute safety. Finally, acceptable quality of stored placental blood must be maintained.42 Because of these concerns, collection and storage of placental blood has not been encouraged.43

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Instead, interest has been renewed in delayed clamping of the umbilical cord immediately following delivery to expand neonatal blood volume and red cell volume/mass to, possibly, improve circulatory hemodynamics and increase tissue perfusion and oxygenation.22,44

Platelet Transfusion Pathophysiology of Neonatal Thrombocytopenia Blood platelet counts of 150,000/µL are present in normal fetuses (as early as 17 weeks of gestation) and in normal neonates. Thus, platelet counts are in the normal range, regardless of gestational age at birth. Low platelet counts indicate potential problems. Preterm neonates commonly have thrombocytopenia (eg, in one neonatal intensive care unit, 22% of infants had platelet counts
150,000/µL).45 In contrast, thrombocytopenia is documented in
1% of term neonates.46 Blood platelet counts
100,000/µL pose significant clinical risks. In one study, neonates with birthweights less than 1.5 kg and a platelet count
100,000/µL were compared with neonates of similar size who did not have thrombocytopenia.47 The bleeding time was prolonged when platelet counts were
100,000/µL, and platelet dysfunction was suggested by bleeding times that were disproportionately long for the degree of thrombocytopenia present. The incidence of intracranial hemorrhage was 78% among thrombocytopenic neonates with a birthweight less than 1.5 kg vs 48% for similar neonates without thrombocytopenia. In addition, the extent of hemorrhage and neurologic morbidity was greater among infants with thrombocytopenia.47 Although multiple pathogenetic mechanisms underlying thrombocytopenia likely are involved in these sick neonates, accelerated platelet destruction frequently is implicated by shortened platelet survival time, increased level of platelet-associated immunoglobulin G, increased platelet volume, normal number of marrow megakaryocytes, and an inadequate response to platelet transfusions.45,48 Another mechanism that contributes to neonatal thrombocytopenia is diminished platelet production. This is evidenced by neonatal megakaryocytes that are smaller and of lower ploidy than those of adults.49 In addition, some investigators have reported decreased numbers of megakaryocyte progenitors,50 while others have not.49 The response to thrombocytopenia in adults is to increase megakaryocyte number, volume, and ploidy—mediated primarily by thrombopoietin—all of which leads to increased platelet production. Thrombocytopenic neonates either fail to increase, or increase inadequately, megakaryocyte number, volume, and ploidy when compared to adults and, consequently, they cannot increase platelet counts sufficiently to correct thrombocytopenia.49,51 Moreover, thrombopoietin seems to increase neonatal megakaryocyte proliferation, but inhibit endoreduplication,52 in contrast, both are increased in adults. As is reminiscent of neonatal red cell and neutrophil production, where basal erythropoiesis and myelopoiesis are adequate,

Chapter 30: Blood Component Transfusions for Infants

basal platelet production and thrombopoiesis are adequate. However, under the stress of cytopenia, none of the three cell lines is capable of increasing production to completely and promptly correct low blood counts (ie, the infants lack sufficient hematopoietic reserve).

Recommendations for Platelet Transfusion during Infancy The relative risks of different degrees of thrombocytopenia in various clinical settings during infancy remain largely unanswered. Prophylactic platelet transfusions to prevent bleeding in preterm neonates were studied systematically several years ago, and the results still seem relevant.53 No randomized clinical trials of therapeutic platelet transfusions have been reported for bleeding thrombocytopenic neonates. Recognizing the need for more data, it seems logical in the interim to transfuse thrombocytopenic neonates per the guidelines presented in Table 30-5. Two firm indications for platelet transfusions are either to control hemorrhage that has already occurred or to prevent it from complicating an invasive procedure. No disagreement exists over using a pretransfusion platelet count of 50,000/µL as a minimum transfusion trigger in these instances. However, platelet transfusions are given at platelet counts 50,000/µL by some physicians to neonates to control bleeding or in hopes of reducing either the threat of, or the worsening of, intracranial hemorrhage in high-risk preterm neonates.47 No data exist to clearly establish the efficacy of platelet transfusions at these relatively high platelet levels. Conventional prophylactic platelet transfusions are given to prevent bleeding when severe thrombocytopenia poses a likely risk of spontaneous hemorrhage. Prophylactic platelet transfusions are given by some physicians to maintain the presence of a normal platelet count in hopes of preventing the neonate from slipping into high-risk situations that might lead to spontaneous bleeding. Regarding the first circumstance, most experts agree that it is reasonable to give platelets to any infant with a platelet count,
20,000/µL because spontaneous hemorrhage is a likely risk at this platelet count. Severe thrombocytopenia occurs most commonly among sick infants who, because of the illness, often receive medications that can compromise the function of their already diminished number of platelets. Because these factors are more pronounced in extremely preterm infants, who are clinically unstable, some neonatologists favor prophylactic platelet transfusion whenever the platelet count decreases to
50,000/ µL, or even to
100,000/µL, in critically ill infants.47 Table 30-5. Guidelines for Neonatal Platelet Transfusions ● ● ● ●

Maintain 50,000 to 100,000/µL platelets for significant bleeding Maintain 50,000/µL platelets for invasive procedures Maintain 20,000/µL platelets prophylactically for clinically stable neonates Maintain 50,000 to 100,000/µL platelets prophylactically for clinically unstable neonates

Words in italics must be defined locally. For example, consider bleeding site, extent and degree of prematurity, and underlying medical condition.

It is not efficacious to maintain a completely normal platelet count (150,000/µL) in preterm neonates without bleeding.53 Moreover, such a practice can place infants at increased risk from more platelet donor exposures. Intracranial hemorrhage occurs commonly among sick preterm infants. Although neither a causative role for thrombocytopenia nor a therapeutic benefit for platelet transfusion has been established in this disorder, it seems logical to presume thrombocytopenia might be a risk factor.54 Accordingly, in a randomized trial designed to address this issue, transfusion of platelets whenever the platelet count decreased below the normal value of 150,000/µL (which maintained the average daily platelet count 200,000/µL) was compared with transfusion of platelets only when the platelet count decreased below 50,000/µL.53 There was no difference in the incidence of intracranial hemorrhage (28% vs 26%) in the two groups. Thus, there is no documented benefit to transfusing “prophylactic platelets” to maintain a completely normal platelet count in preterm neonates vs transfusing “therapeutic platelets” to treat thrombocytopenia when it actually occurs.

Platelet Product to Transfuse The ideal goal of platelet transfusions for many thrombocytopenic neonates is to increase the low pretransfusion platelet count to a posttransfusion count 50,000/µL and for sick preterm infants to 100,000/µL. This can be achieved consistently by transfusing 5 to 10 mL/kg of unmodified platelet concentrates (withdrawn from a unit of platelets collected either by centrifugation of fresh units of whole blood or plateletpheresis and transfused directly). Platelet concentrates should be transfused as rapidly as the neonate’s condition allows, certainly within 2 hours. Routinely reducing the volume of platelet concentrates for infants by means of additional centrifugation steps is both unnecessary and unwise—unless a specific reason exists to do so. To illustrate, transfusion of 10 mL/kg platelet concentrate, taken directly from the unit and transfused, provides approximately 10,000/µL platelets. If the blood volume of an infant is 70 mL/kg body weight and the plasma volume is 40 mL/kg, the platelet dose of 10 mL/kg increases the platelet count 100,000 to 150,000/µL above the pretransfusion baseline—assuming a posttransfusion platelet recovery of 60%. This calculated increment is consistent with the increment actually achieved after transfusing this dose as reported in clinical studies.53 With modest thrombocytopenia, a 5 mL/kg dose may be sufficient. In general, 5 to 10 mL/kg is not an excessive transfusion volume even for sick neonates, as long as the intake of other intravenous fluids, medications, and nutrients is adjusted. If volume reduction is to be achieved, the method of reduction and the efficacy of platelet transfusions after reduction must be validated locally to ensure the quantity and quality of platelets (both ex vivo and in vivo) remaining after modification. In the selection of platelet units for transfusion, it is desirable for the infant and the platelet donor to be of the same ABO blood group. It is important to minimize repeated transfusions of group O platelets to group A or B recipients, because large quantities of

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passive anti-A or anti-B can lead to hemolysis. This should be avoided easily, with the exception of a directed-donor situation in which the infant receives—usually after an emotional rather than medical decision—platelet transfusions from an out-of-group donor. Directed-donor platelets should not be used in this situation; ABO-identical platelets from the general inventory should be selected. Proven methods exist to reduce the volume of platelet concentrates when truly warranted (eg, multiple transfusions anticipated in which several doses of passive anti-A or anti-B may lead to hemolysis, or failure to respond to a transfusion of 10 mL/ kg of unmodified platelet concentrate). However, additional processing should be performed with great care—using a method that is well-validated locally—because of probable platelet loss, clumping, and dysfunction caused by the additional handling.

Approach to Infants with Thrombocytopenia Thrombocytopenia exists wherever the blood platelet count is
150,000/µL. Every infant with thrombocytopenia needs an evaluation—if nothing more than a repeated complete blood count, a review of the medical history, and a physical examination. Definitive management of thrombocytopenia depends on the underlying disorder and is beyond the scope of this discussion (see Chapter 23). Correction of the thrombocytopenia per se by means of platelet transfusions is based on maintaining a platelet count deemed appropriate for the infant’s clinical condition (Table 30-5). There are no alternatives to platelet transfusion in the care of neonates with thrombocytopenia. Recombinant thrombopoietin (c-Mpl ligand or megakaryocyte growth and differentiation factor) and interleukin-11 are promising agents in older patients. However, neither is recommended for use during infancy, and both have potential toxicities that can preclude their use in the care of sick preterm infants. Thrombopoietin has broad actions on the early precursors of all three major lineages in the marrow and may produce effects in excess of those expected on megakaryocytes and platelets. Moreover, its interactions with fetal/neonatal megakaryocytes may be quite different from those with adults.52 Interleukin-11 may cause anemia. Clearly, these agents must not be prescribed in the treatment of infants, except in experimental settings with parental consent and institutional oversight. As noted in the plasma transfusion section, recombinant activated Factor VII has been used “off-label” to treat lifethreatening bleeding during infancy.

Plasma Transfusion Pathophysiology of Neonatal Clotting Proteins Hemostasis in a neonate is quantitatively and qualitatively different from that in an older child or adult, and the risk exists of either serious hemorrhage or thrombosis. Thus, some important facts about neonatal clotting proteins are summarized before issues of plasma transfusion are addressed. (See Chapters 17, 20, and 28 for more detailed discussions of hemostasis.)

478

Table 30-6. Guidelines for Neonatal Plasma Transfusions ●

High risk of bleeding because of acquired deficiency of clotting proteins Exchange transfusion Extracorporeal membrane oxygenation Cardiac bypass surgery Disseminated (consumptive) coagulation

●

Bleeding because of congenital clotting protein deficiency, if specific concentrate is unavailable

●

Bleeding because of vitamin K deficiency

●

Thrombosis because of anticoagulant protein deficiency

Dose depends on severity of the deficiency, but a satisfactory starting dose is 10 to 15 mL/kg body weight of Fresh Frozen Plasma or Plasma Frozen within 24 Hours After Phlebotomy.

Maternal clotting factors do not cross the placenta. Therefore, fetal levels depend on endogenous production. Clotting proteins are synthesized by the fetus beginning in the first trimester, with concentrations gradually increasing throughout gestation.55 At birth, the mean levels of the contact factors (Factors XII and XI, prekallikrein, and high-molecular-weight kininogen) are approximately 40% to 50% of adult values in term neonates and approximately 30% to 40% of adult values in preterm neonates. The vitamin-K-dependent factors (II, VII, IX, and X) are present at approximately 40% to 50% of adult values in term neonates and at approximately 20% to 50% of adult values in preterm neonates. Strikingly low levels are present in very immature neonates. Neonatal levels of Factors VIII and XIII and fibrinogen are comparable with adult levels, although the level of Factor XIII can be quite low in some infants at birth. The natural anticoagulant proteins (antithrombin and proteins C and S) are 30% to 50% of adult values. Fibrinolysis is less well studied but probably is diminished to a considerable degree because the plasminogen level is moderately low.

Plasma Transfusion Practices Clotting times are prolonged in neonates compared with results of older children and adults because of developmental deficiency of clotting proteins. Plasma should be transfused only after reference to normal values that take into account the birthweight and age of the infant. Guidelines for neonatal transfusion of plasma are presented in Table 30-6. Fresh Frozen Plasma (FFP) and Plasma Frozen within 24 Hours after Phlebotomy (PF24), whether prepared from whole blood units or by plasmapheresis, are used interchangeably. PF24 has up to a 25% loss of Factor VIII and less loss of Factor V; neither has clinical importance for the vast majority of patients receiving plasma transfusions. The indications for plasma transfusion include reconstitution of RBC concentrates to simulate whole blood for use in massive transfusions (eg, exchange transfusion, extracorporeal membrane oxygenation, or cardiovascular surgery), provision of multiple clotting factors for bleeding caused by disseminated intravascular coagulation or vitamin K deficiency, and treatment of congenital factor deficiencies when more specific treatment

Chapter 30: Blood Component Transfusions for Infants

(purified and virus-inactivated factor concentrates) is not available or the diagnosis of a specific factor deficiency has not been made (ie, multiple factors may need replacement). The use of prophylactic plasma transfusions to prevent intraventricular hemorrhage in preterm neonates is not recommended. Use of plasma as a suspending agent to adjust the hematocrit of RBC concentrates before small-volume transfusions (15 mL/kg) should be discouraged because plasma offers no medical benefit over simply transfusing red cells suspended in the preservative solution in which they are stored. Similarly, the use of plasma in partial exchange transfusion for the management of neonatal hyperviscosity syndrome (erythrocytosis) is unnecessary, because safer colloid solutions (eg, 5% albumin) are available. In the treatment of bleeding neonates, cryoprecipitate often is considered as an alternative to plasma because of its small volume. However, cryoprecipitate contains only fibrinogen, von Willebrand factor, and Factors VIII and XIII. It is not effective for managing the more extensive multi-factor clotting deficiencies that are commonly encountered—despite the desirability of a small infusion volume. Discussion of the transfusion of clotting factor concentrates is presented in detail in Chapter 28. However, it is important to point out that recombinant activated Factor VII has been effective in treating life-threatening bleeding in infants with deficiency and/or dysfunction of clotting proteins or platelets.56

Neutrophil Transfusion Pathophysiology of Neonatal Neutropenia Neonates are unusually susceptible to severe bacterial infections, and multiple abnormalities of neonatal body defenses contribute to their pathogenesis. Neonatal neutrophils have both quantitative and qualitative abnormalities related to the increased incidence, morbidity, and mortality of bacterial infections. These abnormalities include absolute and relative neutropenia, diminished chemotaxis, abnormal adhesion and aggregation, defective cellular orientation and receptor capping, decreased deformability, inability to alter membrane potential during stimulation, imbalances of oxidative metabolism, and a diminished ability to withstand oxidant stress.57 Neutropenia can occur during fulminant bacterial infection. Because physiologic neutrophilia occurs soon after birth, it is quite unusual for the absolute blood neutrophil count to decrease to less than 2000/µL during the first week of life. Although an abnormally low neutrophil count can occur in neonates with disorders as diverse as sepsis, asphyxia, and maternal hypertension, suspicion of severe bacterial infection must always be high whenever relative neutropenia (neutrophil count
2000/µL) occurs in a sick neonate. The mechanisms responsible for abnormal neonatal granulopoiesis are only partially defined. One factor is that the postmitotic marrow neutrophil storage pool (metamyelocytes and mature, segmented neutrophils) is inadequate during fulminant infection. The neutrophil storage pool accounts for 26% to 60% of all nucleated cells in the marrow of normal

neonates, whereas neonates with sepsis may have a storage pool numbering less than 10% of nucleated marrow cells. Thus, they have severely diminished marrow neutrophil reserves.58 Second, storage pool neutrophils are released at an excessively rapid and apparently poorly regulated rate from the marrow during stress. Third, committed (clonogenic) neutrophil precursors in neonatal marrow are fewer in number in neonates than in older patients, and most of these cells are actively proliferating even when studied at an apparently basal state.58,59 Thus, neonatal marrow functions at near capacity in basal state and is unable to either rapidly expand production or release stored neutrophils to meet the increased demands of infection. Additional discussion of fetal and neonatal hematopoiesis is presented in Chapter 25.

Neutrophil Transfusion Practices Because both quantitative and qualitative abnormalities of neonatal neutrophils occur, neutrophil transfusions have been reported to successfully treat neonatal sepsis.60 Neutrophil transfusions generally were given to neonates with fulminant sepsis and relative neutropenia (neutrophil count
2000 to 3000/µL during the first week of life or
1000/µL thereafter). Unfortunately, neutrophil transfusions have not provided a satisfactory solution to neonatal sepsis, and they are used rarely, if at all, for several reasons.60 Although neutrophil transfusion is efficacious for some infants with neutropenia and fulminant sepsis, only neutrophil concentrates obtained by means of automated leukapheresis for transfusion have demonstrated effectiveness. The controlled trials contain scientific flaws, and in many instances, conventional supportive care with antibiotics seemed equally efficacious.

Approach to Infants with Sepsis and Neutropenia Each institution must assess its own experience with neonatal sepsis. If nearly all infants survive without apparent long-term morbidity when treated only with antibiotics, neutrophil transfusions are unnecessary, and attention should be focused on prompt diagnosis and optimal antibiotic therapy. If the outcome of standard therapy is not optimal, alternative therapies must be considered to improve the outlook. Such therapies include intravenous immunoglobulin (IVIG) and/or myeloid cytokines such as granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Most studies evaluating prophylactic IVIG to prevent neonatal infections have shown either little or only modest benefit, whereas several therapeutic studies have shown a benefit to adding IVIG to antibiotics to treat neonatal infections.61 Data are insufficient to justify the use of IVIG as a standard of care for all preterm neonates to prevent or manage sepsis. However, it seems reasonable to give “physiologic” doses of IVIG (0.3 to 0.4 g/kg) to septic very-low-birthweight neonates because they are likely to have hypogammaglobulinemia as a result of extremely premature birth (ie, being born before major placental transport of immunoglobulin G has taken place). To date, properly designed clinical studies of recombinant myeloid growth factors given to

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Section II: Part II

human neonates are limited,62,63 and firm recommendations cannot be made for use of these agents in the management of neonatal neutropenia or sepsis. There is no universally accepted role for neutrophil transfusions, IVIG, or myeloid growth factors as a standard of practice to treat neonatal sepsis. Until more information becomes available, it seems reasonable to manage fulminant sepsis in neonates with neutropenia (neutrophil counts
2000/µL during the first week of life or
1000/µL thereafter) as follows. For infants born before 30 weeks of gestation, give one dose of 0.4 g/kg of IVIG to correct possible hypogammaglobulinemia plus 5 µg/kg of G-CSF or 10 µg/ kg GM-CSF on 3 consecutive days. For infants born at 30 weeks of gestation or after, give 5 µg/kg of G-CSF or 10 µg/kg GM-CSF on 3 consecutive days. This therapy should be adjunctive to optimal antibiotic and supportive care, and it must be given with parental consent and the understanding that its efficacy and potential toxicity are not completely understood. Actually, because of high plasma levels of endogenous G-CSF already present in some septic neonates, it has been cautioned that adding additional exogenous recombinant G-CSF offers no benefit.64

Acknowledgment This work was supported by National Institutes of Health grants P01 HL46925 and RR00059.

Disclaimer The author has disclosed no conflicts of interest.

References 1. Strauss RG. Current issues in neonatal transfusions. Vox Sang 1996;51:1-9. 2. Stockman JA. Anemia of prematurity: Current concepts in the issue of when to transfuse. Pediatr Clin North Am 1986;33:111-28. 3. Widness JA, Madan A, Grindeanu LA, et al. Reduction in red blood cell transfusions among preterm infants: Results of a randomized trial with an in-line blood gas and chemistry monitor. Pediatrics 2005;115:1299-306. 4. Madan A, Kumar R, Adams MM, et al. Reduction in red blood cell transfusions using a bedside analyzer in extremely low birth weight infants. J Perinatol 205;25:2-25. 5. Stockman III JA, Graeber JE, Clark DA, et al. Anemia of prematurity: Determinants of the erythropoietin response. J Pediatr 1984;105:786-92. 6. Brown MS, Garcia JF, Phibbs RH, et al. Decreased response of plasma immunoreactive erythropoietin to “available oxygen” in anemia of prematurity. J Pediatr 1984;105:793-8. 7. Rhondeau SM, Christensen RD, Ross MP, et al. Responsiveness to recombinant human erythropoietin of marrow erythroid progenitors from infants with “anemia of prematurity.” J Pediatr 1988;112:935-40. 8. Dame C, Fahnenstich H, Freitag P, et al. Erythropoietin mRNA expression in human fetal and neonatal tissue. Blood 1998;92:3218-25. 480

9. Snijders RJ, Abbas A, Melby O, et al. Fetal plasma erythropoietin concentration in severe growth retardation. Am J Obstet Gynecol 1993;168:615-19. 10. Widness JA, Susa JB, Garcia JF, et al. Increased erythropoiesis and elevated erythropoietin in infants born to diabetic mothers and in hyperinsulinemic rhesus fetuses. J Clin Invest 1981;67:637-42. 11. Ohls RK, Li Y, Trautman MS, Christensen RD. Erythropoietin production by macrophages from preterm infants: Implications regarding the cause of the anemia in prematurity. Pediatr Res 1994;35:169-70. 12. Widness JA, Veng-Pedersen P, Peters C, et al. Erythropoietin pharmacokinetics in premature infants: Developmental, nonlinearity, and treatment effects. J Appl Physiol 1996;80:140-8. 13. Strauss RG. Managing the anemia of prematurity: Red blood cell transfusions versus recombinant erythropoietin. Transfus Med Rev 2001;15:213-23. 14. Bell EF, Strauss RG, Widness JA, et al. Randomized trial of liberal versus restrictive guidelines for red blood cell transfusions in preterm infants. Pediatrics 2005;115:1685-91. 15. Kirpalani H, Whyte RK, Andersen C, et al. The Premature Infants in Need of Transfusion (PINT) study: A randomized, controlled trial of a restrictive (low) versus liberal (high) transfusion threshold for extremely low birth weight infants. J Pediatr 2006;149:301-7. 16. Strauss RG. Red blood cell transfusion practices in the neonate. Clin Perinatol 1995:22:641-55. 17. Ramasethu J, Luban NL. Red blood cell transfusions in the newborn. Semin Neonatol 1999:4:5-16. 18. Stockman JA, Clark DA. Weight gain: A response to transfusion in selected preterm infants. Am J Dis Child 1984;138:828-35. 19. Phillips HM, Holland BM, Abdel-Moiz A, et al. Determination of red-cell mass in assessment and management of anaemia in babies needing blood transfusion. Lancet 1986;i:882-4. 20. Jones JG, Holland BM, Veale KE, Wardrop CA. “Available oxygen,” a realistic expression of the ability of the blood to supply oxygen to tissues. Scand J Haematol 1979;22:77-82. 21. Alverson DC, Isken VH, Cohen RS. Effect of booster blood transfusions on oxygen utilization in infants with bronchopulmonary dysplasia. J Pediatr 1988;113:722-6. 22. Strauss RG, Mock DM, Johnson K, et al. Circulating red blood cell (RBC) volume, measured using biotinylated RBCs, is superior to the hematocrit to document the hematologic effects of delayed versus immediate umbilical cord clamping in preterm neonates. Transfusion 2003;43:1168-72. 23. Strauss RG, Villhauer PJ, Cordle DG. A method to collect, store and issue multiple aliquots of packed red blood cells for neonatal transfusions. Vox Sang 1995;68:77-81. 24. Luban NLC, Strauss RG, Hume HA. Commentary on the safety of red blood cells preserved in extended storage media for neonatal transfusions. Transfusion 1991;31:229-35. 25. Strauss RG. Data-driven blood banking practices for neonatal RBC transfusions. Transfusion 2000;40:1528-40. 26. Strauss RG, Burmeister LF, Johnson K, et al. AS-1 red blood cells for neonatal transfusions: A randomized trial assessing donor exposure and safety. Transfusion 1996;36:873-8. 27. Strauss RG, Burmeister LF, Johnson K, et al. Feasibility and safety of AS-3 red blood cells for neonatal transfusions. J Pediatr 2000;136:215-19. 28. Strauss RG, Burmeister LF, Johnson K, et al. Randomized trial assessing feasibility and safety of biological parents as red blood cell donors for their preterm infants. Transfusion 2000;40:450-6.

Chapter 30: Blood Component Transfusions for Infants

29. Walsh TS, McArdle F, McLellan SA, et al. Does the storage time of transfused red blood cells influence regional or global indexes of tissue oxygenation in anemic critically ill patients? Crit Care Med 2004;32:364-71. 30. Weiskopf RB, Feiner J, Hopf H, et al. Fresh blood and aged stored blood are equally efficacious in immediately reversing anemiainduced brain oxygenation deficits in humans. Anesthesiology 2006;104:911-20. 31. Strauss RG, Mock DM, Widness JA, et al. Post-transfusion 24-hour recovery and subsequent survival of allogeneic red blood cells in the bloodstream of newborn infants. Transfusion 2004;44:871-6. 32. Hilsenrath P, Nemechek J, Widness JA, et al. Cost-effectiveness of a limited donor blood program for neonatal RBC transfusions. Transfusion 1999;39:938-43. 33. Vamvakas EC, Strauss RG. Meta-analysis of controlled clinical trials studying the efficacy of recombinant human erythropoietin in reducing blood transfusions in the anemia of prematurity. Transfusion 2001;41:406-15. 34. Donato H, Vain N, Rendo P, et al. Effect of early versus late administration of human recombinant erythropoietin on transfusion requirements in premature infants: Results of a randomized, placebo-controlled, multicenter trial. Pediatrics 2000;105:1066-72. 35. Yeo CL, Choo S, Ho LY. Effect of recombinant human erythropoietin on transfusion needs in preterm infants. J Paediatr Child Health 2001;37:352-8. 36. Meyer MP, Sharma E, Carsons M. Recombinant erythropoietin and blood transfusion in selected preterm infants. Arch Dis Child Fetal Neonatal Ed 2003;88:F41-F45. 37. Maier RE, Obladen M, Müller-Hansen I, et al on behalf of the European Multicenter Erythropoietin Beta Study Group. Early treatment with erythropoietin β ameliorates anemia and reduces transfusion requirements in infants with birth weights below 1000 g. J Pediatr 2002;141:8-15. 38. Avent M, Cory BJ, Galpin J, et al. A comparison of high versus low dose recombinant human erythropoietin versus blood transfusion in the management of anemia of prematurity in a developing country. J Trop Pediatr 2002;48:227-33. 39. Franz AR, Pohlandt F. Red blood cell transfusions in very and extremely low birthweight infants under restrictive transfusion guidelines: Is exogenous erythropoietin necessary? Arch Dis Child Fetal Neonatal Ed 2001;84:F96-F100. 40. Amin AA, Alzahrani DM. Efficacy of erythropoietin in premature infants. Saudi Med J 2002;23:287-90. 41. Anderson A, Fangman J, Wager G, Uden D. Retrieval of placental blood from the umbilical vein to determine volume, sterility, and presence of clot formation. Am J Dis Child 1992;146:36-9. 42. Bifano EM, Dracker RA, Lorah K, Palit A. Collection and 28-day storage of human placental blood. Pediatr Res 1994;36:90-4. 43. Strauss RG. Autologous transfusions for neonates using placental blood. A cautionary note. Am J Dis Child 1992;146:21-2. 44. Mercer JS, Vohr BR, McGrath MM, et al. Delayed cord clamping in very preterm infants reduces the incidence of intraventricular hemorrhage and late-onset sepsis: A randomized, controlled trial. Pediatrics 2006;117:1235-42. 45. Castle V, Andrew M, Kelton J, et al. Frequency and mechanism of neonatal thrombocytopenia. J Pediatr 1986:108:749-55. 46. Castro V, Kroll H, Origa AF, et al. A prospective study on the prevalence and risk factors for neonatal thrombocytopenia and platelet

47. 48. 49.

50.

51.

52.

53.

54. 55.

56. 57.

58.

59.

60. 61.

62.

63.

64.

alloimmunization among 9332 unselected Brazilian newborns. Transfusion 2007;47:59-66. Andrew M, Castle V, Saigal S, et al. Clinical impact of neonatal thrombocytopenia. J Pediatr 1987;110:457-64. Castle V, Coates G, Kelton JG, Andrew M. 111In-oxine platelet survivals in thrombocytopenic infants. Blood 1987;70:652-6. Sola-Visner MC, Christensen RD, Hutson AD, Rimsza LM. Megakaryocyte size and concentration in the bone marrow of thrombocytopenic and nonthrombocytopenic neonates. Pediatr Res 2007;61:479-84. Murray NA, Roberts IA. Circulating megakaryocytes and their progenitors in early thrombocytopenia in preterm neonates. Pediatr Res 1996;40:112-19. Sola MC, Calhoun DA, Hutson AD, Christensen RD. Plasma thrombopoietin concentrations in thrombocytopenic and nonthrombocytopenic patients in a neonatal intensive care unit. Br J Haematol 1999;104:90-2. Pastos KM, Slayton W, Rimsza LM, et al. Differential effects of recombinant thrombopoietin and bone marrow stromal conditioned media on neonatal vs adult megakaryocytes. Blood 2006;108:3360-2. Andrew M, Vegh P, Caco C, et al. A randomized trial of platelet transfusions in thrombocytopenic premature infants. J Pediatr 1993;123:285-91. Lupton BA, Hill A, Whitfield MF, et al. Reduced platelet count as a risk factor for intraventricular hemorrhage. Am J Dis Child 1988;142:1222-4. Reverdiau-Moalic P, Delahousse B, Body G, et al. Evolution of blood coagulation activators and inhibitors in the healthy human fetus. Blood 1996;88:900-8. Mathew P, Young G. Recombinant factor VIIa in paediatric bleeding disorders—a 2006 review. Haemophilia 2006;12:457-72. Rosenthal J, Cairo MS. Neonatal myelopoiesis and immunomodulation of host defenses. In: Petz LD, Swisher SN, Kleinman S, et al, eds. Clinical practice of transfusion medicine. 3rd ed. New York: Churchill Livingstone, 1995:685-704. Christensen RD, MacFarlane JL, Taylor NL, et al. Blood and marrow neutrophils during experimental group B streptococcal infection: Quantification of the stem cell, proliferative, storage and circulating pools. Pediatr Res 1982;16:549-54. Erdman SH, Christensen RD, Bradley PP, Rothstein G. Supply and release of storage neutrophils: A developmental study. Biol Neonate 1982;41:132-7. Strauss RG. Current status of granulocyte transfusions to treat neonatal sepsis. J Clin Apher 1989;5:25-9. Jenson HB, Pollock BH. Meta-analyses of the effectiveness of intravenous immune globulin for prevention and treatment of neonatal sepsis. Pediatrics 1997;99:E2. Schibler KR, Osborne KA, Leung LY, et al. A randomized, placebocontrolled trial of granulocyte colony-stimulating factor administration to newborn infants with neutropenia and clinical signs of early-onset sepsis. Pediatrics 1998;102:6-13. Cairo MS, Agosti J, Ellis R, et al. A randomized, double-blind, placebo-controlled trial of prophylactic recombinant human granulocyte-macrophage colony-stimulating factor to reduce nosocomial infections in very low birth weight neonates. J Pediatr 1999;134:64-70. Calhoun DA, Lunøe M, Du Y, et al. Granulocyte colony-stimulating factor serum and urine concentrations in neutropenic neonates before and after intravenous administration of recombinant granulocyte colony-stimulating factor. Pediatrics 2000;105:392-7.

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PART III

Oncology Patients

31

Transfusion Support for the Oncology Patient Christopher A. Tormey1 & Edward L. Snyder2 1

Instructor, Department of Laboratory Medicine, Yale University School of Medicine, Yale-New Haven Hospital, New Haven, Connecticut, USA 2 Professor of Laboratory Medicine, Yale University School of Medicine, and Director, Blood Bank/Apheresis Service, Yale-New Haven Hospital, New Haven, Connecticut, USA

Transfusion support for patients with cancer is a complicated, multifaceted medical therapy meant to overcome complications related to chemotherapy, radiation, transplantation, or widespread metastatic disease. Oncology patients are often substantially immunosuppressed and may require chronic transfusion support. For these patients, greater attention must be paid to the preparation, modification, and response to blood components to help ensure better outcomes. Furthermore, with recent evidence that drugs such as erythropoietin may be particularly harmful to oncology patients, heavier reliance on allogeneic blood transfusion may be necessary in the immediate future. This chapter highlights the most challenging aspects of transfusion therapy for oncology patients encountered by clinicians and transfusion services on a routine basis.

Red Cell Transfusion Indications Red cell transfusions should be used to increase the oxygencarrying capacity of whole blood for oncology patients with anemia. Unfortunately, there are no oncology-specific evidencebased target laboratory criteria for the transfusion of Red Blood Cell (RBC) units. Therefore, oncology patients should be transfused on a symptomatic basis or at predetermined hemoglobin or hematocrit measurements as defined by oncology services. When clinicians apply standards used in other critically ill populations, and consider the underlying comorbid diseases, most oncology patients should tolerate hemoglobin levels of 7 to 10 g/dL without the need for allogeneic transfusions.1 There is no evidence to show that sustained increases in hemoglobin over 10 g/dL provide any therapeutic benefit for ill patients; in fact, “hypertransfusion” strategies may actually be harmful to patient outcome.2 Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

482

Selection of ABO Group for RBC Transfusion For the majority of oncology patients, no specific consideration is given to selection of ABO group of RBC units so long as the unit is ABO compatible with the recipient. However, for patients who have undergone allogeneic hematopoietic stem cell transplantation (HSCT), the choice of ABO group can be difficult, particularly if there is an ABO mismatch between hematopoietic progenitor cell (HPC) donor and recipient. ABO incompatibility, although not a barrier to HSCT, does complicate posttransplant management. Patients receiving ABO-mismatched transplants are known to require more transfusions because of delayed cellular engraftment and red cell aplasia. These patients are also at risk for acute and delayed red cell hemolysis caused by ABO incompatibility during HPC reinfusion and as engraftment takes place.3-7 Despite these complications, numerous studies have shown that ABO-mismatched HSCT does not adversely affect long-term survival or outcome in either pediatric or adult populations.3,8,9 Although ABO mismatch does not seem to have long-term drawbacks, providing appropriate blood component support immediately following transplantation can be a complex endeavor for a transfusion service. In the setting of allogeneic transplantation, there are several possibilities for ABO antibody mismatch. Patients may have a minor mismatch, wherein the donor possesses antibodies against recipient cells. This would occur when a patient with group A red cells receives HPCs from a group O donor. Another possibility is a major mismatch, wherein a recipient harbors antibodies directed against donor cells. This would occur when a patient with group O red cells receives HPCs from a group A donor. For minor incompatibility, a strategy to reduce hemolysis during HPC infusion is plasma reduction to remove, or significantly lower, titers of offending antibodies.10 Similarly, for major mismatches, techniques for red cell depletion can prevent immediate hemolysis during infusion.11 After transplantation, the selection of blood components can be difficult, because antibodies can persist for weeks or months after stem cell rescue.12,13 Additionally, following HSCT,

Chapter 31: Transfusion Support for the Oncology Patient

patients may present as chimeras with two distinct blood group populations seen on routine type and screening tests.12 Thus, strategies have been developed to provide the most appropriate ABO component for these patients. For major incompatibility, with recipient anti-A or anti-B directed against donor red cells, it is necessary to use recipient-type RBC units until ABO antibodies are no longer detectable.10,14,15 For minor incompatibility, recipient-type plasma and platelets are necessary until the recipient’s red cells are no longer detectable. Donor-type RBC units can be used immediately after HSCT for minor incompatibility.10,14,15 For a patient with both major and minor incompatibility (“two-way” incompatibility), group O RBC units and group AB plasma and platelets should be used until offending antibodies and cells are no longer detectable. Tables 31-1 and 31-2 summarize major and minor incompatibility by blood group and provide a transfusion protocol for ABO-mismatched HSCT.10,14,15 Table 31-2 provides a suitable protocol to follow.

Alloimmunization to Red Cell Antigens Despite significant immunosuppression, patients with malignancies may still mount immune responses to “foreign” red cell antigens.16 This not only complicates transfusion, but

Table 31-1. Compatibility by ABO Group in Hematopoietic Stem Cell Transplantation Donor ABO Blood Group Recipient ABO Blood Group

O

A

B

AB

O

C

M

M

M

A

m

C

Mm

M

B

m

mM

C

M

AB

m

m

m

C

C ⫽ compatible; M ⫽ major incompatibility; m ⫽ minor incompatibility; mM ⫽ both major and minor incompatibility (“two-way” incompatibility).

also may increase the risk for hemolysis in patients undergoing allogeneic HSCT.17 Rates for red cell alloimmunization have been most extensively studied in patients with hematologic malignancies undergoing HSCT and range from 2% to 8% in these populations.18,19 Other retrospective or prospective studies are necessary to better define the red cell alloimmunization rate for patients with solid tumors and other oncologic problems.

Alternatives to Allogeneic Red Cell Transfusion For patients unwilling, or unable, to undergo allogeneic transfusion, several alternatives are available (see Chapter 37). In theory, red cell alternatives should reduce infectious and immunologic complications of allogeneic transfusion while providing similar therapeutic benefit. Several perioperative techniques have been developed to curb allogeneic blood use in the operative setting (see Chapter 38). Acute normovolemic hemodilution (ANH) involves the removal of whole blood immediately before a surgical procedure with crystalloid or colloid volume replacement.20 If bleeding occurs, the blood is readily available for reinfusion at the bedside. ANH has been shown to reduce the need for allogeneic transfusion in multiple studies.20 Intraoperative blood recovery—a process whereby shed whole blood is centrifuged, washed of contaminants and debris, and then reinfused to the patient—is also of benefit in reducing allogeneic blood usage.21 Although some concerns about promotion of tumor cell metastasis with the use of blood recovery and reinfusion have been raised, several trials conducted in the setting of surgery for hepatobiliary and genitourinary cancers have shown no evidence of decreased survival in these patients.22-24 Furthermore, irradiation of the recovered blood has also been shown to prevent poor outcome in oncology patients undergoing surgery.25 The use of preoperatively donated autologous whole blood can also be considered, although this is often not feasible for oncology patients who are too anemic and who can require surgery at unpredictable times. Despite the irregular scheduling of oncologic surgery, autologous whole blood donation has been successfully utilized for patients with prostate cancer and

Table 31-2. Transfusion Protocol for HSCT Patients Recipient

Donor O

Donor A

Donor B

Donor AB

RBC type

FFP/PLT type

2nd choice PLT

RBC type

FFP/PLT type

2nd choice PLT

RBC type

FFP/PLT type

2nd choice PLT

RBC type

FFP/PLT type

2nd choice PLT

0

O

O

A or

O

A

O

O

B

O

O

AB

A

A

O

A

O

A

A

O

O

AB

A

A

AB

A

B

O

B

O

O

AB

A

B

B

O

B

AB

B

AB

O

AB

A

A

AB

A

B

AB

B

AB

AB

A or B

NOTE: Rh positive recipients with Rh negative donors receive Rh negative products. Rh negative recipients with Rh positive donors receive Rh positive products.

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Section II: Part III

gynecologic malignancies.26,27 It is recommended, however, that the use of preoperative autologous donation be restricted to oncology patients undergoing major surgery, because it is not necessarily a cost-effective therapy for all patients.28 The routine administration of erythropoietin-stimulating agents (ESAs) to patients with cancer-related anemia was a strategy developed to reduce allogeneic transfusion within oncology populations.29 However, the chronic use of ESAs for anemia in patients with cancer, as well as for those with other critical illnesses, has come under scrutiny. Several large trials have demonstrated that ESA administration may lead to decreased patient survival and increased risk for thrombosis.30-32 It has been postulated that erythropoietin administration accelerates tumor growth, accounting for decreased survival trends.33 It has also been found that ESAs do not necessarily significantly decrease the need for allogeneic transfusion in critically ill patients.30-32,34 The results of these trials prompted the Food and Drug Administration (FDA) to issue a “black box warning” for the use of ESAs in the setting of cancer-related anemia.35 More data and clinical trials will be necessary to ascertain the ultimate safety and efficacy profile for the use of ESAs in oncology patients.

Component Modification: Irradiation and Leukocyte Reduction Because of their immunosuppressed state, many oncology patients are at high risk for transfusion-associated graft-vs-host disease (TA-GVHD), wherein donor lymphocytes generate a profound

immune response against the recipient’s cells.36 The causes and manifestations of TA-GVHD are discussed below (see Adverse Reactions to Blood Transfusion). Prevention of TA-GVHD can be accomplished with gamma irradiation of cellular blood components.36 Table 31-3 summarizes disorders for which gamma irradiation is prudent.37 Some within the field of transfusion medicine have argued for a policy of universal blood component irradiation. The process has few side effects and broad application of irradiation may help to eliminate TA-GVHD in the occasional patient who is at risk for TA-GVHD, but whose high-risk status is not known to the physicians or other caregivers. The practicality and cost-effectiveness of such a policy has not been determined.38 Irradiation of red cells does have the disadvantage of inducing a red cell membrane potassium leak, which shortens the shelf life of the RBC unit to no more than 28 days from the date of irradiation. In addition to irradiation, blood collection centers and blood banks have implemented prestorage leukocyte reduction techniques to improve transfusion outcomes. Leukocyte reduction of blood components is particularly beneficial for oncology patients. By decreasing the absolute numbers of white cells in RBC units, alloimmunization to HLA antigens is reduced. Leukocyte reduction has also had an ameliorating effect on immunologic refractoriness to platelet transfusions, a significant problem for chronically transfused oncology patients.39,40 Unfortunately, prestorage leukocyte reduction has not shown a benefit in the reduction of red cell antigen alloimmunization.41 Beyond alloimmunization, leukocyte reduction has the potential

Table 31-3. Indications for Gamma Irradiation of Cellular Blood Components Disease State

Hematologic malignancies

Acute myelogenous leukemia Acute lymphocytic leukemia Allogeneic marrow transplantation Autologous marrow transplantation Myelodysplastic syndrome Hodgkin lymphoma Non-Hodgkin lymphoma

Nonhematologic malignancies

Glioblastoma multiforme Neuroblastoma Rhabdomyosarcoma

Immunosuppressed states

Congential immunodeficiency High-dose chemotherapy (eg, fludarabine) Solid organ transplantation

Blood Components

Red cells

All components for children ⬍4 months All directed donations

Platelets

All components for children ⬍4 months All directed donations All apheresis products, including HLA-matched platelets

Granulocytes

484

All granulocyte products

Chapter 31: Transfusion Support for the Oncology Patient

to prevent passage of some tranfusion transmitted diseases— cytomegalovirus (CMV) and other pathogens residing within white cells are efficiently filtered via leukocyte reduction.39 Furthermore, studies have shown a reduction in febrile transfusion reactions associated with leukocyte filtration of blood components.39 According to the 2007 National Blood Collection and Utilization Survey, approximately 72% of all RBC units in the United States in 2006 were leukocyte reduced, most of them before storage.42

transfusion criteria, a recent study suggests that persistent and profound thrombocytopenia leads to greater patient mortality. This retrospective analysis of patients following HSCT showed greater risk for mortality for those individuals who received platelets according to stringent (ie, ⬍10,000/µL) criteria.55 Mortality in these cases, not associated with bleeding episodes, may imply some other protective benefit to increased platelet counts in oncology patients. Prospective studies on long-term patient survival and outcome with conservative approaches to platelet transfusion may yet again change the landscape for the treatment of thrombocytopenic oncology patients.

Platelet Transfusion Indications

Selection of ABO Group and Rh Type for Platelet Transfusion

Platelet transfusion for oncology patients is intended to stop or prevent bleeding in the setting of thrombocytopenia. The use of platelet transfusion for acute hemorrhage in the patient with low platelet counts, or receiving antiplatelet medication, is a first-line therapy.43 Much greater controversy exists, however, in the consideration of platelet transfusion for bleeding prophylaxis. Historically, the practice of providing platelet transfusions on a prophylactic basis arose out of studies involving patients with leukemia who were noted to have decreased bleeding episodes following platelet transfusion.44 Until the early 1990s, routine bleeding prophylaxis included platelet transfusions for oncology patients with platelet counts less than 20,000/µL.45 However, as clinical experience and evidence mounted, it was found that oncology patients could tolerate much lower platelet counts without development of severe bleeding. A number of clinical trials, beginning in 1991, demonstrated that spontaneous hemorrhage was unlikely even at platelet counts as low as 5000 to 10,000/µL. Bleeding episodes and morbidity in these patients were no worse than for those individuals transfused at higher “trigger” platelet counts.46-51 These studies also found that stringent transfusion criteria led to a significant reduction in transfused platelet products, thereby reducing the risks associated with chronic transfusion exposure. The evidence provided by these clinical trials led to the implementation of more conservative platelet transfusion triggers. Most oncology and hematology guidelines now recommend platelet transfusion only in patients with platelet counts less than 10,000/µL in the absence of bleeding.52,53 It is important to recognize that clinical history, presentation, and comorbid states influence the decision to transfuse platelets. For instance, a patient undergoing a major surgical procedure or one who has a history of platelet dysfunction may require higher baseline platelet counts or more frequent platelet transfusion. For patients undergoing major invasive procedures (eg, central venous catheter placement before HPC collection) goals of platelet counts of 40,000 to 50,000/µL are an appropriate target, while procedures such as marrow aspiration can be safely performed at platelet counts less than 20,000/µL.54 Of note, although clinical trials have shown no increased bleeding complications associated with stringent platelet

The ABO antigens are expressed on several platelet proteins and lipids, although the interactions between these antigens and circulating host antibodies do not mediate clinically significant transfusion reactions.56 Thus, most blood banks will transfuse ABO-mismatched platelets to adults without significant concern for incompatibility. However, for the oncology patient, the use of ABO-mismatched platelet products could result in poor outcome and decreased therapeutic benefit. For instance, a form of platelet refractoriness is mediated by ABO antibodies, with mismatched platelets cleared from circulation minutes to hours after infusion.56 Furthermore, there is some evidence to suggest that ABO incompatibility can promote the development of HLA alloimmunization in multitransfused patients.56 Another concern is the possibility for hemolytic transfusion reactions secondary to high-titer or high-avidity ABO antibodies present in the plasma fraction of platelet pools. Although rare, there are several case reports of high-titer ABO antibodies mediating severe hemolysis in patients with hematologic and oncologic diseases receiving apheresis platelet products.57-60 Children in particular may demonstrate clinically significant hemolysis to ABO-incompatible platelet transfusions given their small blood volume. Therefore, it is advisable that oncology patients with low platelet counts receive ABO-compatible platelet products if at all feasible. For populations undergoing HSCT, other considerations regarding major and minor mismatches are also relevant. Table 31-2 summarizes guidelines for platelet transfusion therapy in the peritransplant period. Although there are no Rh antigens on platelets, the concern for Rh alloimmunization during platelet transfusion arises secondary to the presence of residual D-antigen-positive red cells in platelet products collected from Rh-positive donors. Even a few milliliters of red cells are sufficient to cause alloimmunization in Rh-negative recipients, because of the immunogenicity of the D-antigen. There is evidence to suggest that, in part due to immunosuppression, both adult and pediatric patients with hematologic malignancies are unlikely to form anti-D responses to Rh-incompatible platelet transfusions.61,62 Thus, the provision of Rh-incompatible platelet products is likely a safe procedure and unlikely to cause D alloimmunization in oncology populations. Nonetheless, it is still advisable to prevent Rh

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alloimmunization in children and females of childbearing potential, because of the consequences of anti-D development in these populations. Alloimmunization can be prevented with a dose of 250 IU (50 µg) of intravenous Rh Immune Globulin provided within 72 hours of D antigen exposure.63

Platelet Refractoriness and Alloimmunization In the chronically transfused oncology patient, increases in platelet count may be lower than expected following transfusion. This phenomenon, known as platelet refractoriness, can be attributed to both immune and nonimmune causes. In oncology patients, approximately 70% to 80% of cases of platelet refractoriness are caused by nonimmunologic factors.43 Table 31-4 provides a list of the most common variables associated with nonimmune platelet refractoriness. For the patient with nonimmunologic platelet refractoriness, few clinical management options exist. Addressing the underlying cause (eg, splenectomy for hypersplenism, removal of an offending medication) can often alleviate refractoriness. For the acutely bleeding patient or for hemorrhage prophylaxis, strategies of continuous platelet infusion (“platelet drip”) have been attempted with moderate success.54,64 The “platelet drip,” wherein a dose of platelet concentrate is infused slowly over a 4-hour period, is intended to provide an ongoing source of platelets to maintain vascular integrity, while addressing the practical concern of blood bank platelet inventory conservation. An underlying antibody to platelet antigens is most often responsible for the remaining 20% to 30% of platelet refractoriness in oncology patients. Platelets express Class I HLA and numerous other platelet-specific antigens. Most commonly, HLA antigens mediate immunologic platelet refractoriness, although the chronically transfused patient may develop alloantibodies to any platelet antigen.43,65 Treatment strategies for immune-mediated platelet refractoriness center on providing antigen-negative components. The first and simplest strategy for managing immune platelet refractoriness is to provide ABO-compatible units. The next option would be

to obtain crossmatch-compatible platelets from the hospital blood bank or donor center. This process involves crossmatching of patient serum against a variety of donor platelets and selection of those products for which no agglutination reaction is observed.66 Platelet count responses to crossmatch-compatible platelets may be sustainable for 2 to 3 days, but this is also dependent on the age of the unit and other underlying clinical factors. If the majority of tested donors are incompatible with recipient serum, HLA typing of the recipient and provision of HLA antigen-matched platelet products is likely necessary.43,65 Polymorphisms of HLA Class I antigens can complicate compatibility testing and make the process of finding fully matched donors very difficult. The Duquesnoy grading system was developed to allow for the most advantageous use of partially mismatched donations.67 Newly developed computerized algorithms, based on characterization of HLA polymorphisms, are paving the way for broader matches that may help reduce the need to provide partially incompatible platelet products.68 Crossmatch-compatible platelets have been proven to be as efficacious as HLA-matched platelets in raising platelet counts in alloimmunized patients.43,65,66 Thus, the decision to provide HLA-matched platelets should depend upon factors such as quality of the HLA match, severity of alloimmunization, and availability of crossmatched platelet products. In many cases, crossmatch-compatible platelets can be more quickly and easily obtained and are available at lower cost than HLA-matched products. The best strategy to reduce immune-mediated platelet refractoriness is prevention of HLA and platelet antigen exposures. The trend toward universal leukocyte reduction has helped reduce the incidence of alloimmune platelet refractoriness by limiting exposure to HLA antigens.40 Conservative transfusion strategies also help to prevent exposure to various HLA and platelet-specific antigens and may play a role in reducing the frequency of alloimmunization. Unfortunately, once alloimmunization has occurred, immune-modulation with corticosteroids, plasmapheresis, and intravenous immune globulin have been of little benefit.69

Table 31-4. Most Common Causes of Refractoriness to Platelet Transfusion Category

Cause

Nonimmuologic refractoriness

Acute bleeding/consumption Fever (any cause) Infection Medications (eg, amphotericin B) Microangiopathy or disseminated intravascular coagulation Splenomegaly

Immunologic refractoriness

ABO-mismatched transfusion HLA and/or platelet-specific alloantibodies Autoimmune thrombocytopenia (eg, immune thrombocytopenic purpura)

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Alternatives to Platelet Transfusion For patients unwilling to undergo platelet transfusion, or for severe platelet refractoriness, several medical therapies can aid in bleeding episodes. A commonly prescribed drug, 1-deamino-8D-arginine vasopressin (DDAVP), acts to stimulate the release of von Willebrand factor from endothelial cells, which can enhance platelet activity even at very low platelet counts.70 DDAVP is also of proven benefit for platelet dysfunction and bleeding associated with uremia.71 Antifibrinolytic therapies have also been employed as an adjunct to platelet transfusions for the bleeding patient. Medications such as aminocaproic acid, tranexamic acid, and aprotinin have all been successfully used to reduce hemorrhage and allogeneic transfusion requirements in the bleeding patient.72,73

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Chemical and cytokine-based stimulation of the marrow to endogenously increase platelet production has also been attempted. Agents such as thrombopoietin (TPO) and megakaryocyte growth factors have been synthesized, but clinical trials with some of these drugs led to the development of neutralizing antibodies and thrombocytopenia in recipients.74,75 TPO and TPO-like growth factors are finishing clinical trials and may be considered for FDA licensure in the near future. One such agent, AMG531 (Nplate, Amgen, Thousand Oaks, CA), has been licenced by the FDA. Interleukin (IL)-11, a cytokine that drives megakaryocyte production and division, also has been approved for use in thrombocytopenia.74,75 The development of other similar cytokine agents is currently under way.74,75

Selection of Platelet Products Platelet products in the United States, Canada, and Europe can be prepared in several ways. In the past, the majority of platelet units available for transfusion were derived, via centrifugation, from whole blood.43 These individual units are pooled in groups of four to five to yield a product intended to raise platelet counts by approximately 50,000/µL. Thus, platelet pools consist of individual units from multiple donors. Since the 1970s, apheresis techniques have been developed to allow for the collection of full doses of platelet products from one donor.76 Apheresis platelets have grown in popularity and usage, and provide a platelet dose virtually equivalent to pooled platelets.43 Thus, the decision to use platelet pools or platelets from one donor relates more to concerns about exposures to multiple donors, risks for alloimmunization, and platelet quality rather than increases in baseline platelet count. The primary consequence of transfusions obtained from multiple donors is an increased risk for transfusion-transmitted disease.77 For the heavily immunosuppressed oncology patient, this fact alone would seemingly favor the use of apheresis platelets. However, with more rigorous donor screening and sensitive nucleic acid testing, the risk of transfusion-transmitted disease is virtually the same between platelet product options. In addition, even with the use of apheresis products, chronically transfused oncology patients are likely to be exposed to a multitude of donors over the course of their disease. Thus, the use of one apheresis platelet unit in an overall transfusion strategy has minimal benefit in reducing infectious disease risks. Thus, from an infectious disease standpoint, apheresis platelets are not necessarily preferred. Another consideration in exposure to multiple donors is increased risk for platelet alloimmunization. In the era before leukocyte reduction, this was a significant concern for pooled products. However, with the advent of leukocyte reduction, a patient is as likely to become alloimmunized to a platelet pool as to a platelet product from an individual donor.40 Thus, there is minimal advantage in using apheresis platelets to prevent alloimmunization. Once a patient has become alloimmunized, the use of platelet pools may be advantageous until crossmatch-compatible or HLA-matched units are available. In this scenario, an

alloimmunized thrombocytopenic patient may be more likely to respond to one of the four or five donors whose units constitute a platelet pool. For the alloimmunized patient, apheresis platelets should be employed only if the unit is crossmatch-compatible or HLA-matched.43 The quality of platelet products does differ somewhat between pooled and apheresis units. Markers of platelet activation, such as P-selectin, are increased in whole-blood-derived platelet concentrates as compared to apheresis platelets.78 These changes, however, have not correlated with poor in-vivo platelet recovery. Thus, current evidence does not suggest that apheresis platelets are a more viable platelet option. From the standpoint of adverse reactions to platelet transfusion, large trials have revealed no difference in adverse events between apheresis and whole-blood-derived products.79 Bacterial contamination does remain an outstanding concern for pooled platelets given the number of donors and various manipulations of the unit before issuance of the final product. Methods to address the contamination of pooled products, such as prestorage pooling with sampling for bacterial cultures, may help to identify contaminated units.80 By comparison, apheresis products do offer a lower risk for bacterial contamination, but the risk for contamination has not been completely eliminated.81,82 Indeed, 7-day apheresis platelets have been reported to be associated with greater bacterial contamination than 5-day apheresis platelets. Thus, from the standpoint of platelet quality, apheresis platelets seem to offer little advantage when compared to platelet concentrate pools.

Platelet Product Modification: Irradiation, Leukocyte Reduction, and Volume Reduction As is the case with RBC units, platelets can be modified to maximize safety for oncology patients. Despite leukocyte reduction, platelet units contain small amounts of white cells. Thus, patients at risk for TA-GVHD should receive irradiated platelets.36 Table 31-3 summarizes those malignant conditions for which gamma irradiation is appropriate. As mentioned previously, leukocyte reduction of platelet products helps to reduce alloimmunization and refractoriness.40 According to the 2007 National Blood Collection and Utilization Survey, approximately 40% of all whole-blood-derived platelet units were leukocyte reduced, and essentially all apheresis platelets were leukocyte reduced.42 The movement toward universal leukocyte reduction in the United States should help ease the burden of alloimmunization in coming years. Volume reduction of platelet products via centrifugation and resuspension in saline should be considered for oncology patients who have experienced severe allergic and/or anaphylactic reactions to platelet products.83 Volume reduction is also efficient at removing the plasma fraction of platelet products to reduce the risk of hemolysis associated with ABO-incompatible transfusion.83 Volume reduction and saline resuspension should be considered for ABO-mismatched platelet transfusions in infants and children with small blood volumes.

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Plasma and Cryoprecipitated AHF Transfusion Indications, Selection of ABO Group, and Product Modification Plasma transfusions should be used to stop or prevent bleeding in patients with coagulopathy caused by vitamin K antagonists, factor consumption, or liver disease.84,85 Cryoprecipitated AHF can be used to treat bleeding associated with uremia or Factor XIII deficiency associated with severe GVHD.71,86 Plasma should not be used as the sole fluid for volume replacement because of limited supplies and the risks associated with blood transfusion. For the majority of oncology patients, no specific additional consideration is given to selection of ABO group for plasma or cryoprecipitate so long as the unit is ABO compatible with the recipient. Donor and recipient Rh types are not a consideration for infusion of plasma or cryoprecipitate. As mentioned previously, for patients who have undergone allogeneic HSCT, the choice of ABO group can be complex and should be based upon consideration of major and minor mismatches. Table 31-2 summarizes guidelines for plasma transfusion therapy in the peri-transplant period; guidelines for cryoprecipitated AHF are identical to those for plasma. Plasma and cryoprecipitated AHF are maintained at temperatures below ⫺20ºC; because they are acellular, there is no need for gamma irradiation.

Alternatives to Plasma Transfusion For some oncology patients, plasma transfusion may be contraindicated, may be ineffective, or (in the setting of acute hemorrhage) may not provide rapid enough reversal of coagulopathic states. For these conditions, there are several alternatives to plasma products, mostly consisting of recombinant coagulation factors. Among the most commonly used agents for acute moderate to severe bleeding is recombinant activated Factor VII, a potent activator of the coagulation cascade.87 Successful use of Factor VIIa has been reported to help control massive bleeding in a variety of oncology patients.88-91 There is also a role for use of Factor VIIa as a bypass agent for those oncology patients who acquire inhibitors to circulating coagulation factors, mainly Factor VIII.92 Factor VIIa failures in bleeding oncology patients have also been reported; a patient with refractory gastrointestinal bleeding associated with GVHD following HSCT showed no improvement of hemorrhage following Factor VIIa administration.93 The instances of Factor VIIa usage cited above highlight the difficulty of recommending broad application of this therapy to the bleeding oncology patient. The most significant problem with making an evidence-based recommendation is that the vast majority of uses of Factor VIIa in the literature are in the form of case reports. Few adequate clinical trials have been conducted to determine the safety and efficacy of this drug in oncology populations. Furthermore, appropriate dosing regimens are imprecise and mostly based on data gathered in trials performed in other critically ill patient populations. Factor VIIa also carries a number of risks, chief of which is the possibility for development

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of severe or even fatal thromboembolism.94 Thus, Factor VIIa may be considered as an alternative to plasma infusion, but should be used only after other interventions have failed.

Intravenous Immune Globulin Intravenous immune globulin (IVIG) has become a very useful product in the management of oncology patients.95 Consisting of human gamma globulins, IVIG has been used to provide passive immunity in highly immunosuppressed individuals. IVIG has also been successfully used as immunomodulatory therapy for patients with conditions such as immune thrombocytopenic purpura (ITP).96,97 For recipients of HPC products, IVIG has been associated with improved immune defense against pathogens such as CMV and has helped to decrease the complications of acute GVHD.98,99 The benefit from use of IVIG for chronic GVHD and infection prophylaxis in marrow transplantation is not clear and warrants further study.99 IVIG infusion is usually tolerated without significant adverse events in oncology patients. The common side effects of administration include myalgias, headache, fever, and fatigue.100 A rare but serious complication of IVIG administration is acute renal failure seen with particular formulations using sucrose as a globulin stabilizer.100 Thus, oncology patients with chronic renal insufficiency should be closely monitored during IVIG administration. Current IVIG products have not transmitted hepatitis B, hepatitis C, or human immunodeficiency virus; there are rare case reports of transmission of viruses such as parvovirus B19, a serious pathogen for oncology patients.101 Plasma is now screened for this virus before being pooled for fractionation.

Granulocyte Transfusion Indications Granulocyte transfusion is a therapeutic option used almost exclusively in oncology patients. Classically, granulocyte infusions have been used to treat severe, antibiotic-refractory bacterial or fungal infections in patients with an absolute neutrophil count of less than 500/µL. Only patients with a reasonable chance at sustainable marrow recovery after resolution of the underlying infection are candidates for granulocyte products.102 Granulocyte infusion may be useful for patients who have received ablative chemotherapy regimens and who are not able to mount an adequate cellular response to infection. In general, to overcome a significant bacterial or fungal infection, humans should produce 2 to 3 ⫻ 1011 polymorphonuclear cells per day, numbers that often cannot be achieved for the immunosuppressed oncology patient.103 The transfusion of exogenous granulocytes can provide additional innate immunity. Doses of 1 ⫻ 1011 granulocytes per square meter of body surface area have been reported to increase neutrophil counts by 1000 to 2000/µL are higher.103 In the past, the most significant barrier to the use of granulocytes was obtaining an adequate dose from a healthy donor. Trials performed in the 1970s and 1980s, using whole blood filtration

Chapter 31: Transfusion Support for the Oncology Patient

and centrifugation techniques, showed little clinical benefit likely secondary to suboptimal granulocyte doses.102 By the mid1980s granulocyte infusion therapy was largely abandoned. The technique gained renewed interest in the early 1990s primarily because of the development of granulocyte colony-stimulating factor (G-CSF).104 The administration of G-CSF to healthy donors allowed for nearly 10-fold increases in granulocyte collection yield. G-CSF has led to higher granulocyte doses and has the added benefit of promoting persistent elevation in recipient white count after infusion, attributable to mobilization of both mature granulocytes and earlier myeloid precursors in donor products.102 Improvements in apheresis technology have also helped to increase white cell collection efficiency. Over the last 10 to 15 years, a number of trials have been performed to evaluate the safety and efficacy of granulocyte transfusion using G-CSF stimulation in healthy donors. Studies performed in both adult and pediatric populations have found that granulocyte transfusion leads to significant improvement in patients with fungal infections refractory to antimicrobial drugs.105–110 Granulocyte doses in these studies were routinely 5 to 10 ⫻ 1010 with resultant increases in peripheral white count of 2000 to 5000/µL. There is also a growing body of literature on the prophylactic use of granulocyte transfusion to prevent onset or recurrence of severe infection.106,111–113 While this therapy may have some efficacy in prevention of fungal infection recurrence, the role of granulocytes as an agent for primary infection prophylaxis remains unclear. To determine the true efficacy of granulocyte transfusion in these settings, future randomized, controlled trials will be necessary.102

Selection of ABO Group and Donor Preparation Healthy donors who qualify for granulocyte transfusion should be ABO-compatible with the intended recipient; products must be crossmatched before infusion because of high red cell content. A popular granulocyte mobilization regimen used in donors consists of the administration of 5 to 10 µg/kg G-CSF subcutaneously, in combination with 8 mg oral dexamethasone, both administered approximately 12 hours before leukapheresis.114 Granulocyte collections can be performed serially over 4 to 5 days to yield multiple doses for neutropenic patients. For granulocyte donors, side effects are usually mild and include bone pain, headache, fatigue, and fluid retention caused by G-CSF administration and apheresis.102,110,114

Alloimmunization The large number of white cells, red cells, and platelets contained within a granulocyte product increases the potential for red cell, HLA, and platelet antigen alloimmunization in oncology patients.115 Unfortunately, most donor centers do not have the time to match donors and recipients according to HLA antigen expression given the urgent need to supply the granulocyte product. There is evidence to show that those patients previously alloimmunized to HLA antigens via chronic transfusion demonstrate reduced response to granulocyte infusion.116 Thus, the

expectation for successful response to granulocyte therapy should be tempered in an individual who has previously demonstrated HLA alloimmunization. For these patients, HLA-antigen matching may be necessary to improve outcome of granulocyte transfusion.

Alternatives to Granulocyte Transfusion The decision to initiate granulocyte transfusion usually represents failure of other forms of therapy. In these cases, few other treatment options exist. The administration of G-CSF to the neutropenic patient may aid in the production of endogenous granulocytes and is a potential treatment strategy for infection prophylaxis.117 For a reduction in fungal infection recurrence, at least one study has shown that the antifungal agent voricanizole is successful in preventing microorganism reactivation.118

Granulocyte Product Storage, Modification, and Infusion Granulocytes must be stored at 20 to 24ºC without agitation.119 Granulocytes must be infused within 24 hours of collection because of the limited life span of neutrophils and preferably should be given within 6 to 8 hours of collection. Before issuance, the granulocyte product should undergo gamma irradiation to prevent recipient TA-GVHD.120 It is important to remind clinical staff that granulocytes should be infused through a 170to 260-micron filter, but that leukocyte reduction filters should never be used.121

Adverse Reactions to Blood Transfusion Oncology patients undergoing transfusion therapy are subject to the same set of adverse reactions as any patient receiving blood components. However, in the oncology patient, discerning between an adverse transfusion event and worsening of an underlying condition can be difficult. Furthermore, the immunosuppressed oncology patient may be at greater risk for transfusion-transmitted infections and TA-GVHD when compared with immune-competent hosts. This section briefly explores those adverse reactions to transfusion that are most relevant to the oncology patient.

Transfusion-Associated Graft-vs-Host Disease The treatment of many malignant diseases relies heavily upon immunosuppressive drugs (eg, purine analogs such as fludarabine) and high-dose ablative chemotherapy. These therapies place oncology patients at particularly high risk for development of TA-GVHD.122,123 In addition to immune suppression, transfusion of blood from HLA homologous donors places the recipient at increased risk for TA-GVHD.124 This can be particularly problematic for HLA antigen-matched blood or directed donations from family members. TA-GVHD is common, with estimates that up to 1% of patients with hematologic malignancies can develop this condition at some point during their care.122 In all cases of TA-GVHD, depletion of T-cell immunity allows donor lymphocytes to generate immune responses

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against host tissue. TA-GVHD usually manifests within 2 weeks of transfusion as acutaneous rash with fever, diarrhea, liver function abnormalities, nausea, and vomiting.125 Unfortunately, after the onset of TA-GVHD, there are few therapeutic options. Administration of corticosteroids, antithymocyte globulin, and cyclosporine have been attempted but to date have shown little efficacy. A diagnosis of TA-GVHD carries mortality approaching 100%.125 As mentioned previously, the best strategy to eliminate TAGVHD is to prevent its occurrence. The most successful and proven mode of prevention is gamma irradiation of blood components. The AABB specifies a radiation dose of 2500 cGy directed at the center of the blood component with a minimum dosage of 1500 cGy achieved in all portions of the unit.126 Only cellular components (ie, RBCs, platelets, and granulocytes) require irradiation. Leukocyte reduction is thought to contribute to prevention of GVHD.122 However, leukocyte reduction must not be relied on as the sole modality for GVHD prophylaxis because the residual white counts at which GVHD is prevented are unknown.122 If such cellular components are given to susceptible patients, the component(s) must be irradiated.

Transfusion-Transmitted Diseases Cytomegalovirus (CMV) is among the most problematic of the transfusion-transmitted agents for the oncology patient. It is a highly prevalent virus, with data indicating that 40% to 50% of healthy adults carry CMV antibodies.127 Within blood donor populations, CMV has been detected in mononuclear cells of both seropositive and seronegative individuals, making the identification of true CMV-negative donors difficult.128 For the immune-competent host, CMV can usually be controlled through various immune mechanisms and is rarely clinically significant. For the oncology patient, however, CMV infection can have serious consequences including pneumonia, gastrointestinal inflammation/infection, and delayed engraftment of myeloid cell lines for recipients of HSCT products. For the oncology patient who has never been exposed to CMV or for the severely immunosuppressed individual, there are several options for blood transfusion. CMV-seronegative blood components can be obtained from blood donor centers, although ABO-compatible components may be hard to find as the majority of urban adults are already CMV positive. Additionally, as mentioned above, even CMV-seronegative donors may harbor residual virus within their white cells. Leukocyte reduction of blood components is thus an excellent alternative to reduce the risk of CMV transmission. Studies have shown that leukocyte removal is equally efficient as CMV-seronegative blood components for prevention of viral transmission.129,130 Despite these data, several other studies suggest that CMV virus transmission can occur even with leukocyte reduction efforts.131,132 Currently, there is general consensus that in the absence of blood components that are definitively seronegative, leukocyte reduction is an acceptable alternative for reducing transmission of CMV to oncology patients.

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Parvovirus B19, a nonenveloped, single-stranded DNA virus, infects erythroid precursors, leading to suppression of red cell production.133 For immunosuppressed patients, parvovirus infection can result in severe erythroid hyperplasia and the development of red cell aplasia.133 Fortunately, active parvovirus is not found in many healthy blood donors. Two recent European studies showed that less than 1% of blood donors actively carry parvovirus in peripheral blood as detected by polymerase chain reaction (PCR) methods.134-136 There have been only occasional case reports documenting transmission of parvovirus B19 to hematology and oncology patients. In these cases, patients develop characteristic red cell aplasia and often remain chronically transfusion dependent.137-139 The rarity of reports of transmission of this virus may indicate low infectivity via transfusion, or may represent lack of disease recognition on the part of clinicians and transfusion service personnel. A large retrospective study showed that the actual risk of parvovirus transmission to oncology patients is quite low. In this study, 1% of all blood components from a hospital blood bank tested positive for parvovirus DNA by PCR. Of all virus-contaminated units, 14 were received by patients with underlying hematologic malignancies and none developed signs, symptoms, or laboratory findings consistent with parvovirus infection.140 Because of the low rates of transmission, the cost efficacy and clinical utility of screening all blood donors for this organism has yet to be determined.134,136 In order to be most prudent in the application of parvovirus testing, screening of units intended for high-risk patients has been suggested as a strategy for prevention of disease transmission.141 West Nile virus (WNV) is a mosquito-borne pathogen that emerged in 1999 as a significant cause of morbidity and mortality in the United States. The presentation of WNV can be highly variable; patients can be completely asymptomatic or experience a spectrum of symptoms ranging from mild fever and fatigue to severe encephalitis.142 In addition to mosquito-spread illness, transmission of WNV via blood transfusion has been repeatedly documented.143 Although any transfusion recipient is susceptible to WNV infection, there is some evidence to suggest that immunosuppressed individuals, such as those undergoing chemotherapy and transplantation, are at higher risk for developing some of the more severe neurologic complications of WNV infection.144 United States and Canadian blood centers have implemented routine nucleic acid testing of blood donors using advanced algorithms to match the seasonal nature of virus infectivity.145 Current screening techniques should help to limit the exposure of oncology patients to WNV. Trypanosoma cruzi, transmitted via the bite of the reduviid bug, is the blood-borne parasite responsible for Chagas’ disease. After initial exposure of the host to the parasite, Chagas’ disease manifests as a transient acute illness.146 Over a number of years, the trypanosome mediates ongoing destruction of multiple organ systems, causing chronic cardiac, gastrointestinal, and neurologic disease resistant to treatment with current antimicrobial drugs.146 Chagas’ disease is endemic in Latin America, but thou-

Chapter 31: Transfusion Support for the Oncology Patient

sands of individuals in North America also carry the disease.146 Transmission of T. cruzi via transfusion has been reported in several patients in the United States and Canada; most reported cases have occurred in severely immunocompromised oncology patients.147–150 The seeming exclusivity of transmission to oncology patients is thought to be caused by underlying host immunosuppression.147 As a result of the growing risk for trypanosome transmission in North America, US and Canadian blood centers have undertaken efforts to broaden laboratory and medical history screening measures to better assess donor risk factors for Chagas’ disease.151,152 As with other pathogens, increased vigilance should help to reduce the devastating effects of trypanosome transmission in oncology patients. In addition to thorough donor history screening and a battery of serologic and nucleic acid tests, there have been several advancements in the field of pathogen inactivation that could be helpful to oncology patients undergoing transfusion.153 For instance, there is growing interest in photochemical treatment of blood components involving the addition of the chemical amotosalen with subsequent long-wave ultraviolet irradiation.154,155 This technique—which causes disruption of pathogen DNA, preventing microorganism activation and proliferation—has shown great efficacy in reducing the infectivity of a number of pathogens harmful to oncology patients including CMV, parvovirus B19, and T. cruzi.154,156,157 If clinical trials and safety data continue to look promising, pathogen inactivation should likely further improve transfusion safety for the oncology patient. As of this writing, amotosalen-treated blood components are not FDA-licensed in the United States.

Transfusion-Related Acute Lung Injury Transfusion-related acute lung injury (TRALI) represents the development of noncardiogenic pulmonary edema in patients undergoing transfusion therapy. TRALI has been theorized to be caused by antibodies that activate neutrophils passing through pulmonary vasculature.158 These antibodies are believed to be directed against HLA antigens and are more frequently found in donor blood than in recipient plasma.158 Oncology patients, per se, are not at increased risk for TRALI as compared to other patient populations. However, particular blood components used in oncology populations, such as granulocytes, can be associated with increased risks for TRALI. Pulmonary problems associated with granulocyte infusion can be particularly severe for patients who previously have been alloimmunized to HLA antigens. There is also some evidence to suggest that concurrent administration of granulocytes and the antifungal agent amphotericin B can lead to higher rates of pulmonary complications.159 Thus, it is recommended that granulocyte infusion and amphotericin B therapy be spaced out over time to avoid pulmonary decompensation.

Acute Hemolytic Transfusion Reactions Acute intravascular red cell hemolysis wherein red cells are destroyed can be a catastrophic complication of blood transfusion, leading to renal failure, shock, hypotension, and death. As

with TRALI, oncology patients are not necessarily at increased risk for hemolysis because of their underlying disease state. Nonetheless, a series of case reports has documented severe hemolysis occurring in oncology patients receiving ABOincompatible apheresis platelet products with high-titer ABO agglutinins.57–60 Thus, it is recommended that oncology patients, particularly children, receive ABO-compatible platelet pools whenever possible. If no other options exist and ABOincompatible platelets must be provided, volume reduction with removal of incompatible plasma can greatly reduce the risk of hemolysis.83

Febrile Nonhemolytic Transfusion Reactions Most of the blood components provided to oncology patients can cause rigors, chills, and transient elevation in body temperature. Unfortunately, it is often difficult to distinguish between the signs and symptoms of a febrile nonhemolytic transfusion reaction (FNHTR) and new-onset fever caused by infection in an immunosuppressed patient. Some of the clinical characteristics of FNHTRs may be helpful in distinguishing between these entities. FNHTRs involve an increase in temperature of at least 1ºC (1.8ºF) during or within 2 hours of the completion of a transfusion.160 Fevers that begin more than 2 hours after the completion of transfusion are most likely due to another cause. Symptoms of FNHTRs typically appear during the latter half of a transfusion as causative cytokines or agents accumulate in circulation.160 Despite clues on presentation, FNTHR ultimately remains a diagnosis of exclusion. For the oncology patient who experiences fever during transfusion, a full evaluation by transfusion service personnel and the clinical team is necessary to rule out other causes for temperature elevation. Another strategy to deal with FNHTRs in oncology populations is to prevent their occurrence. In patients who have had repeated febrile reactions to blood transfusion, an antipyretic agent (eg, acetaminophen) given 30 minutes before transfusion can decrease adverse febrile events.160 Aspirin or aspirincontaining products should not be given to thrombocytopenic patients. Aspirin acetylates the platelet enzyme cyclooxygenase and this induces a thrombocytopathy, preventing the platelet release reaction. Prestorage leukocyte reduction of blood components has also been shown to reduce the incidence of FNHTRs.161

Transfusion-Associated Immunomodulation The administration of allogeneic blood components can have consequences for immune function in all patient populations; most notable among these are apparent decreases in cellular immunity. These deleterious immune effects have been reported in several non-oncologic populations wherein transfusion is noted to suppress the immune system.162,163 The immunomodulatory effects of allogeneic transfusion for oncology patients are slowly being unraveled. Retrospective studies indicate that allogeneic transfusion in cancer patients could lead to immunosuppression, promoting solid tumor recurrence

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and increased susceptibility to primary and reactivated viral infection.164-166 Conversely, pediatric literature suggests that allogeneic transfusion has little negative effect on outcome for children with acute lymphoblastic leukemia; other groups of pediatric oncology patients have not been extensively studied.167 Several animal studies have recapitulated the tumor recurrence effects of allogeneic transfusion seen in adults.168-172 These studies, and others performed in vitro, have identified several candidates that may be responsible for the immunosuppressive effects including ubiquitin, fas and fas-ligand, soluble HLA peptides, and other mediators derived from the mononuclear elements of blood.163,171,173 Particular focus has been placed on the relationship between leukocyte reduction of blood components and their immunomodulatory effects. Prestorage leukocyte reduction appears to reduce the immunosuppressive effects of transfusion, perhaps by prophylactic removal of soluble immune mediators secreted by white cells that would otherwise accrue during storage.169 Future models and randomized, controlled trials may help clinicians better classify this immunologic phenomenon and address its potential causes.

Adverse Effects of Hematopoietic Progenitor Cell Infusion The majority of allogeneic and autologous HPCs are cryopreserved using solutions containing 10% dimethylsulfoxide (DMSO).174 DMSO is a unique chemical modifier that allows for the controlled freezing and thawing of mononuclear cells without development of membrane lysis.174 Traditionally, the adverse events associated with HPC infusion have been associated with the DMSO content of the progenitor product. DMSO is a toxic substance and has been linked to fever, nausea, vomiting, and chills during or immediately after HPC infusion; patients receiving fresh, noncryopreserved cells generally experience these symptoms to a much less degree.175 DMSO has also been linked to pulmonary and cardiovascular problems during HPC infusion including dyspnea, hypotension, bradycardia, and arrhythmia.174,175 The number of HPC units being infused and the size of the individual receiving the infusion are believed to enhance DMSO toxicity. Small children and those patients receiving multiple HPC units are more susceptible to DMSO-related problems.174 As clinical experience and evidence have been compiled, researchers have begun to evaluate other components of the HPC product as they related to infusion toxicity. Several recent studies have correlated adverse events to the numbers of granulocytes present in HPC products.176,177 These studies found that even after DMSO depletion in the cellular product, 50% to 60% of patients undergoing HPC infusion experienced an adverse event such as fever, rigors, or dyspnea. It is thus possible that the dyspnea associated with HPC infusion is not entirely caused by DMSO, but rather is mediated by granulocytes through TRALIlike mechanisms. These studies argue that a reduction in granulocyte content of the final HPC product leads to better tolerance of infusion. The challenge would be to remove the unwanted granulocytes while leaving behind the lifesaving CD34⫹ cells.

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In addition to the adverse events associated with DMSO and granulocyte content, patients undergoing HPC infusion often experience symptoms similar to those seen after traditional blood component transfusion. TRALI and anaphylactic reactions during autologous and allogeneic HPC infusions have been reported.178,179 These reactions are rare, but must be considered part of the differential diagnosis for a patient presenting with shortness of breath or cardiovascular complications following HPC infusion. Bacterial contamination of HPC products, occurring during collection or processing phases, is also of concern.180 HPCs are routinely cultured and, if bacterially contaminated, are provided to patients in conjunction with prophylactic broadspectrum antibiotics before infusion. A recent study showed that of 13 patients who received bacterially contaminated HPCs, only one developed evidence of bacteremia180 Thus, with appropriate vigilance and antibiotic therapy, bacterial contamination of an HPC product need not be an absolute barrier to infusion. Many efforts are under way to reduce the toxicity and side effects that are associated with HPC infusion. The largest target has been DMSO; many studies have shown that reducing the content of this additive from 10% to 5% of the final product volume does not negatively affect HPC viability during storage, thawing, or reinfusion.174 There are also devices undergoing evaluation that can be used to “wash” HPC products, effectively reducing DMSO content before infusion.181 With regard to DMSO-related symptoms, premedication with antipyretic and antihistamine as medications 30 minutes before infusion may help alleviate unpleasant side effects such as fever and rigors and lives. Adequate field sterilization practices during stem collection and the processing of HPC products in sterile areas with air-controlled fume hoods can also help to reduce risks of bacterial contamination.182

Summary Transfusion therapy plays a major role in the care of patients with hematologic or oncologic disorders; however, blood transfusion carries risks for these patients that are generally of less concern for other general hospital populations. For oncology patients, RBC transfusions are given for increased oxygen-carrying capacity, platelets for the cessation or prevention of bleeding due to thrombocytopenia, plasma for coagulopathies, and granulocytes for pervasive bacterial and fungal infections. All of these components can create special problems for oncology patients including TA-GVHD, transfusion-transmitted diseases, alloimmunization to blood cell antigens, pulmonary decompensation, and immunomodulation. Patients undergoing HPC transplantation are a unique group and present complex concerns related to transfusion, including major and minor ABO incompatibility and chimeric blood cells. Transfusion for patients undergoing treatment with cellular therapies requires careful blood component selection. The process of HPC infusion itself carries many risks including DMSO toxicity and hemolytic reactions. In all areas of transfusion therapy, new advances such as pathogen inactivation and synthetic alternatives

Chapter 31: Transfusion Support for the Oncology Patient

to whole blood should help to increase the safety and tolerance of blood component infusion within oncology populations.

12.

Acknowledgment The authors acknowledge the outstanding work of the late Margot S. Kruskall, MD, whose original text for the 3rd edition of Rossi’s Principles of Transfusion Medicine formed the template for this chapter in the 4th edition.

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Disclaimer C. Tormey has disclosed no conflicts of interest. E. Snyder has disclosed financial relationships with Fenwal, CaridianBCT, Terumo, Pall Corporation.

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products for the prevention of transfusion-associated CMV infection after marrow transplant. Blood 1995;86:3598-603. Narvios A, Lichtiger B. Bedside leukoreduction of cellular blood components in preventing cytomegalovirus transmission in allogeneic bone marrow transplant recipients: A retrospective study. Haematologica 2001;86:749-52. Vamvakas E. Is white blood cell reduction equivalent to antibody screening in preventing transmission of cytomegalovirus by transfusion? A review of the literature and meta-analysis. Transfus Med Rev 2005;19:181-99. Nichols WG, Price TH, Gooley T, et al. Transfusion-transmitted cytomegalovirus infection after receipt of leukoreduced blood products. Blood 2003;101:4195-200. Parsyan A, Candotti D. Human erythrovirus B19 and blood transfusion—an update. Transfus Med 2007;17:263-78. Henriques I, Monteiro F, Meireles E, et al. Prevalence of parvovirus B19 and hepatitis A virus in Portuguese blood donors. Transfus Apher Sci 2005;33:305-9. Zaaijer H, Koppelman M, Farrington C. Parvovirus B19 viraemia in Dutch blood donors. Epidemiol Infect 2004;132:1161-6. Bonvicini F, Gallinella G, Gentilomi GA, et al. Prevention of iatrogenic transmission of B19 infection: Different approaches to detect, remove or inactivate virus contamination. Clin Lab 2006;52: 263-8. Yilmaz S, Oren H, Demircio lu F, et al. Parvovirus B19: A cause for aplastic crisis and hemophagocytic lymphohistiocytosis (case report). Pediatr Blood Cancer 2006;47:861. Plentz A, Hahn J, Holler E, et al. Long-term parvovirus B19 viraemia associated with pure red cell aplasia after allogeneic bone marrow transplantation. J Clin Virol 2004;31:16-9. Cohen BJ, Beard S, Knowles WA, et al. Chronic anemia due to parvovirus B19 infection in a bone marrow transplant patient after platelet transfusion. Transfusion 1997;37:947-52. Plentz A, Hahn J, Knöll A, et al. Exposure of hematologic patients to parvovirus B19 as a contaminant of blood cell preparations and blood products. Transfusion 2005;45:1811-5. Brown KE, Young NS, Alving BM, Barbosa LH. Parvovirus B19: Implications for transfusion medicine. Summary of a workshop. Transfusion 2001;41:130-5. Hayes E, Gubler D. West Nile virus: Epidemiology and clinical features of an emerging epidemic in the United States. Annu Rev Med 2006;57:181-94. Pealer LN, Marfin AA, Petersen LR, et al for the West Nile Virus Transmission Investigation Team. Transmission of West Nile virus through blood transfusion in the United States in 2002. N Engl J Med 2003;349:1236-45. Kumar D, Humar A. Emerging viral infections in transplant recipients. Curr Opin Infect Dis 2005;18:337-41. Vamvakas EC, Kleinman S, Hume H, Sher GD. The development of West Nile virus safety policies by Canadian blood services: guiding principles and a comparison between Canada and the United States. Transfus Med Rev 2006;20:97-109. Moncayo A, Ortiz-Yanine M. An update on Chagas disease (human American trypanosomiasis). Ann Trop Med Parasitol 2006;100:663-77. Young C, Losikoff P, Chawla A, et al. Transfusion-acquired Trypanosoma cruzi infection. Transfusion 2007;47:540-4. Cimo P, Luper W, Scouros M. Transfusion-associated Chagas’ disease in Texas: Report of a case. Tex Med 1993;89:48-50.

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149. Nickerson P, Orr P, Schroeder ML, et al. Transfusion-associated Trypanosoma cruzi infection in a non-endemic area. Ann Intern Med 1989;111:851-3. 150. Grant IH, Gold JW, Wittner M, et al. Transfusion-associated acute Chagas’ disease acquired in the United States. Ann Intern Med 1989;111:849-51. 151. Comeau P. Canadian Blood Services to screen for Chagas’ disease. CMAJ 2007;77:242. 152. Blood donor screening for Chagas’ disease—United States, 2006-2007. MMWR Morb Mortal Wkly Rep 2007;56:141-3. 153. Snyder E, Dodd R. Reducing the risk of blood transfusion. Hematology Am Soc Hematol Educ Program 2001;433-42. 154. Roback JD, Conlan M, Drew WL, et al. The role of photochemical treatment with amotosalen and UV-A light in the prevention of transfusion-transmitted cytomegalovirus infections. Transfus Med Rev 2006;20:45-56. 155. Snyder E, McCullough J, Slichter SJ, et al for the SPRINT Study Group. Clinical safety of platelets photochemically treated with amotosalen HCl and ultraviolet A light for pathogen inactivation: The SPRINT trial. Transfusion 2005;45:1864-75. 156. Sawyer L, Hanson D, Castro G, et al. Inactivation of parvovirus B19 in human platelet concentrates by treatment with amotosalen and ultraviolet A illumination. Transfusion 2007;47:1062-70. 157. Castro E, Gironés N, Bueno JL, et al. The efficacy of photochemical treatment with amotosalen HCl and ultraviolet A (INTERCEPT) for inactivation of Trypanosoma cruzi in pooled buffy-coat platelets. Transfusion 2007;47:434-41. 158. Bux J, Sachs U. The pathogenesis of transfusion-related acute lung injury (TRALI). Br J Haematol 2007;136:788-99. 159. Dutcher JP, Kendall J, Norris D, et al. Granulocyte transfusion therapy and amphotericin B: Adverse reactions? Am J Hematol 1989;31:102-8. 160. Heddle N. Febrile nonhemolytic transfusion reactions. In: Popovsky M, ed. Transfusion reactions. 3rd edition. Bethesda, MD: AABB Press, 2007:57-103. 161. Paglino JC, Pomper GJ, Fisch GS, et al. Reduction of febrile but not allergic reactions to RBCs and platelets after conversion to universal prestorage leukoreduction. Transfusion 2004;44:16-24. 162. Blajchman M. Transfusion immunomodulation or TRIM: What does it mean clinically? Hematology 2005;10:208-14. 163. Vamvakas E, Blajchman M. Transfusion-related immunomodulation (TRIM): An uupdate. Blood Rev 2007;21:327-48. 164. Blumberg N, Heal J. Transfusion-induced immunomodulation and its possible role in cancer recurrence and perioperative bacterial infection. Yale J Biol Med 1990;63:429-33. 165. Chung M, Steinmetz O, Gordon P. Perioperative blood transfusion and outcome after resection for colorectal carcinoma. Br J Surg 1993;80:427-32. 166. Vamvakas E. Perioperative blood transfusion and cancer recurrence: Meta-analysis for explanation. Transfusion 1995;35:760-8. 167. Freiberg AS, Hancock ML, Kunkel KD, et al. Transfusions and risk of failure in childhood acute lymphoblastic leukemia. Leukemia 1994;8:1220-3.

168. Blajchman MA, Bardossy L, Carmen R, et al. Allogeneic blood transfusion-induced enhancement of tumor growth: Two animal models showing amelioration by leukodepletion and passive transfer using spleen cells. Blood 1993;81:1880-2. 169. Bordin J, Bardossy L, Blajchman M. Growth enhancement of established tumors by allogeneic blood transfusion in experimental animals and its amelioration by leukodepletion: The importance of the timing of the leukodepletion. Blood 1994;84:344-8. 170. Clarke P, Burton R, Wood K. Allogeneic blood transfusion reduces murine pulmonary natural killer (NK) activity and enhances lung metastasis of a syngeneic tumour. Int J Cancer 1993;55:996-1002. 171. Hashimoto MN, Kimura EY, Yamamoto M, Bordin JO. Expression of Fas and Fas ligand on spleen T cells of experimental animals after unmodified or leukoreduced allogeneic blood transfusions. Transfusion 2004;44:158-63. 172. Okuyama M, Motoyama S, Saito S, et al. Soluble and cell-associated forms of some yet to be identified factor in transfused blood which promotes solid tumor growth in mice. Surg Today 2004;34:673-77. 173. Patel M, Proctor K, Majetschak M. Extracellular ubiquitin increases in packed red blood cell units during storage. J Surg Res 2006;135:226-32. 174. Fleming K, Hubel A. Cryopreservation of hematopoietic and nonhematopoietic stem cells. Transfus Apher Sci 2006;34:309-15. 175. Alessandrino P, Bernasconi P, Caldera D, et al. Adverse events occurring during bone marrow or peripheral blood progenitor cell infusion: Analysis of 126 cases. Bone Marrow Transplant 1999;23:533-7. 176. Calmels B, Lemarie C, Esterni B, et al. Occurrence and severity of adverse events after autologous hematopoietic progenitor cell infusion are related to the amount of granulocytes in the apheresis product. Transfusion 2007;47:1268-75. 177. Cordoba R, Arrieta R, Kerguelen A, Hernandez-Navarro F. The occurrence of adverse events during the infusion of autologous peripheral blood stem cells is related to the number of granulocytes in the leukapheresis product. Bone Marrow Transplant 2007;40:1063-7. 178. Essayan D, Schilder R, Kagey-Sobotka A, et al. Anaphylaxis during autologous peripheral blood progenitor cell infusion. Bone Marrow Transplant 1997;19:749-52. 179. Urahama N, Tanosaki R, Masahiro K, et al. TRALI after the infusion of marrow cells in a patient with acute lymphoblastic leukemia. Transfusion 2003;43:1553-7. 180. Kelly M, Roy DC, Labbe AC, Laverdiere M. What is the clinical significance of infusing hematopoietic cell grafts contaminated with bacteria? Bone Marrow Transplant 2006;38:183-8. 181. Calmels B, Houze P, Hengesse JC, et al. Preclinical evaluation of an automated closed fluid management device: Cytomate, for washing out DMSO from hematopoietic stem cell grafts after thawing. Bone Marrow Transplant 2003;31:823-8. 182. Lowder J, Whelton P. Microbial contamination of cellular products for hematolymphoid transplantation therapy: Assessment of the problem and strategies to minimize the clinical impact. Cytotherapy 2003;5:377-90.

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Hematopoietic Growth Factors (Cytokines) W. Conrad Liles1 & David C. Dale2 1

Professor and Vice-Chair of Medicine, Director, Division of Infectious Diseases, University of Toronto, Toronto, Ontario, Canada 2 Professor of Medicine, Department of Medicine, University of Washington, Seattle, Washington, USA

Hematopoietic growth factor (HGF) is an inclusive term for the family of glycoproteins that regulate proliferation and differentiation of hematopoietic cells.1-5 Many of these factors also influence cell function. The term cytokine, a natural product of cells that influence other cells, is sometimes used as a synonym for growth factor. Some cytokines that function as HGFs are also called interleukins (ILs), regulatory factors produced by leukocytes that influence functions of other leukocytes. Colony-stimulating factor (CSF) is used in naming some cytokines that stimulate hematopoietic cells to grow in vitro in culture systems, such as granulocyte CSF (G-CSF), granulocyte-macrophage CSF (GM-CSF), and macrophage CSF (M-CSF). Another CSF, formerly called multiCSF, is now most commonly called IL-3. The HGFs, their names, and principal functions are outlined in Table 32-1.

Cell Biology and Physiology The HGFs share some general structural features among themselves and with other cytokines, but each is the product of a different gene and has a distinctive amino acid sequence, secondary and tertiary structures, receptor-binding domain, and glycosylation sites.2,3 Some HGFs, such as erythropoietin, are produced by relatively few cells at selected tissue sites (eg, the juxtaglomerular cells of the kidney). Others, such as G-CSF, are produced almost ubiquitously by endothelial cells, fibroblast cells, and many other cell types. The factors governing production and secretion also vary substantially. The feedback regulation of erythropoietin in relation to tissue oxygenation is well characterized. Although the relationships governing leukocyte production and deployment are less well understood, it is known that complete absence of G-CSF or its receptor leads to severe neutropenia.6-8 Improved immunoassay systems have greatly aided understanding of the pharmacokinetics and tissue binding of HGFs. Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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Neutrophilia occurs with many acute inflammatory conditions and in some malignant diseases, and is largely attributable to the increased production and levels of G-CSF.9-11 Levels of G-CSF are elevated in patients with severe neutropenia who have fever and infection.10 However, assay systems for G-CSF are not sensitive enough to determine whether there is a true direct feedback relationship between plasma levels of G-CSF and blood neutrophil counts. The presence of high-affinity receptors for the CSFs on both immature and mature neutrophils further confounds the study of these relationships. Levels of some other cytokines that influence neutrophil formation (eg, IL-1 and IL-6) also increase with inflammation.12 In contrast, serum levels of GM-CSF, another factor that stimulates neutrophil formation, do not correlate with the presence of neutropenia or infection.10 Levels of thrombopoietin (TPO), the platelet growth factor made primarily in the liver, vary inversely with platelet counts and are reduced with advanced liver disease.3,13 Proliferation, differentiation, and stimulation of hematopoietic cells occur through factor-specific cell surface receptors composed of dimers or trimers of transmembrane proteins.1-3 There are general structural similarities in the extracellular and intracellular domains of receptors for many HGFs, and there are common components to the receptors for some factors (eg, a common β chain of the receptors for IL-3, IL-5, and GM-CSF). Specificity results from the unique, or private, components of the receptor for each factor. Receptor binding and cellular activation lead to conformational changes in the receptor, activation of intracellular kinases, and phosphorylation of specific proteins that are transferred to the cell nucleus.2

Pharmacokinetics and Pharmacodynamics The pharmacologic properties of HGFs depend on the dose, route of administration, affinity of binding to receptors, and complex circulatory and distribution factors, as with other drugs. There are extensive pharmacokinetic and pharmacodynamic data on erythropoietin, G-CSF, and GM-CSF (the growth factors

Chapter 32: Hematopoietic Growth Factors (Cytokines)

Table 32-1. Hematopoietic Growth Factors Factor

Other Name

Cell Source

Chromosome Location

Function

EPO

Erythropoietin

Juxtaglomerular cells

7q

Stimulates erythrocyte formation and release from marrow

TPO

Thrombopoietin, MGDF

Hepatocytes, renal and endothelial cells, fibroblasts

3q 27

Stimulates megakaryocyte proliferation and platelet formation

G-CSF

Granulocyte colony-stimulating factor; filgrastim; lenograstim

Endothelial cells, monocytes, fibroblasts

17q 11.2 q21

Stimulates formation and function of neutrophils

GM-CSF

Granulocyte-macrophage colonystimulating factor

T lymphocytes, monocytes, fibroblasts

5q 23-31

Stimulates formation and function of neutrophils, monocytes, and eosinophils

M-CSF

Macrophage colony stimulating factor; colony- stimulating factor-1 (CSF-1)

Endothelial cells, macrophages, fibroblasts

5q 33.1

Stimulates monocyte formation and function

IL-1 α and β

Endogenous pyrogen hemopoietin-1

Monocytes, keratinocytes, endothelial cells

2q 13

Proliferation of T, B, and other cells; induces fever and catabolism

IL-2

T-cell growth factor

T cells (CD4⫹, CD8⫹), large granular lymphocytes (NK cells)

4q

T-cell proliferation, antitumor and antimicrobial effects

IL-3

Multicolony-stimulating factor; mast cell growth factor

Activated T cells; large granular lymphocytes (NK cells)

5q 23-31

Proliferation of early hematopoietic cells

IL-4

B-cell growth factor; T-cell growth factor II; mast cell growth factor II

T cells

5q 23-31

Proliferation of B and T cells; enhances cytotoxic activities

IL-5

Eosinophil differentiation factor; eosinophil colony-stimulating factor

T cells

5q 23.3-q32

Stimulates eosinophil formation; stimulates T- and B-cell functions

IL-6

B-cell stimulatory factor 2; hepatocyte stimulatory factor

Monocytes, tumor cells, B and T cells, fibroblasts, endothelial cells

7p

Stimulates and inhibits cell growth; promotes B-cell differentiation

IL-7

Lymphopoietin 1; pro-B-cell growth factor

Lymphoid tissues and cell lines

8q 12-13

Growth factor for B and T cells

IL-11

Plasmacytoma stimulating activity

Fibroblasts, trophoblasts, cancer cell lines

19q 13.3-13.4

Stimulates proliferation of early hematopoietic cells; induces acute phase protein synthesis

IL-12

Natural killer cell-stimulating factor

Macrophages, B cells

5q 31-33; 3p 12-q13.2

Stimulates T-cell expansion and IFN-γ production; synergistically promotes early hematopoietic cell proliferation

LIF

Leukemia inhibitory factor

Monocytes and lymphocytes; stromal cells

22q

Stimulates hematopoietic differentiation

SCF

Kit ligand; steel factor

Endothelial cells; hepatocytes

4q11-q20

Stimulates proliferation of early hematopoietic cells and mast cells

MGDF ⫽ megakaryocyte growth and development factor; IL ⫽ interleukin; NK ⫽ natural killer; SCF ⫽ stem cell factor.

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approved for clinical use) but relatively little data on the other factors. Intravenously administered recombinant erythropoietin has a circulating half-life of 4 to 13 hours in patients with chronic renal failure and approximately 20% less in healthy volunteers.14 Therapeutically effective levels are maintained for at least 24 hours, after a single intravenous or subcutaneous injection. An increase in reticulocyte counts occurs within 10 days if erythropoietin is given daily. Exogenous erythropoietin accelerates erythropoiesis, but detectable increases in hematocrit and hemoglobin level usually take approximately 2 weeks to develop.14 Increasing the degree of glycosylation or the attachment of polyethylene glycol to the erythropoietin molecule increases its size, delays its renal clearance, and extends the duration of its biological effects.15-16 For most clinical applications, erythropoietin is administered weekly or biweekly. Granulocyte colony-stimulating factor can be administered intravenously or subcutaneously. After intravenous administration, the elimination half-life is approximately 3.5 hours.17 Blood levels are influenced by the neutrophil count, because neutrophils actively bind the injected drug.18 G-CSF stimulates proliferation of neutrophil progenitors and rapidly mobilizes mature neutrophils from the marrow reserves. It increases blood neutrophils threefold to fourfold in hematologically healthy persons within 4 to 6 hours.19-20 A sustained increase in blood neutrophils occurs if administration of G-CSF is continued on a daily basis.20 Granulocyte colony-stimulating factor accelerates neutrophil formation, which leads to an increased percentage of circulating band neutrophils with more deeply staining primary granules. These changes are similar to those commonly seen with bacterial infection. Granulocyte-macrophage colony-stimulating factor can be administered intravenously or subcutaneously, but intravenous administration has been associated with toxicity that includes fever, dyspnea, and acute respiratory distress syndrome.21,22 These effects seem to be dose related and to occur less frequently with subcutaneous administration. Similar to G-CSF, GM-CSF stimulates neutrophil production and release of cells from the marrow. When given repeatedly for several days, GMCSF increases total white cell count, including eosinophils and monocytes as well as neutrophils.23 The leukocyte count returns to the baseline level when administration of G-CSF or GM-CSF is discontinued. Results of in-vitro studies show that both G-CSF and GMCSF influence the function of neutrophils.5,24,25 Both types of CSF “prime” neutrophils to increase their responsiveness to other stimulators of the neutrophil oxidative (respiratory) burst, such as opsonized particles or bacteria. Neutrophils and other leukocytes produced in vivo in response to G-CSF or GM-CSF stimulation in general have normal or increased, but not decreased, responsiveness to exogenous stimulators of neutrophil function. The clinical significance of these findings is not clear. Substantial clinical data and numerous animal experiments indicate that neutrophils produced in vivo in response to these cytokines are

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capable of killing bacteria and accelerating recovery from bacterial infection.5 For some pathogens, in-vivo G-CSF treatment may enhance the microbicidal activity.26 The pharmacologic and physiologic properties of other HGFs are less well understood, but some generalizations are possible. The factors influencing the early phases of hematopoiesis tend to have multiple side effects, such as fever and flu-like symptoms, and a narrower range of tolerable doses than do factors that influence the later phases of hematopoiesis. Examples of early factors with substantial side effects include IL-1, IL-2, IL-3, IL-4, IL-6, and stem cell factor. The predominantly late-acting factors such as erythropoietin, G-CSF, GM-CSF, and M-CSF are much better tolerated.

Clinically Approved Agents and Products in Development Erythropoietin is approved for treatment of anemia in several clinical settings: to treat anemia of chronic renal failure, anemia in chronic zidovudine-treated human immunodeficiency virus (HIV)-infected patients, anemia in cancer patients on chemotherapy, and to reduce allogeneic blood transfusions in anemic surgery patients. Erythropoietin is available as a glycosylated recombinant protein from multiple manufacturers and as an identical 165-amino acid protein (darbepoetin) that differs from recombinant human erythropoietin in containing five N-linked oligosaccharide chains, whereas recombinant human erythropoietin contains three. Darbepoetin has delayed clearance and a longer biological effect permitting less frequent administration; it has been widely investigated as a stimulus to erythropoiesis.27,28 A pegylated erythropoietin was shown to be effective in an open-label, parallel group, noninferiority trial involving 1115 patients with the anemia of chronic renal failure.16,29 Currently, the long-term safety of all forms of erythropoietin is under review. An increase in the risk of venous thrombosis has been recognized from the earliest trials; recent questions also relate to an increased risk of malignancies observed as a secondary endpoint in some clinical studies.30-32 G-CSF and GM-CSF have been approved by the United States Food and Drug Administration (FDA) for the management of neutropenia in specific clinical situations. G-CSF is indicated for cancer patients receiving myelosuppressive chemotherapy, patients with acute myeloid leukemia receiving induction or consolidation chemotherapy, cancer patients receiving marrow or progenitor cell supportive therapy, and patients with various forms of severe chronic neutropenia. A pegylated form of G-CSF (PEG G-CSF) has a much longer blood half-life and longer biological effects and can be used once per chemotherapy cycle rather than on a daily basis. Chapter 15 describes the role of G-CSF in granulocyte collection and transfusion, a use similar to that for collection of hematopoietic progenitor cells (HPCs) but not yet approved by the FDA. GM-CSF is indicated following induction chemotherapy in acute myeloid leukemia,

Chapter 32: Hematopoietic Growth Factors (Cytokines)

for mobilization of marrow progenitor cells, and to accelerate marrow engraftment after hematopoietic transplantation. Interleukin-11 is currently approved to prevent the need for platelet transfusions following myelosuppressive chemotherapy in adult patients with nonmyeloid malignancies at risk of severe thrombocytopenia. Adverse effects including fever, edema, atrial arrhythmias, and syncope have limited its use. Several promising new agents are in development for treatment and prevention of thrombocytopenia, but none are currently approved by the FDA. The native (TPO) molecule was first identified and cloned in 1994 by several groups almost simultaneously.33-37 Its receptor (TPO-R) is expressed on megakaryocytes, platelets, and primitive HPCs.3,13 Soon after this discovery, preclinical data indicated that recombinant human TPO (rHuTPO) and a truncated version of TPO coupled to polyethylene glycol called megakaryocyte growth and development factor (PEG-rHuMGDF) stimulated a twofold to fivefold increase in platelet count with a peak occurring 10 to 14 days after beginning the stimulus.38 Phase I and II clinical trials demonstrated the effectiveness of PEG-rHuMGDF to raise platelet counts, but the development of neutralizing antibodies and significant thrombocytopenia in some subjects led to discontinuation of clinical trials with these specific agents.39 A recombinant TPO-R protein with a carrier Fc domain to increase its size and reduce renal clearance (currently referred to as AMG531) has recently undergone Phase I, II, and III testing and appears to be safe and effective for treatment of chronic refractory idiopathic thrombocytopenic purpura (ITP) in trials for up to 96 weeks with weekly administration.40-42 In a randomized, doubleblind, placebo-controlled Phase III trial involving 63 patients with ITP who had previously had splenectomies, responses with either a durable or transient increase in platelet counts were observed in 33 of 42 treated patients and 0 of 21 patients receiving placebo.42 The therapy was well tolerated; no neutralizing antibodies were observed. One subject had reversible evidence of increase marrow reticulin. A venous thrombosis occurred in one patient, but this did not prevent continuation of AMG531 treatment.42 This drug has been licensed by the FDA for use in Chronic ITP. Eltrombopag is an orally active TPO-R chrome agonist shown to increase platelet counts in healthy adults, and in patients with ITP43 or thrombocytopenia caused by hepatitis C infection and cirrhosis of the liver.44 Eltrombopag is a small-molecule, nonpeptide oral agonist to the TPO-R.45,46 The clinical trial involving 118 ITP patients with platelet counts less than 30,000/µL showed that 80% of patients receiving 50 or 75 mg/day responded to this therapy.43 The results in the hepatitis C trial were similar; headaches were the most common adverse event in both trials. Other oral agents (eg, AKR-501 and SB-559448) are under investigation.47 Despite several clinical trials, there are no approved uses for IL-3 or M-CSF.

Use of Myeloid Growth Factors to Mobilize Peripheral Blood Stem Cells For years it has been recognized that circulating HPCs are capable of forming hematopoietic colonies in vitro in culture systems

and of repopulating the marrow after total ablation. These cells appear to function normally to sustain hematopoiesis and to allow expansion in states of hematopoietic stress. Before the availability of HGFs, several investigators reported that the number of circulating HPCs increases during the recovery period after hematotoxic chemotherapy. This occurs usually after about 10 to 16 days, when marrow recovery is in progress.48,49 It was subsequently learned that a similar response occurs in hematologically intact persons given HGFs after about 5 to 7 days.50,51 Numerous studies have shown that the number of these progenitor cells, also called peripheral blood stem cells (PBSCs) or peripheral blood progenitor cells, predictably increases in the blood as much as 100-fold during recovery from chemotherapy or 4 to 6 days after administration of HGF is begun.52-57 The quantity and quality of cells are sufficient for PBSCs autologous reconstitution after myeloablative therapy and use of PBSCs is rapidly replacing marrow for that purpose.58 Peripheral blood stem cells are preferred because of the relative ease of procurement, more rapid restoration of blood counts compared with marrow infusion, and the possible recovery of these cells from patients who previously received intensive chemotherapy or radiation therapy and from patients with tumors involving the marrow.58 An unexpected benefit of the development of PBSC support came with the recognition that transfusion of PBSCs accelerated recovery of platelet counts and thus obviated the need for many platelet transfusions.53 This finding suggests that the persisting thrombocytopenia historically seen 3 to 6 weeks after conventional marrow transplantation was caused by the limited number or function of precursor cells infused rather than deficiencies in humoral or environmental factors. The basic physiologic principles governing mobilization of PBSCs probably involve breakage of the molecular bonds between key adhesive marrow elements, ie, the receptor-ligand pairs, which hold the progenitor cells in the extravascular space of the marrow juxtaposed to the marrow stromal cells. In primates, antibodies to the adherence protein VLA-4 can induce more rapid PBSC mobilization.59 The chemokine IL-8 has similar effects.60 Treatment with G-CSF is recognized to expand the total myeloid and progenitor mass, increase the number of circulating blood neutrophils, and also release neutrophil-associated proteases into the marrow and blood.61,62 There is good evidence that the released proteases disrupt key adhesive bonds holding the progenitors in the marrow, including the receptor-ligand pair CXCR-4 on the progenitors and stromal-cell-derived factor 1 (SDF-1) on the marrow stromal cells. The CXCR-4 antagonist AMD 3100 has similar effects in that this molecule also disrupts these bonds and promotes release of progenitor cells into the peripheral blood.63-65 All mobilizing agents cause leukocytosis with an increase in blood neutrophils as well as increased numbers of circulating HPCs, but they do not proportionately increase the numbers of circulating promyelocytes, myelocytes, or metamyelocytes. With G-CSF the kinetics of the response (delay of a few days before

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the increase in PBSCs occurs) suggests that early hematopoietic cells may need to expand in numbers before they “spill over” or are “released” into the blood. The increase in PBSCs in the recovery phase from chemotherapy generally occurs when the marrow is still relatively hypocellular, thus implying that marrow cell density is not the simple explanation for mobilization.65 In the initial development of support with PBSCs, many investigators questioned whether these cells are truly hematopoietic stem cells. Most of the cells collected and transfused after treatment with HGFs or chemotherapy undoubtedly are differentiated precursor cells. However, results of experiments with mice, dogs, and nonhuman primates as well as with humans support the contention that preparations of PBSCs do contain longlived precursor cells along with many differentiated cells.

Technical Considerations Quantitation Peripheral blood stem cells are quantified according to the cell surface expression of cluster determinant 34 (CD34) or granulocyte-macrophage colony-forming unit (CFU-GM) as measured with an in-vitro colony-forming assay. Because the invitro bioassay requires approximately 2 weeks to complete and is highly dependent on the expertise of the tissue culture laboratory, it is used largely for quality control and for research studies. Flow cytometric analysis for CD34⫹ cells can be performed quickly and easily on the day of collection and is essential to determine the adequacy of PBSC collection. Total mononuclear cell numbers also are useful. Cell numbers that usually correlate with engraftment are 4 ⫻ 108 or more mononuclear cells per kilogram, 2 ⫻ 106 or more CD34⫹ cells per kilogram, and 2 ⫻ 105 or more CFU-GMs per kilogram. Because this antigen is expressed on cells that have differentiated beyond the colonyforming stage, the number of CD34⫹ cells is 10-fold greater than the required number of CFU-GMs.66 Cytokine Doses Currently, G-CSF is the cytokine most frequently used to mobilize PBSCs. At doses of 5 µg/kg or less per day, the harvest of PBSCs usually is inadequate. Doses of approximately 10, 16, 24, and 32 µg/kg/day have been used in clinical trials. Although the yield may be slightly increased at higher doses, the usual dose is 10 to 16 µg/kg/day.58 Granulocyte-macrophage colony-stimulating factor, IL-3, and stem cell factor (c-kit ligand) also mobilize PBSCs, but have greater side effects. The role of thrombopoietin in mobilization is still uncertain.67 Combinations of G-CSF with each of these cytokines or with chemotherapy have been investigated at many centers. In general, it seems that a regimen of chemotherapy plus G-CSF (with or without a second cytokine) yields the greatest quantity of CD34⫹ cells and allows for the fewest leukapheresis procedures, but there is not yet a standard approach. The timing of the response to chemotherapy is quite variable, making this approach more difficult for patients and their families. Cyclophosphamide, carboplatin, paclitaxel, etoposide, and ifosfamide are the chemotherapeutic agents that

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have been studied most extensively for mobilization and harvesting of PBSCs. Collection and Processing of Peripheral Blood Stem Cells Currently, PBSCs are collected and used for both autologous and allogeneic hematopoietic stem cell transplantation.53,56 Under these circumstances, the type and intensity of previous chemoor radiotherapy affect collection of PBSCs. Extensive previous treatment may render adequate PBSC collection difficult, if not impossible. Most collections are performed using techniques and procedures evaluated by committees of the AABB, Foundation for the Accreditation of Cellular Therapy, International Society for Cellular Therapy, and American Society for Apheresis. When patients are treated with HGFs alone, collection usually is begun on treatment Day 4. Up to 20 liters of blood are usually processed to separate predominantly mononuclear cells, and the CD34⫹ cell collections from the first and succeeding days are counted to determine the total number of leukapheresis procedures to be performed. Tumor Cell Contamination Both marrow aspirated for transplantation and apheresiscollected PBSCs may contain tumor cells. The number of contaminating cells seems to be determined according to marrow involvement, stage of disease, tumor type, and previous therapy. Previously untreated patients tend to have greater marrow reserves or mobilizable tumor cell burdens. Techniques for detecting tumor cells in these preparations are rapidly improving. It is unclear what the minimal tumor cell contamination must be to avoid the risk of tumor cell engraftment with PBSCs mobilized by administration of HGFs.68-70 Selection for Peripheral Blood Stem Cells Because CD34 is a useful marker of early hematopoietic cells, several methods have been devised in which antibodies to CD34 are used for selective collection of PBSCs. The goal is to select PBSCs and eliminate tumor cells from the materials to be transfused. At present, these technologies do not eliminate all tumor cells completely but reduce the numbers markedly. Studies are still in progress to determine the clinical advantage of these procedures. Cryopreservation Peripheral blood stem cells collected after treatment with HGFs usually are preserved by means of controlled rate freezing with 10% dimethylsulfoxide (DMSO). Results of some studies suggest that HGFs may inhibit spontaneous apoptosis, but it is unclear whether this is clinically important for preservation of PBSCs.

Clinical Trials of Growth-Factor-Mobilized Peripheral Blood Stem Cells Numerous uncontrolled trials and a few randomized, controlled clinical trials have shown the efficacy of HGF-mobilized PBSCs to accelerate marrow recovery after intensive chemotherapy or

Chapter 32: Hematopoietic Growth Factors (Cytokines)

myeloablative chemoradiation therapy. The results of these studies have established that administration of more than 4 ⫻ 106 CD34⫹ PBSCs per kilogram mobilized by G-CSF results in neutrophil recovery to more than 500/µL and platelets to 20,000/µL in approximately 10 to 12 days.62 These recovery times probably can be achieved without treating the recipient with HGFs after infusion of PBSCs. These times for recovery with PBSCs are considerably shorter than those after autologous marrow transplantation, particularly for platelets. Platelet counts recover to 20,000/µL by approximately Day 12 after treatment with PBSCs without G-CSF or GM-CSF therapy and to 20,000/µL on approximately Day 20 or later after autologous marrow transplantation.

Alternative Agents Although the use of HGFs to mobilize PBSCs has greatly facilitated hematopoietic transplantation, there is a need to further improve this procedure. The discovery that SDF-1 and CXCR4 play critical roles in hematopoietic cell homing was soon followed by identification of an antagonist to the binding of this receptor-ligand pair. AMD 3100 is a small organic chemical inhibitor of this interaction that was discovered in the process of screening for agents with anti-HIV properties.64 AMD 3100 is administered subcutaneously and stimulates the semiselective release of CD34⫹ cells from the marrow to the blood in a dose-dependent fashion.71-73 Clinical studies in human volunteers have established that AMD 3100 increases CD34⫹ cells at 6 to 10 hours after a single subcutaneous dose sufficient for collection of greater than 5 ⫻ 106 cells/kg by standard leukapheresis. Clinical trials in normal volunteers and patients with multiple myeloma and non-Hodgkin lymphoma have demonstrated that AMD 3100 used in combination with G-CSF is more effective than either alone.73 The additive effects of AMD 3100 to G-CSF are especially important for patients who are poor mobilizers because of their underlying disease or previous chemotherapy.74 FDA approval of AMD 3100 is pending, as of this writing. Other similarly acting new agents are in development. Other Considerations Many laboratories have intensively studied the expansion of marrow cells and PBSCs using in-vitro incubation of the cells with a mix of growth factors, including stem cell factor, IL-3, IL-6, GM-CSF, G-CSF, and erythropoietin. This approach is a promising way to expand the number of differentiated progenitor cells for supportive care. It also may prove useful for providing undifferentiated and stem cell support if, eventually, cells can be expanded without stimulating differentiation. Paralleling these efforts, many investigators are studying genetic alteration of PBSCs mobilized by HGF administration with the intent to introduce therapeutically important genes into PBSCs. This gene therapy strategy may prove useful for the management of genetic, infectious, and malignant diseases. This work is still at an investigational stage.

Granulocyte Colony-Stimulating Factor and GranulocyteMacrophage Colony-Stimulating Factor for Neutropenia Associated with Chemotherapy and Hematopoietic Stem Cell Transplantation For many years, the myelosuppressive effect of chemotherapy has been the dominant factor determining treatment doses and schedules in therapy for hematologic and nonhematologic malignant diseases. It is generally believed that recovery of blood leukocyte and platelet counts is delayed because of the time required for cell proliferation and differentiation as well as for the generation of endogenous factors regulating cell recovery. The potency of G-CSF and GM-CSF to stimulate recovery, and the effectiveness of prophylactic administration of G-CSF to stimulate recovery were easily recognized in the first clinical trials.75,76 In two randomized, controlled trials,77,78 the effectiveness of prophylactic administration of G-CSF was definitively demonstrated in the care of patients with small-cell lung cancer. In these studies, patients treated with cyclophosphamide, doxorubicin, and etoposide received either placebo or G-CSF at a dose of 5 µg/kg/day. The incidence of fever and documented infections was reduced in the G-CSF-treated group both in the first cycle and for the overall six cycles of treatment. Antibiotic use and days of hospitalization also decreased. The results of this trial have been replicated in a number of studies.79 Meta-analyses and comprehensive reviews have confirmed the benefit to prevent febrile neutropenia and to prevent deaths from sepsis in adults treated with G-CSF.80,81 Randomized clinical trials also have shown that marrow recovery after transplantation is accelerated by GM-CSF and G-CSF.82 The results of these trials have led to a number of conclusions and practice guidelines.83-85 Granulocyte colony-stimulating factor effectively stimulates marrow recovery after chemotherapy for many malignant diseases, including myeloid and nonmyeloid leukemia. It has proven to be difficult to establish whether GM-CSF is equally effective. In healthy subjects and patients, GM-CSF is less potent as a stimulus to increase neutrophil production and thus to raise blood neutrophil counts.86 In addition, GM-CSF may cause fever and inflammatory symptoms, making it difficult to use the occurrence of febrile neutropenia as the primary endpoint for clinical trials. The greatest value of CSF treatment is in the care of patients with sustained neutropenia and a high likelihood of developing febrile neutropenia and severe infections. Conversely, it has been easier to show the clinical benefit of CSF treatment in trials of single-agent chemotherapy with a lower risk of febrile neutropenia.87 This apparent paradox is attributable to effects of the chemotherapy on the hematopoietic tissues. More chemotherapy causes more severe and protracted neutropenia, but it also reduces the amount of target tissue for the CSF. The net effect is that the benefits of CSF treatment, particularly to accelerate hematopoietic recovery, are greater with milder chemotherapy treatments. For this and other reasons, the American Society of Clinical Oncology and other groups have revised their original guidelines to set a 20% risk of febrile neutropenia as the level for demonstrable benefit of prophylactic CSF.83,84 In any patient with documented febrile neutropenia after one course of chemotherapy, the use of these factors to avoid

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infectious complications while maintaining dose intensity is generally indicated unless a dose-reduction strategy is more appropriate because of the disease, stage of the disease, comorbidities, or personal choice of the patient. The use of G-CSF and GM-CSF in the management of myeloid malignant disease has been studied extensively. It appears that these agents can be used safely to accelerate marrow recovery after chemotherapy, as in the management of nonmyeloid malignant disease. The timing and dosing of G-CSF or GM-CSF are largely based on schedules used in randomized clinical trials, but many questions remain to be answered. Because of the potential for the CSF to increase hematotoxicity if given before or concomitant with chemotherapy, they are generally recommended to be given the day following chemotherapy. The duration of CSF treatment is also not well established. The best evidence of their effectiveness comes from the large randomized clinical trials that proved their benefits. In these trials the CSF was given until the blood counts were normal or above normal.77-80 Conversely, the treatment period may have been longer than necessary in these trials. In general it would appear that treatment should be started without delay approximately 24 hours after completion of any course of chemotherapy and continued until there are clear signs of increasing blood neutrophils before stopping this supportive therapy. Uncertainty also exists about optimal dosing. For GM-CSF, dosing may be limited by side effects. For G-CSF, the acceptable dose range is much broader, but evidence suggests that doses greater than 5 µg/kg/day do not significantly accelerate marrow recovery. The G-CSF dose of 5 µg/kg/day is the current standard of practice.83-85

Colony-Stimulating Factors for Idiosyncratic Drug-Induced Neutropenia A wide array of drugs induce transient neutropenia, presumably on an immune or toxic basis. The penicillins, sulfonamides, and antithyroid and psychotropic drugs often are implicated. With respect to treatment, the offending (or presumably offending) agent should be discontinued, and patients with a fever should be treated with broad-spectrum antibiotics. Numerous case studies and small series have suggested that HGFs may accelerate marrow recovery in this setting. However, the evidence is largely anecdotal, and practices differ considerably. The sickest patients with documented infections and delayed marrow recovery are the ones most likely to benefit from CSF treatment.88

Colony-Stimulating Factors in the Management of Chronic Neutropenia The clinical benefit of long-term G-CSF therapy for patients with severe congenital neutropenia (including Kostmann syndrome), severe idiopathic neutropenia, or cyclic neutropenia, was established in a randomized clinical trial.89 In a study of patients with severe chronic neutropenia, the occurrence of fever, oropharyngeal ulcers, infections, hospitalization, and antibiotic use were all significantly reduced with dose-adjusted, daily G-CSF treatment. The quality of life and activity profiles of these patients also improved. The principal side effect of G-CSF therapy was musculoskeletal pain early in

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the course of treatment, which became less severe with long-term therapy. Data from the Severe Chronic Neutropenia International Registry have shown that the subgroup of patients with severe congenital neutropenia are at risk of developing myelodysplasia and acute myeloid leukemia.90,91 The risk appears to be disease specific with a substantially lower, or no risk for patients with cyclic or idiopathic neutropenia. At present, hematopoietic transplantation is the only available alternative therapy, and it is limited largely by the availability of a suitable well-matched marrow donor. For these reasons, careful follow-up evaluation of patients receiving long-term G-CSF therapy, especially patients with severe congenital neutropenia, is indicated with regular blood counts and clinical observation and annual marrow examination.

Colony-Stimulating Factors in the Management of Nonneutropenic Infectious Disease Considerable research efforts have been devoted to the possible use of CSFs as adjunctive therapy for nonneutropenic infectious conditions. The rationale for these studies was to enhance the number and functional activities of neutrophils to promote recovery from, or prevention of, local or systemic infections. On the basis of results of Phase II studies, a set of large Phase III trials were conducted. In a double-blind, controlled, multicenter trial, 756 patients with community-acquired bacterial pneumonia were enrolled to receive intravenous antibiotics plus either G-CSF (300 µg/day for up to 10 days; n ⫽ 380) or placebo (n ⫽ 376).92 Outcome measures included time to resolution of morbidity, 28-day mortality, length of hospital stay, and occurrence of adverse events. A microbial cause of pneumonia was identified for 56% of patients, and an independent review group judged use of antimicrobial agents appropriate in the care of 98% of the patients. Administration of G-CSF increased the peripheral blood neutrophil count threefold, but the primary endpoint—time to resolution of morbidity, mortality, and length of hospitalization—was not affected. Subgroup analysis showed that the time of resolution of infiltrates was significantly more rapid in the G-CSF-treated group. Administration of G-CSF was safe and well tolerated. Further studies of more severely ill patients with pneumonia also showed similar results, with no significant effect on the primary endpoint; however, there were intriguing findings with the secondary and subgroup analyses.93 Because of a lack of neutrophilia and impaired superoxide generation in neutrophils from patients with diabetes, G-CSF appears to be a reasonable candidate for adjunctive therapy in the management of severe infections in these patients. In a small randomized, double-blind, placebo-controlled trial, 40 patients with insulin-dependent diabetes and foot infections were assigned to receive either G-CSF (n ⫽ 20) or placebo (n ⫽ 20) for 7 days.94 Both groups were treated with similar antibiotic and insulin regimens. After 7 days of G-CSF treatment, there was earlier eradication of pathogens, quicker resolution of cellulitis, shorter hospital stay, and shorter duration of antibiotic treatment. Other trials, however, have not confirmed these findings.95 These results await corroboration in a multicenter trial before adoption of use of G-CSF for this purpose in routine clinical practice.96

Chapter 32: Hematopoietic Growth Factors (Cytokines)

Colony-stimulating factors may have therapeutic potential in the treatment of patients with HIV. Granulocyte-macrophage colony-stimulating factor has been shown to partially correct HIV-1-mediated impairment in the ability of monocyte-derived macrophages to phagocytose Mycobacterium avium complex in vitro.97 In a study involving 258 patients with HIV-1 infection and moderate neutropenia, G-CSF treatment significantly reduced the incidence of severe neutropenia and bacterial infection.98 Patients treated with G-CSF also had 54% fewer episodes of severe bacterial infection and needed 45% fewer days of hospitalization for management of bacterial infection than did the control group. The use of G-CSF to prevent or treat infections has been suggested in numerous other small trials, but randomized, controlled trials are necessary to establish the benefits of such practices.

Granulocyte Colony-Stimulating Factor for Mobilization of Neutrophils in Granulocyte Transfusion Therapy Granulocyte transfusion therapy would appear to be a rational approach to the management of severe bacterial and fungal infections in patients with prolonged neutropenia. Potential clinical efficacy, however, has long been limited by insufficient donor stimulation regimens and suboptimal leukapheresis techniques. Methodologic progress, in particular for mobilization of neutrophils in healthy donors by means of administration of G-CSF and dexamethasone, has greatly enhanced leukapheresis yields. Granulocyte transfusion therapy is discussed in detail in Chapter 20.

Summary The discovery and clinical development of HGFs have had great effect on the use of blood components in the supportive care of patients with many hematologic and malignant diseases. Hematopoietic growth factors are providing new opportunities for the provision of blood services, such as collection of PBSCs, autologous erythrocyte transfusion for elective surgical procedures, and G-CSF mobilization of neutrophils for granulocyte transfusion therapy.

Disclaimer W. C. Liles has disclosed no conflicts of interest. D. C. Dale has disclosed financial relationships with Amgen, Genzyme, and Cellerant.

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Hematopoietic Progenitor Cells: Biology and Processing Thomas Leemhuis1 & Ronald A. Sacher2 1

Director, Cellular Therapies Division, Hoxworth Blood Center, University of Cincinnati Academic Health Center, Cincinnati, Ohio, USA 2 Director, Hoxworth Blood Center, University of Cincinnati Academic Health Center, Cincinnati, Ohio, USA

Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) used for hematopoietic reconstitution after myeloablative therapy were among the first cellular therapy products shown to have a real clinical benefit. The ability of the HSC to engraft and differentiate to produce all blood cell lineages, and the ethical issues surrounding the much publicized advances in embryonic stem cell research, have both prompted experimentation into additional therapeutic uses for HSCs and HPCs. A partial list of potential clinical uses of these hematopoietic cells is presented in Table 33-1. Many have not yet been proven safe or effective. As the field of hematopoietic transplantation has progressed, much knowledge has been gained concerning the complex interactions between cell populations contained in the donor’s marrow, mobilized peripheral blood progenitor cell (PBPCs), or umbilical cord blood (UCB) graft and the recipient’s cells. As a result, much has been learned in the laboratory about how to selectively deplete or enrich for subpopulations of donor cells before infusion. These cells then can serve functions other than conventional marrow replacement or rescue. They can be used for adoptive immunotherapy,1,2 as vehicles for gene therapy,3 and perhaps even to induce tolerance of an organ transplant from the same donor.4 There are several clinical situations where such in-vitro manipulations have been shown to provide significant clinical benefit. Transfusion medicine and clinical laboratory technology have grown to include cell processing techniques that concentrate, separate, isolate, purify, enrich, deplete, cryopreserve, and quantitate cellular components contained within marrow, peripheral blood, and UCB. The more common, clinically relevant processing techniques used to prepare hematopoietic grafts for infusion are reviewed in this chapter. The focus is on the rationale and the basic principles behind the techniques, but select technical details and literature references are also provided to assist laboratory professionals with the task of preparing standard operating Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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Table 33-1. Potential Therapeutic Uses of Hematopoietic Stem and Progenitor Cells Hematopoietic cell transplantation Autologous; allogeneic Myeloablative, nonmyeloablative Adoptive immunotherapy Cardiac repair Revascularization of microcirculation Repair of damaged cardiac muscle Peripheral vascular disease Bone fracture Muscle/tendon repair Gene therapy Fanconi anemia Chronic granulomatous disease Severe combined immunodeficiency

procedures (SOPs). This chapter concludes with a discussion of the quality assurance and regulatory compliance principles that apply to any laboratory performing cell manipulations for therapeutic purposes.

Sources of Hematopoietic Progenitor Cells Human HSCs and HPCs reside primarily in the marrow, but can also be obtained in clinically relevant cell doses from cytokinemobilized peripheral blood, and UCB. These cells can also be found in other locations, such as fetal liver, but their usefulness is limited by either the low cell numbers that can be obtained or by ethical issues. A partial list of the cellular characteristics of each of these source tissues is presented in Table 33-2. Clinical-scale marrow collections often contain a significant amount of peripheral blood, thus they contain all hematopoietic cell types, from the most immature stem cells that enable long-term engraftment to the most mature granulocytes and erythrocytes with naturally short life spans. Marrow usually

Chapter 33: HPCs: Biology and Processing

Table 33-2. Cellular Characteristics of Various Stem Cell Sources Marrow

Mobilized Peripheral Blood

Umbilical Cord Blood

Stem cell content

Adequate

Good

Low

Progenitor cell content

Adequate

High

Low

T-cell content

Low

High

Very low

Risk of tumor contamination

High

Low

Not applicable

contains between 0.5% and 1% CD34⫹ cells. Optimal posttransplant hematopoietic recovery requires 2 to 5 ⫻ 106 CD34⫹ cells/kg.5 Therefore a clinical-scale marrow harvest should contain 2 to 5 ⫻ 108 total nucleated cells/kg. This generally requires a 1- to 1.5-liter collection for an adult recipient. Their large volume and complex cellular makeup make most selection procedures difficult. ABO compatibility is essential, unless a red cell or plasma depletion procedure is performed before infusion. Mobilized PBPC products are routinely used as an alternative source of HSCs and HPCs for transplantation.6-8 The CD34⫹ repopulating cells can be mobilized into the peripheral blood and then collected by apheresis following myelosuppressive chemotherapy and/or stimulation with 10 to 20 µg/kg granulocyte colony-stimulating factor (G-CSF).9,10 Because an apheresis procedure is used to collect PBPC products, they contain very few erythrocytes or granulocytes, compared to marrow, and are primarily composed of mononuclear cells (MNCs). PBPC products also contain larger numbers of HPCs than either marrow or UCB, and therefore facilitate faster engraftment and shorter hospital stays. Patients who have been heavily pretreated with multiple rounds of chemotherapy or radiation therapy often mobilize poorly and require multiple collection episodes.11 PBPC products contain larger numbers of T cells than marrow collections, and thus present a greater risk of causing graftvs-host disease (GVHD) in the allogeneic setting, although the rate of acute GVHD is less than originally feared.12-16 Mobilized peripheral blood is easier to collect than marrow and, therefore, presents a lower risk to the volunteer donor. However, the longterm risks of G-CSF administration remain unclear. The availability and relative ease of collection of cord blood stem cells, along with the reported decreased rate of GVHD, makes UCB transplantation an attractive option. Because of the limited number of HPCs that can be acquired from this source, however, most recipients have been children.17-19 UCB contains a high proportion of HSCs and HPCs, but only approximately 10% of the cell dose of a typical marrow harvest; therefore, engraftment is often delayed with UCB transplants relative to marrow or PBPC transplants.20-22 Some centers have begun to test whether infusing 2 UCB units would improve engraftment kinetics.23 It appears that either the number or the alloreactivity of the T cells are reduced in UCB grafts, enabling the use of partially mismatched unrelated donors with relatively low rates of GVHD.24-26 With its high proliferative

potential, UCB may be a good target for gene therapy,27 and/or exvivo expansion.28 However, results of studies of the ability of ex-vivo expanded portions of UCB grafts to shorten the time to engraftment have generally been disappointing.29-32 Even though each of these tissue sources contain HPCs capable of long-term engraftment, the products vary dramatically in terms of red cell content, granulocyte and platelet content, T-cell number, and CD34 number. Thus, they present different processing objectives and challenges to the processing laboratory. No two HPC products received for processing are the same, in terms of donor age, disease status, tumor cell infiltration, infectious disease status, or prior chemotherapy exposure. Therefore, processing results (purity, recovery, viability) will often vary for unknown reasons. There are distinct advantages and disadvantages to using a particular HPC source tissue for transplantation. The decision regarding which source to use for a particular patient is best left to the patient’s physician so that disease status and donor availability can also be factored into the decision. The preferred, sought-after donors of allogeneic HPCs have customarily been sibling donors who are HLA identical at the HLA-A, -B, and -DR loci. When such a match cannot be found, related donors mismatched at one or two HLA loci are used, as are HLA-matched unrelated volunteer donors from the international registries.

Collection of Progenitor Cells Marrow Harvests Marrow harvests are invasive surgical procedures performed in an operating room under general anesthesia. Approximately 1000 to 1500 mL of marrow is collected, 3 to 10 mL at a time, from multiple punctures of both posterior iliac crests, using specially designed stainless steel beveled needles and syringes. The marrow is collected into a disposable collection apparatus that contains a 500micron filter and a 200-micron filter to remove most of the bone chips, clots, fat, and fibrin before the marrow is drained into a collection bag that contains an anticoagulant. Target cell doses often range between 2 and 5 ⫻ 108 nucleated cells/kg and between 2 and 5 ⫻ 106 CD34⫹ cells/kg. Collection protocols vary, but most often the syringes are heparinized in advance with 1 mL of a 100 U/mL heparin solution. The marrow is drained into a collection bag containing the same heparin solution, such that the heparin concentration is at least 10 U/mL in the final product. Additional heparin may be required if there is to be a long-distance transport or overnight hold of the marrow before processing. Although the marrow donor usually remains in the hospital overnight, it is possible to perform the marrow harvest as an outpatient procedure with local anesthesia. Complications from the general anesthesia or from infection at the anatomic site of the harvest can occur.

Peripheral Blood Collections via Apheresis With the development of recombinant colony stimulating factor (CSFs), it has become possible to mobilize large numbers of

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Table 33-3. Instrument Settings for Peripheral Blood Progenitor Cell Collections Instrument

RBC Content (%)

Flow Rate (mL/min)

Anticoagulant to Whole Blood Ratio

Cycle Volume (mL)

Number of Cycles

Spectra

1 to 3

60 to 150

1:12 to 1:15

Continuous

N/A

Amicus

6 to 8

40 to 75

1:12 to 1:15

1000 to 1400

7 to 14

COM.TEC

6 to 8

40 to 60

1:10 to 1:14

300 to 500

25 to 40

progenitor cells into the peripheral circulation so that they can be collected by a much less invasive procedure. By stimulating the donor with either hematopoietic growth factors, or chemotherapy and growth factors, a sufficient number of circulating stem cells for marrow rescue can be collected in one to three apheresis procedures.10 Although the peripheral blood of healthy individuals contains fewer than 0.1% HSCs, this number increases dramatically during recovery from cytotoxic therapy and even more so when recombinant CSFs such as G-CSF are administered. Peak counts usually are obtained 5 days after stimulation with G-CSF (10 to 20 µg/kg/day) or 10 to 14 days after chemotherapy and GCSF. Daily monitoring of peripheral blood CD34 counts is useful for determining when to start leukapheresis after chemotherapy and G-CSF mobilization. Most centers begin leukapheresis when the peripheral CD34 count reaches or exceeds 10 CD34⫹ cells/ µL. Stem cell factor, thrombopoietin, and flt-3 ligand have also been reported to successfully mobilize HPCs.33-35 Peripheral blood stem cells can be used as a source of stem cells for both autologous and allogeneic marrow transplants. A PBPC collection is performed with a cell separation device originally developed for therapeutic plasmapheresis, leukapheresis, and platelet donation procedures. This apheresis device uses a centrifuge to separate and collect MNCs, including peripheral HSCs, from the blood. In order to achieve a target cell dose of 2 to 5 ⫻ 106 CD34⫹ cells/kg, it is necessary to process 12 to 25 liters of blood or 2.5 to 6.0 times the patient’s calculated blood volume. Investigators have reported that the yield of CD34⫹ cells increases continuously as more blood volumes are processed.36 Although up to six times the donor’s blood volume can be safely processed, some donors are not able to tolerate 4 or 6 hours being connected to an apheresis machine. Therefore, these donors require repeated collections on sequential days once the peripheral CD34 count has increased to acceptable levels (⬎10 CD34⫹ cells/µL) for collection. There are currently three different commercially available apheresis instruments. In each case, instrument settings such as inlet flow rate, centrifuge speed, collect pump flow rate, and anticoagulant:whole blood ratio vary, depending on the target cell type to be collected. The three instruments operate differently. The Amicus (Baxter, Deerfield, IL) and the COM.TEC (Fresenius SE, Bad Homburg, Germany) are more automated, computercontrolled instruments; however, the Spectra (CaridianBCT, Lakewood, CO) is more widely used. Suggested settings for the collection of CD34⫹ progenitor cells using these instruments are presented in Table 33-3. Note that the red cell content setting for the Spectra is set with a colorgram. Targeting a colorgram of 1% to 3% results in a product with approximately 6% red cells,

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whereas this parameter can be entered directly into the other two instruments. Also, because the final product is more concentrated when collected by the Amicus, adding 100 to 150 mL of autologous plasma is recommended to rinse the collection line and to improve viability during storage. A high red cell content in PBPC collections can cause problems with patient/donor ABO incompatibility in allogeneic transplants, and cause processing issues when performing cell selection procedures. The CD34⫹ (MNC) layer is just below the platelet layer. Going deeper into the white cell layer (closer to the red cell layer) increases the collection of granulocytes (PMNs), which can cause increased clumping and clotting in the products. In order to achieve the desired product hematocrit, the collect valve may be closed until the desired interface is established. Two donor-related issues require special consideration. First is the issue of priming of the apheresis machine. Even when devices to that minimize extracorporeal volume are used, priming of the apheresis machine with red cells is required for individuals in whom the volume of the tubing set exceeds 10% to 15% of the total blood volume. This step prevents unacceptable dilutional anemia during the procedure and prevents fluid overload associated with the return of red cells from the centrifuge chamber at the end of the procedure. Second is the use of acid-citratedextrose (ACD) anticoagulant. The citrate component of ACD poses a risk of symptomatic hypocalcemia, which can limit the rate of blood processing and/or the duration of the procedure. Calcium replacement should be strongly considered to prevent adverse events during the apheresis collection procedure. The anticoagulant:whole blood ratio is important to both the product and the donor. Increasing the ratio can reduce the collection time, but it also reduces the amount of anticoagulant being mixed with the donor’s whole blood and thus increases the potential for clotting inside the tubing set and/or the product bag. When making adjustments to this ratio, one must consider the donor’s platelet count from the day of collection.

Umbilical/Placental Cord Blood Collection Harvesting of UCB is the least invasive of all HSC collection techniques. The cord is clamped, and a 16-gauge needle attached to a standard blood donor set containing a preservative, usually ACD formula A (ACD-A) or citrate-phosphate-dextrose-adenine (CPDA-1), is inserted into the umbilical vein. Cord blood collection can either be performed by the obstetrician with the placenta in situ, or immediately after delivery by a trained team that can process the placenta in a location in close proximity to the delivery room. If cells are collected after delivery, the placenta

Chapter 33: HPCs: Biology and Processing

can be suspended from a specially constructed frame to aid in drainage of the cord blood. The usual volume collected is less than 170 mL.37 After collection by gravity, some additional blood remaining in the placenta can be expressed by means of physical massage of the organ. A device that applies pressure to the placenta in a funnel-like framework has been reported to provide increased yields in total volume and CD34⫹ content.38

that an adequate number of viable cells will remain after processing to provide for robust engraftment in a reasonable amount of time. Second, the processing technique must not place the component at undue risk of contamination with microorganisms. All procedures must be performed under aseptic conditions, preferably in a closed system, with appropriate assays for detection and identification of infectious agents at critical steps. Several of the more commonly used cell processing procedures are listed in Table 33-4, and described below.

Processing Procedures Red Cell Depletion A few general principles must be followed to preserve product integrity and to protect the operator from harm. Collection of a good quality unit is the ultimate goal. ● Practice universal precautions. All products are potentially infectious and if handled carelessly, can spread disease to the operator, or to another product. Donors should be tested for transfusion-transmitted diseases in advance of the procedure. ● Follow aseptic processing guidelines. Closed systems are of course preferable, but if not available, adherence to strict aseptic processing guidelines, such as use of a biosafety cabinet, is essential to protect the product from environmental contaminants. ● Prepare. Time is a critical factor with these products; however, it is important to perform the procedure in a deliberate manner. Preparation, including taking time to prepare materials, reagents, workspace, worksheets, and labels in advance of product arrival frequently reveals problems that can be solved in advance, thereby improving product quality. ● Follow the SOP. The SOPs serve as a step-by-step guide during processing. These procedures need to be written clearly, kept in a convenient location, updated easily, version-controlled, and consulted frequently during processing to prevent errors—even if the procedure is being performed by experienced personnel. Assuming the procedures have been validated to result in the desired endpoint, they must be followed as written. ● Observe. Although it is important to follow the SOP as written, it is also important to be alert for product abnormalities or unexpected outcomes that might necessitate a procedural deviation. ● Document. The benefits of having detailed documentation on file for each procedure performed cannot be overstated. Such documentation is needed for accreditation by voluntary standardsetting organizations [AABB, Foundation for the Accreditation of Cellular Therapy (FACT), College of American Pathologists (CAP), The Joint Commission], regulatory review by federal agencies [Clinical Laboratory Improvement Act (CLIA) and Food and Drug Administration (FDA)], and for best practice analyses during any internal or external quality systems/outcome reviews. As new techniques are developed, or existing procedures modified, the specific aims will of course vary. However, there are two common goals of all in-vitro manipulations of HPCs. First, there must be minimal loss of, or damage to, the progenitor cells. The operator needs to ensure that the cells are collected and processed in accordance with the existing standard of care, so

When the donor and recipient have a major ABO incompatibility (patient has antibodies to donor red cells), infusing more than 20 mL of incompatible red cells can cause a serious hemolytic transfusion reaction. Often, red cells are removed from marrow and UCB products to debulk the product before cryopreservation or as a prerequisite to subsequent processing. In the latter case, the absolute residual red cell content is less important, so a simple buffy coat preparation may suffice. Red cell depletion of PBPC products is rarely necessary because the apheresis procedure results in relatively low level red cell content. Red cells can be depleted from marrow or UCB products by use of several different techniques. Historically, red cell depletion strategies were either based on density differences between cell types,39 or a differential sensitivity to hypotonic solutions. More recently, hetastarch has been used to cross-link red cells such that they rapidly settle out of solution. Exposure of red cells to a 1:5 v:v ratio of 6% hetastarch results in the formation of cross-links between multiple red cells, significantly increasing their weight and causing them to rapidly settle to the bottom of a V-bottom container (bag).40 Within 45 to 60 minutes, the leukocyte-rich plasma layer can be expressed into a separate collection bag or the red cell pellet can be drained into a waste container. Some marrow contains an excessive number of red cells; thus normalizing the hematocrit before adding the hetastarch can be beneficial. In the authors’ experience, this method results in a good leukocyte recovery (⬎90%) with approximately 95% reduction in red cell content, resulting in ⬍20 mL residual red cells. This maneuver allows for safer product transfusion to an ABO-incompatible recipient. A somewhat less pure product results from centrifugation followed by removal of the leukocyte-rich buffy coat layer.

Plasma Depletion Plasma depletions are also commonly performed on allogeneic PBPC and marrow products to prevent donor antibodies to recipient ABO antigens from causing a transfusion reaction following product infusion or to simply reduce the volume of the product in order to prevent volume overload. The product is divided into multiple 300-mL or 600-mL transfer packs and centrifuged at approximately 900 ⫻ g for 10 minutes. The plasma is extracted, and the infranatant fluids are combined into one container. The addition of a heparinized isotonic buffer solution containing a small amount of protein (human serum albumin, HSA) helps preserve cell viability and improves the infusion flow rate.

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Table 33-4. Commonly Used Cell Processing Procedures Aim

Rationale

Methods

Volume reduction

Ease of storage and infusion

Centrifugation

Plasma depletion

Reduce volume of ABO-incompatible plasma

Red cell depletion

●

●

T-cell depletion

● ●

Centrifugation

Reduce number of ABO incompatible red cells Debulk product

●

Reduce GVHD in allogeneic recipients Eliminate autoreactive T cells from autografts (autoimmune disorders)

●

● ●

● ●

● ●

Malignant cell depletion

Eliminate tumor from autografts

● ● ● ●

Stem cell enrichment

● ● ●

Donor lymphocyte infusions

● ● ●

Centrifugal elutriation Antibody ⫹ complement lysis Solid-phase monoclonal antibodies Stem cell selection Immunotoxins Antibody ⫹ complement lysis Stem cell selection Immunotoxins Cell culture

Reduce tumor burden Reduce GVHD Facilitate expansion/gene marking

Stem cell selection

Reduce graft rejection Increase donor chimerism Facilitate GVL response

CD3 aliquots

Cryopreservation

HPC storage

Gene transfer

● ●

Hetastarch sedimentation Ficoll-hypaque Buffy coat NH4Cl lysis

Controlled-rate freezing with cryoprotectants

Correct defective HPC Confer drug resistance

● ●

Retroviral transduction Lentiviral transduction

GVHD ⫽ graft-vs-host disease; GVL ⫽ graft-vs-leukemia; HPC ⫽ hematopoietic progenitor cell

One must be careful, however, that infusion volumes don’t exceed approximately 20 mL/kg.

Mononuclear Cell Enrichment Marrow and UCB (and to a lesser extent PBPCs) are heterogeneous collections of cellular and noncellular components. The cells that lead to hematopoietic reconstitution are mononuclear; other cell populations in the graft may be superfluous, or cause harm. For example, most of the cells in a marrow or UCB product are mature red cells and granulocytes, which lyse and release potentially harmful enzymes during freezing and thawing. The CS3000 (Baxter) and the Spectra (CaridianBCT) use an automated differential centrifugation technique to remove approximately 95% of the red cells from a marrow product, while approximately 75% of the MNCs are recovered in a final product that contains more than 80% MNCs.41

CD34 Selection Immature progenitor cells possess a surface marker (CD34) that gradually disappears as the hematopoietic cells mature and

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differentiate.42,43 CD34 selection is used for the purpose of tumor purging, T-cell depletion, or to provide a starting population for gene therapy or stem cell expansion procedures. Two different CD34 selection devices are approved for clinical use in the United States or Europe. Both positively select antibody-coated CD34⫹ cells by immunomagnetic reactions that allow the CD34⫹ cells to be retained in the device while the antigen-negative cells are eluted. However, the details of the selection methods differ. The Isolex 300i System (Baxter Oncology, Deerfield, IL) is the only CD34 selection device currently approved by the FDA for use in autologous PBPC transplantation. This system is almost fully automated and consists of four steps: 1) labeling cells with an anti-CD34 monoclonal antibody (MoAb); 2) mixing sensitized cells with paramagnetic beads coated with sheep anti-mouse IgG, whereby CD34⫹ cells form rosettes with the beads; 3) attaching the rosettes to a magnet and washing CD34⫺ cells from the system; and 4) releasing the cells from the beads using a peptide mimetope (a synthetic polypeptide designed to mimic the CD34 antigen) that competes with the MoAb binding site of the CD34 molecule.44-46 The magnetic field retains the beads and residual

Chapter 33: HPCs: Biology and Processing

antibody/peptide complexes in the separation chamber while the CD34⫹ cells are collected in a separate bag. All washes (including the initial wash to remove platelets) are performed within the closed system apparatus by using a unique spinning membrane technology. Reagent additions, incubations times, cell washing, and other steps in the selection are all software controlled. Depending on the protocol, the selected cells are then prepared either for immediate infusion, cryopreservation, or further manipulation. The CliniMACS system is based on the immunomagnetic cell selection technology developed by Miltenyi Biotec.47,48 This device is approved for clinical CD34⫹ cell selection in Europe, Canada, and some other countries and is currently undergoing clinical trials in the United States. In this partially automated system, the PBPC or red-cell-depleted marrow product is washed manually to remove platelets and plasma, and is then treated with a murine anti-CD34 that is conjugated to a stable colloidal suspension of super-paramagnetic particles composed of iron oxide and dextran. The cells are placed into the disposable tubing set in the selection device and the CD34⫹ cells are selected on a high-gradient magnetic separation column. The magnetically labeled cells are held in the column while unbound cells are washed through. The magnetic field is removed and the selected cells are collected. Buffer additions and flow rates are controlled via a computer-controlled system of peristaltic pumps, fluid sensors, and pinch valves. The retained antibody/particle complex does not affect the functional capacity of the selected cells. Other antibody-antigen systems can be employed for clinical application if performed under the appropriate FDA-approved investigational new drug (IND) protocol. CD34 selection is considerably more efficient when starting with PBPC apheresis products than it is with either marrow or UCB, or frozen/thawed products. It is possible to routinely achieve greater than 90% CD34 purity and greater than 70% CD34 recovery when selecting CD34⫹ cells from PBPC, but recovery and purity both suffer when selecting CD34⫹ cells from either marrow or UCB. Preselection stem cell enrichment techniques such as density centrifugation or red cell depletion are helpful for achieving a better purity and recovery. Adding a small amount of a clinical grade DNAse and magnesium chloride to older products delayed in transit or frozen/thawed products, greatly reduces clumping and improves postselection purity and recovery. Even with fresh PBPC, the starting product quality can affect selection performance. Products with low hematocrits and low granulocyte content are preferable, as are products with low to normal platelet counts.49

T-Cell Depletion The process of T-cell depletion (TCD) removes potentially hostreactive immunocompetent T cells from an allogeneic graft, resulting in a dramatic reduction in the incidence of GVHD. Unfortunately, if too many T cells are removed, this process may also increase the incidence of graft failure and leukemia relapse, suggesting that some residual T cells are beneficial in an allogeneic product. Rigorous depletion of 4 logs also significantly delays T-cell immune reconstitution, especially CD4⫹

T cells, after transplantation. Conversely, if not enough T cells are removed, GVHD may occur, particularly if the donor and recipient are not fully HLA-matched. Some centers prefer to use the most rigorous depletion procedure available (immunomagnetic, as discussed below), and then add back defined doses of T cells or T-cell subsets to combat graft failure or speed immune reconstitution. Other centers have chosen to do a less complete TCD and use additional posttransplant immune suppression to prevent GVHD. Thus, the choice of TCD procedure to employ should be based on the desired target dose for infusion. The critical number of T cells needed to support the graft-vs-leukemia effect has not yet been determined. However, transplantation with a T-cell dose of 1 ⫻ 105/kg body weight allows engraftment and minimizes the severity of GVHD. TCD may also be used to remove autoreactive T cells from autografts used to treat patients with various severe autoimmune diseases. This approach appears promising but is currently considered to be experimental. Early T-cell depletion techniques were based on the phenomenon by which T cells form rosettes around sheep red cells.50 After rosetting, the T cells can be removed from the suspension by means of density gradient centrifugation. This technique is tedious and labor intensive, but surprisingly effective. More recently, most centers that perform T-cell depletions utilize MoAbs to T-cell antigens to remove these cells from allogeneic grafts. Methods have been developed in which antibodies to antigens such as CD3, CD4, CD5, CD6, and CD8 are used in conjunction with complement, immunotoxins, or magnetic microspheres to remove T cells from allogeneic grafts.51-53 Not all TCD methods target the total CD3⫹ T-cell population. Depleting only the CD8⫹ subset of CD3⫹ T cells may achieve similar outcomes to total TCD without as many of the negative side effects. Some centers even target only those donor T cells that respond to the recipient alloantigens.54 Performing a CD34 selection also produces an infusion product with 4 log fewer T cells than were contained in the original product.55-57

Donor Lymphocytes Donor T cells that are reactive to recipient minor histocompatibility antigens can cause a graft-vs-leukemia response and thereby reduce relapse rates.58,59 Donor leukocyte infusion (DLI), has been used to obtain complete remissions in relapsed acute myeloid leukemia, acute lymphocytic leukemia, multiple myeloma, non-Hodgkin lymphoma, myelodysplastic syndrome, and chronic myeloid leukemia.60-62 It is most successful with chronic myelogenous leukemia. Approximately 65% of patients achieve complete remission in comparison with 25% of patients with acute myelogenous leukemia or myelodysplastic syndrome.63 Donor lymphocyte infusions have been most effective when administered early in the relapse. The risks associated with DLI are GVHD and marrow suppression. The risk is minimized if treatment begins with a small dose that is increased if there is no response. Allogeneic T lymphocytes are now commonly cryopreserved either before, or as part of, the HPC collection, especially in the unrelated donor setting. The donor CD3⫹ T cells contained in

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marrow, mobilized PBPC products, nonmobilized PBPC products, or small-volume PBPC donations are enumerated and can be given to the recipient fresh, in specified doses, or else cryopreserved in CD3⫹ dose aliquots. Typically, the T-cell doses infused range from 1 ⫻ 106/kg to 5 ⫻ 107/kg. Physicians typically request that multiple doses be cryopreserved for later use, in case of relapse. The same cryopreservation procedure used for HPC products is most commonly used for these small-volume products.

Cryopreservation Transplantation of autologous marrow and PBPC and allogeneic UCB requires a means of efficient preservation of the progenitor cells, sometimes for months or even years before transplantation. As cryopreservation techniques have improved, more allogeneic products are being cryopreserved for donor convenience, or to reduce the risk of a collection failure or transport error while a patient is immunocompromised. Cryopreservation is among the most critical manipulations performed in clinical cell processing laboratories. Techniques vary slightly among institutions; however, in all cases, the objective is to adjust the volume to the container of choice, add a cryoprotectant solution and then freeze the cells at a controlled rate, in a manner that preserves cell viability and proliferative potential after thawing. It is essential that the cryopreservation procedure prevent ice crystal formation, prevent the formation of toxic solute concentrations that can result from cell dehydration, and stabilize the cell membrane in order to prevent damage during the thawing process. Mononuclear cells tolerate freezing relatively well, so minor procedural deviations are not usually detrimental. Even so, process variables such as volume:surface area ratio and the rate that the cryoprotectant is added should be well controlled, and monitored regularly. Many centers prefer to perform a buffy-coat procedure to reduce the red cell content in UCB and marrow products before cryopreserving them.37 There are numerous cryoprotectant solutions being used with good results. All contain a colligative agent such as dimethylsulfoxide (DMSO); one or more polymeric agents such as HSA, autologous plasma, or hetastarch; an anticoagulant; and an isotonic diluent or culture media. The standard freezing mixture contains 20% DMSO (final concentration, 10%), ACD-A (final concentration 10%), an isotonic electrolyte solution, and a source of protein, usually autologous plasma or HSA, although several laboratories prefer a lower DMSO concentration.64-67 DMSO is toxic to cells if exposed for long periods at room temperature; thus, it is important to keep the cell suspension and all reagents chilled before and after the addition of the DMSO.68 In addition, diluting DMSO in aqueous solutions is an exothermic reaction. Therefore, it is important to dilute the DMSO with an aqueous buffer and refrigerate the solution before adding it to the cells to minimize the DMSO toxicity. The addition of the DMSO solution to the cells needs to occur slowly for similar reasons. Product handling procedures that occur after DMSO addition need to be gentle and need to avoid exposing the cell membranes to excessive shear forces.

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Stiff et al69 were the first to incorporate hydroxyethyl starch (HES) as an additional cryoprotectant. The addition of HES to the cryopreservation media allows for the reduction of the DMSO concentration, thereby decreasing the unpleasant side effects and adverse events occasionally experienced during or after DMSO infusion.70,71 However, it is important to be aware of possible complications, such as hypertension, that can result from infusing large volumes of HES. A 2.5% to 6% HES concentration in combination with 5% DMSO is an effective cryoprotectant for cryopreserving hematopoietic cells.72,73 Whichever cryoprotectant is used, the freezing rate is also important for viable cell recovery. A controlled freezing rate of ⫺1ºC to ⫺2ºC per minute is described as optimal in most reports. Thus, the most effective freezing profiles use a liquid nitrogen freezer that is programmed to maintain a constant freezing rate of between ⫺1ºC and ⫺2ºC, despite the heat of fusion that occurs as the solution begins to solidify.74 Slightly different profiles are required for different container sizes. A typical freezing profile is shown in Fig 33-1. The program that the freezer used to generate this freezing profile is presented in Table 33-5. After the cells reach ⫺90ºC, they are transferred to a liquid nitrogen storage freezer and are stored in the vapor phase, at ⭐⫺160ºC. Some centers do not use a programmable freezer, but instead place the products into a –80ºC mechanical freezer for 18 to 24 hours before transferring them to liquid nitrogen storage. There are several reports in the literature claiming equivalence to controlledrate freezing if the packaging configuration is correct.66,75-79 Although the storage of HSCs at ⫺80ºC does not seem to cause obvious damage during short periods, there is evidence that complete cessation of enzymatic and metabolic activity does not occur until a temperature of at least ⫺135ºC has been reached.69 Once a particular method has been validated to result in rapid engraftment and an acceptable postthaw viability (using 7-amino-actinomycin D), it is important to keep variables such as product volume, container size, and maximum cell concentration consistent so that the temperature distribution is uniform.80 The optimal cell concentration is still not clear, so many laboratories choose to allow this parameter to vary, up to a maximum of between 1 and 3 ⫻ 108 cells/mL, so that the product volume per bag can be consistent from patient to patient.81

Storage When the freezing program is completed, the bags can be removed from the chamber and placed in prechilled and labeled protective canisters for storage. The storage temperature should be low enough to slow the metabolism and block all the enzymatic pathways; thus, the lower the storage temperature the better. Mechanical freezers can achieve temperatures as low as ⫺120ºC; however, liquid nitrogen freezers are much more reliable and provide even lower temperatures. Vapor phase liquid nitrogen storage (⫺160ºC) has the advantage of preventing product cross-contamination and minimizes occasional bag breakage during thawing. Vapor phase storage is most frequently

Chapter 33: HPCs: Biology and Processing

60

40

20

Reference point

Temperature in degrees celsius

0

⫺20

⫺40

Sample

⫺60

⫺80

Chamber ⫺100

Figure 33-1. Typical freezing curve from the cryopreservation of a PBPC product in a controlledrate freezer. This curve was generated using the freezing profile shown in Table 33-5.

⫺120

Time in minutes

⫺140

10

Table 33-5. Typical Profile for Hematopoietic Cell Cryopreservation Profile

70-mL bag

Step No.

Rate (degrees per min.)

End Temp. (degrees C)

Trigger

1

Hold

4

Chamber temp

2

⫺1

⫺8

Sample temp

3

⫺25

⫺60

Chamber temp

4

⫹10

⫺30

Chamber temp

5

⫺1

⫺30

Sample temp

6

⫺10

⫺90

Sample temp

7

Hold

⫺90

Chamber temp

used for short-term (⬍1 year) storage.82,83 Liquid phase storage (⫺196ºC) is less prone to occasional temperature fluctuations as freezers are accessed, and provides a larger safety margin if the liquid nitrogen source is depleted; thus, it is frequently used for long-term storage. In either case, continuous temperature monitoring, remote alarm notification, and automatic fill features are essential to maximize product safety during storage. Overwrap bags provide an extra layer of protection against product crosscontamination, and are advisable for high-risk products. Short-term storage of products before or after processing is best accomplished by providing nutrients, buffering the pH, and augmenting the anticoagulant solution, if appropriate. Viability is best maintained if the cells are stored at a cool temperature in a gas-permeable bag. Because it is not clear how long HPC products remain effective when stored under these conditions, it is best to minimize storage times whenever possible.

20

30

40

50

60

Transportation Product transport is essentially short-term product storage under duress, yet it is a critical maneuver for those centers that process tissues from distant clinical sites. Even the best planned courier systems fail in bad weather, or when air travel is involved, requiring last-minute adjustments in staffing schedules. Such situations have the potential to compromise the product if delays are excessive. Diluting the cells with autologous plasma and a buffer solution at the collection site, adding extra heparin for long-distance transport, and shipping at a cool temperature in a well-insulated container help significantly to protect the product. Adding a source of DNAse upon arrival in the laboratory for products more than 24 hours old can also improve processing results by eliminating the free DNA released from cells that have died during transport. All product shipment procedures need to comply with current biohazard product packaging and labeling regulations. Fresh HPC products need to be packaged in sealed, puncture-proof containers. Transportation of frozen HPC products requires the use of a liquid nitrogen dry shipper that contains absorbent material in the wall of the device to absorb the liquid nitrogen and eliminate the risk of spillage. These devices are designed to hold temperatures below ⫺160ºC for 8 to 10 days, but the liquid nitrogen evaporation rate needs to be checked before each use.

Thawing/Infusion Cryopreserved cells are less prone to injury when thawed rapidly. The bags of cell suspension are thawed just outside the patient’s room in sterile overwrap bags in a 37ºC waterbath. The bags are thawed one at a time and immediately, but slowly, infused through a central venous catheter or intravenous access, as soon as the product reaches approximately 15ºC. It is generally best if

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laboratory personnel perform the thawing procedure and clinical personnel perform the infusion. The bag of cells may be hung and allowed to drip through an administration set, or the cell suspension can be withdrawn from the bag with a syringe and injected directly into the catheter port. A few centers wash the cell suspension to reduce the volume or to remove DMSO before infusion, particularly for pediatric patients or for patients with renal insufficiency. Because of the fragility of the cells after thawing, any procedure must treat the cells gently and not subject them to vigorous shaking or centrifugation, which can cause the suspension to clump, resulting in poor cell recovery. Slowly diluting the cell suspension with approximately 1:5 cold dextran and allowing the cell membranes to stabilize before centrifugation improves viability and recovery. If any postthaw processing is anticipated, it is advisable to consider the addition of DNAse during either freezing or thawing to reduce the clumping caused by single-stranded DNA from the cells that did not survive the freeze-thaw process. Platelets and plasma proteins also can cause clumping of progenitor cells after thawing.

Quality Control Quality control testing plans need to be protocol-specific, and designed to demonstrate postprocessing product integrity and product safety. Product safety is the primary concern, especially for Phase I clinical trial products. However, demonstration of product integrity (potency) is also important for interpreting clinical findings.

Safety The tests most often performed in order to demonstrate safety are donor infectious disease testing, bacterial and fungal product sterility testing, endotoxin testing, and mycoplasma testing (for cultured products). Additional safety tests are often required by the FDA for complex products—for example, testing for the presence of replication-competent retrovirus in the final product is required when using a retroviral vector to genetically alter an HPC product. Products need to be tested for sterility at receipt and at the end of processing. Final product testing is mandated; testing at receipt provides a way to determine whether any of the techniques or materials used have compromised the purity of the product. Even if every effort is made to maintain sterility during collection and processing of an HPC product, there is a risk of contamination with microorganisms, especially when multiple manipulations are performed. Contamination can result from microorganisms in the skin or bloodstream of the patient or donor, improperly sterilized equipment or materials, environmental contaminants, or faulty handling of the graft.84,85 Sterility testing should be performed with a validated method for detecting bacterial (aerobic and anaerobic cultures) and fungal contamination. Procedures need to be in place whereby the

516

patient’s physician is promptly notified if a sterility test result is positive. For products released before even preliminary results of sterility testing, it is often difficult to obtain proof of product safety before infusion. It is still important to perform the cultures and to document product safety after the fact. Same-day procedures such as Gram’s staining and endotoxin assays can also be helpful.

Integrity Integrity testing provides the patient’s physician with important product purity and potency information and can be valuable for evaluating process improvements and deviations. The most widely used potency assay for progenitor cell components is the number of viable CD34⫹ cells (or their subsets) present at the end of the procedure. Numerous reports in the literature have concluded that the time to hematopoietic recovery after transplantation is optimal with CD34 doses of at least 5 ⫻ 106 per kg.86-88 The total nucleated cell dose is obtained using an automated hematology analyzer. The percentage of MNCs can be determined by performing a differential count on a stained smear. Cell viability and phenotypic characterization (CD45, CD34, CD3) are best accomplished using flow cytometry.89-93 Functional assays such as colony-forming ability, long-term culture-initiating ability, or animal model in-vivo hematopoietic reconstitution assays are essential for establishing the therapeutic potential of new products, but are impractical for everyday use.94

Release Criteria Minimal acceptance criteria for nucleated cell count, viability (FDA recommends ⬎70%), target cell content, progenitor cell content, etc, should be established in advance. If a product does not meet release criteria, it must be further processed (to bring it into compliance), discarded, supplemented with additional product, or released with the approval of appropriate laboratory and clinical personnel. In principle, product release criteria should have clinical relevance. With cell therapy products, however, the products are so extremely valuable to their intended recipients that physicians almost always want to infuse the cells even if they do not meet predefined acceptance criteria. Often, it is not possible to discard a contaminated product because the patient will not survive the myeloablative therapy without receiving the product. The patient’s physician is notified when a product does not meet expectations and the procedures used to produce the product are thoroughly investigated. Product release decisions should be based on objective criteria that are more encompassing than just the review of final product test results. Most release criteria are safety oriented; therefore, proper procedural documentation, raw material testing, and defined acceptance criteria for in-process test results all contribute to user confidence in product quality and should be incorporated into the quality assurance plan. The best way to design relevant release criteria is to determine under what circumstances the product should not be infused. What if the product wasn’t properly labeled? What if it were known to contain human immunodeficiency virus? What if the purity was

Chapter 33: HPCs: Biology and Processing

so low that the chance of achieving the goal of the trial is too remote to justify the risks of proceeding? Such issues need to be addressed among the transplant team members.

Table 33-6. Quality Plan Essentials

Quality Assurance

Materials Management

Process Improvement

Supplier qualification Human Resources Personnel training Staff competency

Deviation management Corrective and preventive action Equipment

Documents and Records Document control Label control Worksheets

Validation Calibration Maintenance Information Management

Facilities

Nonconforming Events

Organization and Leadership Process Control Standard operating procedures

Accreditation Voluntary standards have been developed by two professional organizations in an effort to improve product quality. The AABB and FACT have published standards for the collection, processing, storage, and infusion of cell therapy products.95,96 Both professional organizations offer on-site accreditation of laboratories acting in accordance with the published standards. Both systems emphasize the prevention of errors and an ongoing process of continuous quality improvement. FACT and AABB accreditation are theoretically voluntary, but are essentially mandatory because most clinical transplant programs are required to be accredited to obtain insurance contracts. Adherence to voluntary standards such as those promoted by these organizations is an excellent yardstick for predicting product performance. Accreditation should be mandated for every cell processing facility.

Federal Regulations All cell therapy products, including hematopoietic stem cells, are biologic products that are currently subject to regulation by the FDA via the Code of Federal Regulations (CFR) under 21 CFR part 1271 or 21 CFR part 211, depending on the product type. All cell processing groups must now be registered with the FDA and must be prepared for ad hoc FDA inspections. Minimally manipulated PBPC and marrow products for homologous use are regulated via FDA’s current good tissue practice (cGTP) regulations that are contained in 21 CFR part 1271. The more complex biologic products, such as cord blood expansion products and gene therapy products, are regulated via FDA’s current good manufacturing practice (cGMP) regulations that are contained in 21 CFR part 211.

Quality Systems The best way to meet all the various standards and regulations is to design and write a quality assurance plan that contains clear instructions for how to accomplish each of the universal quality assurance principles listed in Table 33-6. Multiple resources are available for writing a quality management plan, including both FACT and AABB standards. In general, quality management plans that focus on error prevention (not error reporting) are the most effective. Many problems can be avoided by strict attention to process control. Control of the product collection, processing, transport, and storage procedures is critical. Standard operating procedures should be written clearly and concisely so that critical control points in the procedure can be monitored. Materials management procedures prevent problems by ensuring that all reagents and supplies are properly acquired, stored, and used. Validation programs are excellent tools for proving proper equipment operation and process performance. A good

Environmental control Environmental monitoring Safety

Monitoring and Assessment Audits QC testing

Customer Focus Communication

document control system ensures consistent SOP conformance and prevents most labeling errors. Environmental monitoring and control systems help prevent accidental contamination errors. Proper deviation reporting and corrective action tracking greatly facilitate continuous process improvement. Perhaps most important, a program of personnel training and ongoing competency assessment prevents the operator errors that are caused by miscommunication or misunderstanding. Once these systems are in place, it can be advantageous to file a type V drug master file with the FDA that can be referenced with each subsequent IND submission to speed the approval process.

Disclaimer T. Leemhuis has disclosed no conflicts of interest. R. Sacher has disclosed professional relationships with Talecris Biotherapeutics and GlaxoSmithKline.

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75. Makino S, Harada M, Akashi K, et al. A simplified method for cryopreservation of peripheral blood stem cells at ⫺80ºC without rate-controlled freezing. Bone Marrow Transplant 1991;8:239-44. 76. Galmes A, Besalduch J, Bargay J, et al. A simplified method for cryopreservation of hematopoietic stem cells with ⫺80 degrees C mechanical freezer with dimethylsulfoxide as the sole cryoprotectant. Leuk Lymphoma 1995;17:81-4. 77. Galmes A, Besalduch J, Bargay J, et al. Cryopreservation of hematopoietic progenitor cells with 5% dimethylsulfoxide at ⫺80ºC without rate-controlled freezing. Transfusion 1996;36:794-7. 78. Hernandez-Navarro F, Ojeda E, Arrieta R, et al. Hematopoietic cell transplantation using plasma and DMSO without HES, with nonprogrammed freezing by immersion in a methanol bath: Results in 213 cases. Bone Marrow Transplant 1998;21:511-17. 79. Katayama Y, Yano T, Bessho A, et al. The effects of a simplified method for cryopreservation and thawing procedures on peripheral blood stem cells. Bone Marrow Transplant 1997;19:283-7. 80. Martin-Henao GA, Torrico C, Azueta C, et al. Cryopreservation of hematopoietic progenitor cells from apheresis at high cell concentrations does not impair the hematopoietic recovery after transplantation. Transfusion 2005;45:1917-24. 81. Cabezudo E, Dalmases C, Ruz M, et al. Leukapheresis components may be cryopreserved at high cell concentrations without additional loss of HPC function. Transfusion 2000;40:1223-7. 82. Tedder RS, Zuckerman MA, Goldstone AQH, et al. Hepatitis B transmission from contaminated cryopreservation tank. Lancet 1995;346;137-40. 83. Fountain D, Ralston M, Higgins N, et al. Liquid nitrogen freezers: A potential source of microbial contamination of hematopoietic stem cell components. Transfusion 1997;37:585-91. 84. Webb IJ, Coral FS, Andersen JW, et al. Sources and sequelae of bacterial contamination of hematopoietic stem cell components: Implications for the safety of hematotherapy and graft engineering. Transfusion 1996;36:782-8. 85. Padley D, Koontz F, Trigg ME, et al. Bacterial contamination rates following processing of bone marrow and peripheral blood progenitor cell preparations. Transfusion 1996;35:53-6.

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86. Weaver CH, Hazelton B, Birch R, et al. An analysis of engraftment kinetics as a function of the CD34 content of peripheral blood progenitor cell collections in 692 patients after the administration of myeloablative chemotherapy. Blood 1995;86:3961-9. 87. Ketterer N, Salles G, Raba M, et al. High CD34⫹ cell counts decrease hematologic toxicity of autologous peripheral blood progenitor cell transplantation. Blood 1998;91:3148-55. 88. Allan DS, Keeney M, Howson-Jan K, et al. Number of viable CD34⫹ cells reinfused predicts engraftment in autologous hematopoietic stem cell transplantation. Bone Marrow Transplant 2002;29:967-72. 89. Keeney M, Chin-Yee I, Weir K, et al. Single platform flow cytometric absolute CD34⫹ cell counts based on the ISHAGE guidelines. Cytometry 1998;34:61-70. 90. Sutherland DR, Anderson L, Keeney M, et al. The ISHAGE guidelines for CD34⫹ cell determination by flow cytometry. J Hematother 1996;5:213-26. 91. Gratama JW, Keeney M, Sutherland DR. Enumeration of CD34⫹ hematopoietic stem and progenitor cells. Curr Prot Cytometry 1999;6:1-22. 92. Brocklebank AM, Sparrow RL. Enumeration of CD34⫹ cells in cord blood: A variation on a single-platform flow cytometric method based on the ISHAGE gating strategy. Cytometry 2001;46:254-61. 93. Chapple P, Prince HM, Wall D, et al. Comparison of three methods of CD34⫹ cell enumeration in peripheral blood: Dual-platform ISHAGE protocol versus single-platform, versus microvolume fluorimetry. Cytotherapy 2000;2:371-6. 94. Verfaillie CM, Ploemacher T, Di Persio J, et al. Assays to determining hematopoietic stem cell content in blood or marrow grafts. Cytotherapy 1999;1:41-9. 95. Padley D, ed. Standards for cellular therapy product services. 3rd edition. Bethesda, MD: AABB, 2007. 96. FACT-JACIE international standards for cellular therapy product collection, processing and administration. 3rd edition. Omaha, NE: Foundation for the Accreditation of Cellular Therapy and the Joint Accreditation Committee of ISCT and EBMT, 2006.

34

Hematopoietic Progenitor Cells: Autologous Transplantation Hooman H. Rashidi1 & Scott D. Rowley2 1

Chief Resident, Department of Pathology and Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut, USA 2 Chief, Adult Blood and Marrow Stem Cell Transplantation Program, Hackensack University Medical Center, Hackensack, New Jersey, USA

The number of patients undergoing hematopoietic stem cell (HSC) transplantation has grown rapidly as the efficacy of this treatment continues to be demonstrated in clinical trials. Transplant registries have collected data on over 65,000 transplant procedures, but this is believed to represent only a portion of all such procedures performed.1 In Europe alone, over 12,000 marrow or peripheral blood stem cell (PBSC) transplant procedures are performed each year.2 Much of this increase can be attributed to the adoption of PBSC products as the primary source of HSCs for transplantation, a change driven by the more rapid engraftment parameters of PBSCs, with resulting lower regimen-related risks of morbidity and mortality, and lower costs. Also, the use of high-dose chemo- and radiotherapy with stem cell rescue has increased because of success in randomized trials of dose-intensive therapy in achieving durable remissions in common dose-responsive malignancies such as multiple myeloma,3 Hodgkin disease,4 and non-Hodgkin lymphoma (NHL).5 It has been 50 years since the discovery that cellular elements of the marrow protect against lethal irradiation.6 Transplantation science during this interval is notable for the development of antineoplastic conditioning regimens achieving effective tumor cell kill, immunomodulation regimens allowing allogeneic transplantation without inevitable fatal graft-vs-host reactions, and simple HSC collection and cryopreservation techniques. An understanding of the hematopoietic and immunologic systems has continued to develop, so that enriched populations of HSCs or immunologic effector cells may be isolated and manipulated ex vivo to generate “somatic cell therapy” products that differ in function from native marrow cells. Historically, HSC transplantation was developed as a technique to deliver high doses of chemotherapy with or without radiation therapy. The collection, storage, and infusion of autologous HSCs was intended to alleviate the iatrogenic marrow failure of the high-dose chemo- and radiotherapy regimen used Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

to treat the patient. The infusion of autologous HSCs, in contrast to allogeneic transplantation, conveys no direct immune antineoplastic benefit to the recipient given that there is no immunologic graft-vs-disease response. Autologous transplantation is, therefore, based on two considerations: ● The dose sensitivity of the disease being treated and the availability of dose-intense regimens capable of ablating this disease. ● The ability to collect and successfully store adequate numbers of HSCs to allow rapid reconstitution of marrow function. Although autologous HSC transplantation does not convey the immunologic graft-vs-disease effect achieved with allogeneic transplantation, the corresponding lack of graft-vs-host disease (GVHD) and the avoidance of toxic immunosuppressive medications required after allogeneic transplantation allows the use of dose-intense therapy for older patients, including patients in their eighth or ninth decade of life, as well as for patients with comorbid conditions that might preclude allogeneic transplantation. Both the dose and combination of agents used for chemotherapy are important in achieving cure of malignant diseases. Malignant diseases such as Hodgkin disease, intermediate-grade NHL, and acute leukemia are potentially curable if multiple chemotherapy agents are administered in a regimen that allows the use of an effective dose of each agent. The importance of combining multiple chemotherapy agents is illustrated by the increase in cure rates for Hodgkin disease treated with effective drugs in combination, in contrast to similar drugs used singly or in sequence. The importance of dose is illustrated by the lack of further increase in the proportion of patients cured by the inclusion of additional chemotherapeutic agents to a regimen, if such addition requires a reduction of dose to avoid regimenrelated toxicities. Thus, the combination of mechlorethamine, vincristine, prednisone, and procarbazine (MOPP), and doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) in alternating schedules that doubles the number of drugs is no more effective than either regimen used alone in the treatment of Hodgkin disease. This is because in combining these regimens, the dose of the individual drugs is reduced by 50% because of

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the alternate-cycle administration of each regimen. Frei et al7 proposed that effectiveness of a chemotherapy regimen can be estimated by calculating the “summation dose intensity” that includes the amount of drug delivered over a period of time. The curative treatment of dose-responsive malignancies such as breast cancer is limited by the lower activity of the chemotherapy agents available and because almost all of the agents are myelosuppressive, requiring dose reduction to accommodate combination chemotherapy. However, even for breast cancer, the importance of dose density (amount of chemotherapy administered over a unit of time) was demonstrated in 1981 by Bonadonna and Valagussa8 in a retrospective analysis of two of their clinical trials with cyclophosphamide, methotrexate, and 5- fluorouracil (5-FU). They found that disease-free survival (DFS) was longer for those patients who received a higher percentage of the intended dose of chemotherapy. This could reflect either the larger amount of drug delivered or that women who tolerated the drugs were also more likely to live longer. Similarly, Kummel et al9 reported a randomized trial of dose-dense therapy in the adjuvant treatment of patients with node-positive breast cancers and found improved disease-free survival and overall survival in the patient group receiving the dose-dense regimen. Wood et al10 showed in a prospective randomized trial that the delivery of higher doses resulted in a significantly better survival of women being treated for early breast cancer. This larger amount of chemotherapy delivered was better regardless if it was delivered in higher doses with fewer cycles or if an equivalent amount of chemotherapy was delivered in lower doses but over a longer period. The authors conclude that these data are consistent with either a dose-response effect or a threshold level of the dose or dose intensity. The importance of dose in the treatment of lung cancer and NHL is also known. The infusion of HSCs allows the administration of a doseintense (higher doses of drugs over a short period) regimen without concern for the effects on hematopoiesis (myeloablative regimens). The importance of dose intensity instead of dose density for many diseases is demonstrated by randomized trials of autologous transplantation for diseases such as multiple myeloma,3 Hodgkin disease,4 and NHL.5 These trials illustrate that dose-intense therapy is more effective than available nontransplant regimens delivered in multiple cycles over time. The delivery of even more chemotherapy using sequential (tandem) transplants has been explored in a small number of clinical trials,11 but careful selection of regimens is required to avoid the nonmyeloid toxicities of the regimens used. The infusion of HSCs has also been demonstrated to facilitate the administration of dose-dense nonmyeloablative regimens, potentially achieving a doubling of the amount of chemotherapy administered.12,13 The potential of this approach was most clearly illustrated by Petengell et al,13 who treated 25 patients with small-cell lung cancer with multiple cycles of ifosfamide, carboplatin, and etoposide (ICE) chemotherapy. Peripheral blood stem cells were collected after each cycle of chemotherapy and used for the support of the following cycle. The investigators

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were able to double the dose-density, although only about 50% of the patients were able to complete the planned therapy. Five patients reverted to standard-dose therapy at a median of chemotherapy cycle four. Three patients died while on study (two of sepsis). Others reverted to standard-dose therapy because of prolonged cytopenias (two patients) and inability to collect cells or intolerance of dimethylsulfoxide (DMSO) (one patient each). One group treated 18 patients with sarcoma with high-dose ifosfamide and doxorubicin using a single PBSC collection split into multiple fractions and cryopreserved.12 The group administered a median of 0.9
106 CD34 cells/kg with each cycle of therapy; the patients experienced a granulocyte count 500/ µL for a median of 4 days and a platelet count 20,000/µL for a median of 2 days. The acceptance of cell support for these dosedense regimens is limited by the logistics of delivering cryopreserved cells in hospitals and offices without easy access to this technology.

Techniques of Autologous HSC Transplantation Patient Eligibility for Autologous HSC Transplantation Careful patient selection and timing of treatment are critical to the success of dose-intense therapy. Patients with bulky tumor masses and patients with chemotherapy-resistant malignancies are much less likely to achieve durable control of malignancy even with the use of dose-intense regimens. The early introduction of dose-intense therapy in the treatment of a patient, before chemotherapy resistance and excessive chemotherapy-induced organ toxicity develop, is paramount. Therefore, treatment algorithms incorporating HSC salvage strategies must be part of the initial therapeutic decisions for each patient with a disease possibly treatable by high-dose therapy. Patients being prepared for autologous transplantation will frequently undergo several cycles of chemotherapy intended to: ● Demonstrate the sensitivity of the disease to chemotherapy. ● Reduce the bulk of the disease before autologous transplantation. ● Facilitate the collection of PBSCs. Involved-field radiotherapy can be used to debulk large tumor masses but is, in general, reserved for administration after recovery from transplantation because of the deleterious effects of this treatment modality on the collection of PBSCs and the increase in organ toxicity within the treatment field with subsequent administration of chemotherapy. Radiotherapy used as consolidation therapy after autologous transplantation sometimes requires the infusion of additional HSCs to offset the marrow-suppressive effects of this therapy. This illustrates the need for comprehensive planning to ensure that reserve quantities of HSCs are available if needed. Patients undergo a detailed evaluation to uncover any organ dysfunction that could preclude the safe administration of this therapy. Although HSC reinfusion rescues the patient from the hematologic toxicity of the conditioning regimen,

Chapter 34: HPCs: Autologous Transplantation

the nonhematologic (organ) toxicity of dose-intense therapies remains. A retrospective review of 383 consecutive transplant procedures noted factors predictive of early transplant-related mortality including an FEV1 (forced expiratory volume in 1 second) less than 78% of predicted, serum creatinine greater than 1.1 mg/dL, and serum bilirubin greater than 1.1 mg/dL.14 Current transplantation techniques using PBSCs minimize the period of neutropenia and the infection risk. Mortality rates reported after autologous HSC transplantation are frequently less than 5%, although this risk increases somewhat for older patients.15 Because of this risk, patients undertaking dose-intense therapy, if possible, should be free of infection and have other comorbid conditions such as diabetes mellitus under optimal medical control.

antibodies to a chemotherapy or TBI-based regimen to specifically target areas of tumor while sparing radiation-sensitive organs such as the liver, lung, and kidney.16 In the study, 21 to 27 Gy of iodine-131-labeled anti-CD20 was added to a conditioning regimen of etoposide (20 mg/kg) and cyclophosphamide (100 mg/kg). This resulted in a 77% complete response rate and a 68% 2-year probability of progression-free survival (PFS) for patients with relapsed NHL. The 2-year PFS was almost double that previously experienced (36%) by similar patients treated with chemotherapy alone. This technique is currently being evaluated in a multicenter study sponsored by the National Heart, Lung, and Blood Institute, National Cancer Institute, and National Institute of Allergy and Infectious Diseases through the Bone Marrow Transplant/Clinical Trials Network.

Selection of Conditioning Regimen

Selection of HSC Source

Autologous HSC transplantation facilitates disease cure through the administration of dose-intensive regimens. Therefore, the conditioning regimen used should include drugs that are effective in the treatment of the particular malignancy and induce minimal nonhematopoietic organ damage at the myeloablative doses used for transplantation. Immunosuppression is not required for engraftment of autologous HSCs; therefore, agents such as antithymocyte globulin or total body irradiation (TBI) are not necessary. Some regimens, such as high-dose melphalan for the treatment of patients with multiple myeloma and carmustine (BCNU) based regimens for the treatment of lymphoma, are commonly used based on the results of multicenter Phase III studies showing the tolerability of these regimens. Relapse is the major cause of failure of autologous HSC transplantation. Therefore, some transplant programs are exploring novel techniques to deliver yet more intensive conditioning to the regimen. One example of this is the addition of radiolabeled

Peripheral blood stem cells have virtually replaced marrow as the HSC component for autologous transplantation and have shown benefit in Phase III studies in allogeneic transplantation.17 Data published from a European transplant registry showed that the vast majority of autologous and all allogeneic HSC transplants were still marrow transplants in 1991.18 By 2003, PBSCs were used in 97% of autologous HSC transplant procedures. The ease of collection and the rapid engraftment kinetics of PBSCs compared to marrow are widely recognized. With PBSCs, median times to achieve an absolute neutrophil count (ANC) 500/µL are typically about 10 to 12 days, and platelet recovery is even faster (Table 34-1).19-23 In one Phase III study of 47 patients treated for germ cell tumor, rapid engraftment kinetics were achieved with PBSC transplantation. A small number of the PBSC recipients achieved a neutrophil count of 500/µL 1 day faster, and a sustained platelet count of 20,000/µL 7 days faster than the patients receiving marrow with all patients receiving

Table 34-1. Relationship Between Mobilization Scheme, Dose of CFU-GM or CD34 Cells Infused, and Engraftment Kinetics After Autologous PBSC Transplantation* Study

Patients

Nademanee20 21,22

39

Sheridan

29

Weaver23

692

Bensinger19

124

Therapy

Progenitor Cell Dose

Engraftment Kinetics (in days)

CFU-GM
104/kg

CD34
106/kg

ANC 500/µL

Platelets 20,000/µL

G-CSF

ND

6.2

10 (7-40)

15.5 (7-63)

G-CSF

21.0

ND

6 (4-10)

11 (9-136)

Chemotherapy†

30.8‡

9.9‡

9 (5-38)

9 (4-53)

ND

9.4

11 (4-20)

10 (6-65)

G-CSF Chemotherapy† G-CSF * Shown are mean values for progenitor cell quantities infused and median values for time to achieve the particular endpoint of engraftment. † CFU-GM cultures performed on thawed cells. ‡ Shown are median values. CFU-GM  colony-forming unit-granulocyte-macrophage; PBSC  peripheral blood stem cell; ANC  absolute neutrophil count; ND  no data; G-CSF  granulocyte colony-stimulating factor.

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granulocyte colony- stimulating factor (G-CSF).24 A similar trial for patients with NHL or Hodgkin disease reported faster platelet recovery (time to a platelet count 20,000/µL) of 16 vs 23 days, and faster granulocyte recovery (time to a neutrophil count 500/µL ) of 11 vs 14 days for recipients of PBSCs compared to recipients of marrow.25 The PBSC recipients also spent fewer days in the hospital and required fewer red cell and platelet transfusions. There are disadvantages to the use of PBSC components as a source of HSCs for autologous transplantation. These include the usual need for multiple days of collection, the current need for sophisticated flow cytometric analysis of the components to ensure adequacy of HSC content, the inability to collect adequate components from all patients, the (minimal) risks associated with administration of hematopoietic cytokines and the apheresis procedures, and the risks of infusion if the multiple components are cryopreserved. Marrow collection and infusion is available to patients for whom adequate PBSC component(s), defined by the number of CD34 cells collected, cannot be harvested and stored. The dose of CD34 cells in the PBSC component(s) required for infusion depends upon the intended treatment regimen. Lower doses of CD34 cells appear satisfactory for nonablative regimens.12,13 For example, one group infused 0.9
106 CD34 cells per cycle for patients treated with high-dose ifosfamide and doxorubicin.12 Despite an overall doubling of the ifosfamide dose in this Phase I study, the median times between cycles did not progressively increase, indicating that adequate marrow stores were maintained. For myeloablative regimens, increasingly higher CD34 doses result in greater likelihood of rapid recovery of PBSC counts. Patients who receive a dose of CD34 cells above an illdefined threshold experience engraftment. At lower doses of CD34 cells there is considerable heterogeneity in engraftment speed, especially for platelet recovery, with some patients experiencing quick engraftment despite low doses of PBSCs. It is not known why this heterogeneity exists but it may reflect host marrow stromal cell damage from previous disease infiltration or chemotherapy, a weakness in the correlation between CD34 cells and the cells responsible for hematologic recovery or, more simply, a greater degree of error in the measurement of CD34 cells at the lower cell concentrations found for “poor mobilizers.” As the dose of CD34 cells increases, the engraftment speed becomes more consistent for the population studied, although the median days of cytopenia and the minimum number of days is not affected.19,23 Several investigators showed more consistently rapid granulocyte and platelet engraftment for recipients of products containing a quantity of CD34 cells above a dose of about 2 to 3
106/kg of recipient weight.26-28 Many of the randomized studies showing an advantage of autologous HSC transplantation over other (nontransplant) therapies used marrow as the source of HSCs. The duration of aplasia predicts the incidence of peritransplant mortality after autologous transplantation.29 Although this may reflect patient

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characteristics rather than graft characteristics, it is possible that use of PBSCs will result in yet a greater difference in the outcome of these therapies.

Expansion of Inadequate HSC Components Hematopoietic stem cells are characterized by the ability to selfrenew and to differentiate to mature blood cells. Collections of HSCs from the marrow or peripheral blood are termed “stem cells” despite the fact that they contain distinct populations of true HSCs and more differentiated progenitor cells with limited proliferative capacity. In-vitro techniques to increase (expand) the numbers of HSCs in components that contain limited or inadequate numbers of HSCs are being developed with the hope that expansion of HSCs will allow simpler collection techniques or the administration of dose-intense therapy to patients from whom adequate marrow or PBSC components cannot be collected. These techniques have been used in Phase I studies involving marrow, PBSCs, or umbilical cord blood.30 The results of these studies are encouraging, but clinical utility has not been conclusively demonstrated and requires further study.

Tumor Cell Purging Hematopoietic stem cell components are a complex mixture of cells, and many of these cell populations affect the outcome of allogeneic or autologous transplantation. The preponderance of evidence demonstrates that malignant cells contained in an HSC graft are capable of causing disease after transplantation. The transmission of malignant cells in an organ graft has been demonstrated in both HSC and solid organ (eg, orthotopic heart, liver, or kidney) transplantation from living or cadaveric donors suffering from occult disease at the time the graft was harvested.31,32 These reports conclusively demonstrate that even very small quantities of malignant cells can engraft when infused into a properly conditioned host. It is not surprising, therefore, that relapse of disease occurs after autologous HSC transplantation for diseases involving or likely to involve the blood or marrow. Tumor cell contamination or minimal residual disease (MRD) of the component contributing to relapse after autologous HSC transplantation was demonstrated in studies involving the transplantation of genetically marked marrow cells from patients with acute or chronic leukemia or neuroblastoma.33- 35 The level of marked cells detected after relapse was very low, and these studies cannot conclude that the cell inoculum was the sole source of malignant cells causing the disease relapse. Nor do these studies demonstrate that purging of tumor cells is necessary or effective. However, the implication of these studies, as with the solid organ transplant experience, is that the infusion of tumor-contaminated HSCs may be detrimental to the recipient and purging, if without undue toxicity and of proven efficacy, may be beneficial to the subset of patients whose HSC components are contaminated by tumor cells. The unanswered question, therefore, is not whether the cell inoculum can be a source of posttransplant relapse but, rather, whether manipulation of the HSC component can affect this outcome.

Chapter 34: HPCs: Autologous Transplantation

It is generally accepted that collections of hematopoietic progenitor cells from the peripheral blood likely contain fewer malignant cells than marrow collections. However, circulating malignant cells have been found for patients with neuroblastoma, breast cancer, and lymphoma.36 Despite the evidence cited above about the potential contribution of MRD to poor transplant outcome, no study to date in any malignant disease demonstrates a benefit from purging of HSC components intended for autologous transplantation. The hypothesis that purging is beneficial for patients with B-cell malignancies such as chronic lymphocytic leukemia (CLL) or NHL is supported by clinical reports. The University of Nebraska program reported that 10 of 11 patients who received marrow from which lymphoma cells could be cultured relapsed after transplantation compared with 2 of 13 patients who received “culture-negative” grafts.37 This group subsequently reported a fourfold greater PFS of 57% vs 17%, for recipients of grafts with no detectable MRD.38 Only the presence of a positive culture from the HSC inoculum predicting for a lower event-free surivival (EFS) was confirmed in a subsequent randomized study conducted by this group comparing PBSCs and marrow as sources of HSCs.39 The Dana-Farber transplant program reported similar differences in DFS based on the presence or absence of detectable MRD in trials involving antibody-mediated purging.40-42 These studies used sensitive polymerase chain reaction (PCR) techniques to monitor the presence of tumor cells before and after purging with monoclonal antibodies and complement. Patients who had documented persistence of tumor cells had a significantly lower probability of DFS than patients who received components that were PCR negative after purging. The difference in DFS remains highly significant even after 10 years of follow-up (Kaplan-Meier estimate of DFS probability of 80% vs 10%).43 These publications provide the strongest evidence to date that complete tumor cell purging (based on the most sensitive tumor cell detection assays available) is both necessary and beneficial. This group reported much less success in achieving PCR negativity in the purging of components from patients with mantle cell NHL and a concomitant high relapse rate after transplantation for that disease.44 Although the number of patients transplanted for CLL was too small for definitive conclusions, the DanaFarber group likewise suggested a higher probability of DFS for those patients who received PCR-negative HSC components.45 These MRD detection techniques are surrogate markers for transplant outcome. It is, therefore, not surprising that other transplant programs have published contradictory reports. For example, one group reported relapses for three of four patients treated for follicular NHL whose marrow components became PCR negative after purging with one antibody and complement but for only 11 of 25 patients who received PCR-positive marrow.46 A multivariate analysis by other investigators to determine the risk of relapse for 24 patients with poor-prognosis NHL who received unpurged PBSCs did not demonstrate any effect from tumor cell contamination on the risk of relapse after transplantation.47 Yet, these investigators did report that the

persistence or return of tumor cells detected by molecular analysis into the blood or marrow after transplantation was predictive of relapse in 81% of the patients, and that patients with detectable disease after transplantation experienced a 24-fold higher risk of relapse compared with patients who became and remained PCR negative. An alternative explanation for the Nebraska and Dana-Farber reports is that the detection of tumor in the graft with or without purging reflects the quantity of tumor cells in the component which, in turn, is proportional to the amount or the aggressiveness of the disease in the patient. Patients with higher body burdens of tumor are less likely to achieve a durable remission after HSC transplantation. Sharp et al,38 however, assigned patients with marrow involvement to PBSC transplantation, so the better PFS observed by them after PBSC transplantation does not support the contention that marrow contamination reflects only overall body burden of tumor. Although many patients relapse at sites of previous disease, this could reflect preferential trafficking into previous sites of disease of malignant lymphocytes reintroduced with the HSC inoculum.48 Recurrence at previous sites of disease is not conclusive evidence that residual disease in the patient is the only source of relapse after autologous HSC transplantation. The discrepancy between the experiences of these various centers could reflect the importance of adequate purging, differences in patient selection, or the efficacy of the conditioning regimen. This discrepancy also illustrates the need for properly designed studies to directly address the benefit and toxicity of purging. A variety of purging agents have been tested in Phase I and II studies. The numbers of patients and financial resouces estimated to be required for definitive Phase III tests of purging efficacy have discouraged the conduct of these studies.

Cryopreservation of HSC Components The primary difficulty in using HSC components to reduce the hematologic toxicity of dose-intense regimens is the necessity for cryopreservation. Three considerations apply. First, current cryopreservation techniques for PBSC components require cryopreservation of both the HSCs that account for about 1% of the nucleated cell population of the component and the mature blood cells that do not contribute to the recovery of marrow function after infusion. These mature cells both increase the volume of the component (thereby increasing the amount of cryoprotectant solution added) and appear to contribute to the postinfusion morbidity experienced by the recipient.49 Second, the recipient of cryopreserved HSC components may experience considerable toxicity from the DMSO used in the cryoprotectant solution. Third, the cryopreservation of HSCs is technically difficult. Long-term cryopreservation of cells using DMSO requires storage and shipping of cells at a temperature below 135ºC, generally using liquid nitrogen containers. The thawing of large numbers of bags of cells is usually performed by a skilled laboratory technologist at the patient’s bedside. A high incidence of generally mild, infusion-related morbidity with the reinfusion of cryopreserved cells has been reported by

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several centers.50-52 Dimethylsulfoxide has a variety of pharmacologic effects.53 The LD50 values (amount of DMSO required to kill 50% of test animals) reported for intravenous infusion of DMSO are 3.1 to 9.2 g/kg for mice and 2.5 g/kg for dogs.54 The acute toxic dose of DMSO for humans has not been determined. If a large amount of cryopreserved material is to be infused (eg, 1 g DMSO/kg of patient weight), the infusion can be separated over 2 days to avoid complications from infusion of excessive amounts of DMSO. Because the osmolality of thawed marrow (with 10% DMSO) is about 1800 mOsm/kg, central venous catheters are the preferred route of administration. The incidence of the more common reactions appears to be related to the volume of the product infused. The most dramatic toxicity is the rare anaphylactic reaction occurring during the initial administration of thawed cells. This appears to be an allergic reaction to DMSO or other components of the solution used for cryopreservation and, after resuscitation of the recipient, the remainder of the cells should be administered cautiously. Nonallergic, profound hypotension can result from the intravenous infusion of DMSO, presumably from histamine-induced vasodilation, especially for patients who were not adequately premedicated with appropriate antihistaminic medications such as diphenhydramine. Skin flushing, dyspnea, abdominal cramping, nausea, and diarrhea, reported to varying degrees after HSC infusion, can also be attributed to DMSOinduced histamine release. These complaints resolve over a few hours and should be treated symptomatically. Dimethylsulfoxide affects the cardiovascular system in a variety of ways. In a series of 82 patients premedicated with diphenhydramine, both increased blood pressure and decreased heart rate, which became maximal about 1 hour after the completion of the marrow infusion, were observed.50 Cardiac arrest or high-degree heart block occurring during or immediately after the infusion of cryopreserved marrow or PBSCs has been described.55,56 In these two series, the incidence of bradycardia (heart rate 60 beats/minute) was 48.8% and 65%, seconddegree heart block was 9.7% and 24%, and complete (thirddegree) heart block was 4.8% and 5.9%. In both reports, the median time of onset was about 3 hours after the completion of the infusion. In one series, the authors noted that the heart block was often episodic, occurring with episodes of emesis.55 In both series, the cardiac rhythm abnormalities resolved spontaneously within 24 hours after infusion. In contrast, another group found no cardiac rhythm changes in a prospective series of 29 patients.57 A slower overall infusion rate may have accounted for the lack of rhythm changes. Headache has been reported in up to 70% of recipients of cryopreserved cells,52 but other central nervous system complications are rare and generally related to the amount of DMSO infused. Two recipients who received HSC components containing a total of 225 mL and 120 mL, respectively, of DMSO developed reversible encephalopathy.58 The first patient underwent plasmapheresis with prompt improvement in mental status; the second patient recovered over 5 days without specific treatment.

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The weights of the patients were not cited in this report, but both patients probably received more than 2 g DMSO per kilogram of body weight. The infusion of large quantities of poorly cryopreserved mature blood cells can cause renal failure.59 Seizures have been associated with the infusion of highly concentrated PBSC products, suggesting that freezing at very high cell concentrations is not an acceptable strategy to avoid large volumes of DMSO.49

Posttransplant Support Hematopoietic growth factors are administered to speed neutrophil recovery after allogeneic or autologous transplantation. Randomized studies of granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF administration after autologous HSC transplantation demonstrated that these hematopoietic cytokines speed granulocyte recovery, decrease the morbidity caused by the transplant procedure, and are associated with a smaller probability of relapse.60-62 But this has not consistently translated into earlier hospital discharge or reduced transplant cost. Administration of cytokines that primarily stimulate the proliferation of mature hematopoietic progenitor cells may not greatly affect the time to initial hematopoietic recovery after transplantation, although the subsequent rate of rise in peripheral blood counts is notably enhanced, suggesting that adequate numbers of cells at the appropriate maturational stage must be present before growth-regulatory effect from these cytokines can be obtained.63 Late administration of cytokine support (starting several days after HSC infusion) may be as effective as early administration while being less costly to the patient.64

Complications of High-Dose Therapy The patient undergoing dose-intense therapy with autologous HSC transplantation experiences a period of marrow hypoplasia that can persist for days or (rarely, with PBSCs) weeks. During this time, the patient requires antibiotic and blood component support, but death from infection or hemorrhage is uncommon. Common nonhematopoietic toxicity that is not lifethreatening includes alopecia, sterility, and varying degrees of mucositis with concomitant mouth pain, inanition, and diarrhea. Rarely, mucositis compromises airway patency, and intubation of the patient is temporarily required for airway protection. Serious organ toxicity includes hepatic veno-occlusive disease (from highdose regimens in general), interstitial pneumonitis (eg, BCNUinduced), cardiomyopathy (eg, cyclophosphamide-induced), and hemorrhagic cystitis (eg, cyclophosphamide-induced). The risk of nonhematopoietic toxicity increases for older patients and for patients who previously received intensive chemotherapy regimens. For these patients, reduction of dose of the transplant conditioning regimen reduces toxicity but possibly increases the risk of relapse of disease. Patients may experience long-term marrow hypoplasia after HSC transplantation despite the infusion of large quantities of HSC, possibly as a result of poor cryopreservation techniques, marrow stromal cell damage, or posttransplant events, and this complication may preclude the subsequent administration of

Chapter 34: HPCs: Autologous Transplantation

chemotherapy or radiotherapy for patients who experience relapse of disease. The development of myelodysplasia or secondary leukemia is being increasingly reported for patients previously treated with autologous HSC transplantation, particularly for patients with the diagnosis of NHL treated with radiation-containing conditioning regimens.65 The incidence of this complication appears to be small in patients treated for leukemia,66 but has been reported above 10% for patients treated for lymphoid malignancies. Recipients of autologous or allogeneic HSC transplantation are at risk of long-term complications of their treatment including hypothyroidism, hypogonadism, organ toxicity (eg, druginduced pneumonitis), and secondary malignancies. Patients treated with HSC transplantation should be enrolled in a program of long-term supportive care.67

Adjuvant Therapy with Autologous HSC Transplantation The incidence of relapse after high-dose therapy leads to the conclusion that high-dose therapy with autologous HSC transplantation, although well tolerated and likely to prolong life, should be viewed as a platform for other approaches that may be effective in eliminating the MRD of patients destined to relapse after the dose-intense therapy is administered. Additional or yet higherdose chemotherapy or radiotherapy, unless directly targeted to the tumor target, increases the risks of nonhematopoietic toxicity and loss of patients from causes other than relapse. Immunologically based therapies are of interest in this regard and include administration of posttransplant cytokines,68 the addition of tumorspecific antibodies used before or after transplantation or both as an in-vivo-purge,69 and the development of tumor-specific vaccines such as with tumor-antigen-pulsed dendritic cells (DCs).70

Diseases Acute Myelogenous Leukemia Induction chemotherapy yields complete remission for most patients with acute myelogenous leukemia (AML), but these patients will rapidly relapse without postinduction consolidation therapy. Randomized studies of consolidation regimens of varying intensity demonstrate that intensity is correlated with probability of durable remissions.71 The role of autologous or allogeneic HSC transplantation as intensive consolidation therapy for patients entering first remission has been explored in several randomized studies that assigned patients with available sibling donors to allogeneic transplantation and randomly assigned other patients to autologous transplantation or nontransplant consolidation therapy.72-76 In general, autologous marrow transplantation has not proven to be more effective than nontransplant intensive consolidation chemotherapy (Table 34-2). In a study sponsored by the Eastern Cooperative Oncology Group (ECOG), patients assigned to allogeneic transplantation achieved a 43% DFS compared with 35% for patients assigned to chemotherapy and 35% for patients assigned to autologous transplantation

using marrow cells purged ex vivo with a cyclophosphamide derivative that caused a delay in hematologic recovery.72 Overall survival (OS) was slightly but significantly higher for chemotherapy-treated patients because of the ability to rescue patients who relapsed, with subsequent autologous transplantation administered in second remission. However, only 54% of patients assigned to autologous transplantation in the ECOG-sponsored study received this treatment, and analysis of outcomes was by intent of treatment, raising the question of whether a difference could have been found with better compliance. Furthermore, transplantrelated mortality was 14% for patients assigned to the autologous marrow transplant group, which is higher than would be expected for PBSC transplantation using nonpurged HSC products. A subsequent analysis of treatment outcome for patients stratified by cytogenetic risk group revealed a particularly poor outcome for patients with adverse risk cytogenetics who underwent either autologous HSC transplantation or nontransplant therapy.77 In contrast, in one study the DFS at 7 years was 54% for recipients of autologous transplantation compared to 40% for recipients of chemotherapy (p  0.04).73 Another study demonstrated a significantly better DFS for recipients of allogeneic transplantation (48% vs 30%, p  0.04) compared to recipients of chemotherapy. The probability of DFS for recipients of autologous marrow was intermediate, but not significantly different from either of the two other study arms.76 Therefore, the role for autologous transplantation in the treatment of patients in first remission is not obvious, and questions remain about the selection of HSC source, value of ex-vivo purging, and timing of transplantation relative to other options for postinduction consolidation. Several Phase II studies of autologous transplantation for the treatment of patients with AML in second or later remission have been published.78,79 Many of these studies reported survival probabilities of 35% or better, which is higher than what would be expected with standard chemotherapy regimens. A review of British registry data described 154 patients who underwent autologous marrow and/or PBSC transplantation in second remission.80 At 10 years, the actuarial probability of OS was 32%

Table 34-2. Multicenter Randomized Studies Comparing Autologous Marrow Transplantation to Intensive Consolidation Chemotherapy Study

Probability of Event-Free Survival (%)

Zittoun76 Cassileth

72

Allogeneic

Autologous

Chemotherapy‡

55*

48

30 (4 year)

43

35

35 (4 year)

ND†

53*

40 (7 year)

Harousseau

44

44

40 (4 year)

Ravindranath75

ND†

38

36 (3 year)

Burnett73 74

* Significantly different compared to chemotherapy treatment arm. † Allogeneic transplantation was not performed as part of this trial. ‡ Shown is the median duration of follow-up at time of analysis.

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Section II: Part III

with PFS. Overall survival was better for patients who experienced a longer duration of first remission and had a younger age at time of transplantation (which may reflect a difference in the biology of this disease for different age groups). Most patients for whom this information was available in this registry had favorable or intermediate risk cytogenetics; only 1% had adverse risk cytogenetics. Autologous HSC transplantation appears to be effective therapy for the patient with AML in second complete remission (CR) who do not have adverse-risk features. Purging of tumor cells from the HSC inoculum may be beneficial, but remains unproven. In nonrandomized studies, patients who received marrow cells treated with chemotherapy agents were less likely to relapse after transplantation.81 Similarly, indirect laboratory assessments of purging efficacy suggest a benefit for patients who are treated with an aggressive purging technique.82,83 The role of purging has not been proven in randomized studies, nor has it been shown to be of benefit for recipients of PBSC.

Acute Lymphoblastic Leukemia Few adults with acute lymphoblastic leukemia (ALL) are currently cured with induction and consolidation regimens.84 Results have improved modestly with more intensive postremission chemotherapy and with tailoring of protocols in individuals with specific subsets of ALL. There are few data, however, supporting the effectiveness of autologous HSC transplantation as currently performed in ALL despite the theoretic potential of dose-intense therapy. Two published reports comparing autologous to allogeneic marrow transplantation reported DFS after autologous transplantation similar to that reported for patients undergoing less intense induction and maintenance chemotherapy (approximately 20% to 30%).85,86 A third study comparing autologous to allogeneic transplantation reported significantly improved probability of long-term survival for patients undergoing allogeneic transplantation.87 Weisdorf et al88 described transplant outcomes after autologous or unrelated donor transplantation for 712 younger (median age 18 years) patients with ALL (517 unrelated donor, 195 autologous) in first or second CR with data in two registries. They reported much higher treatment-related mortality for recipients of unrelated donor transplants, but more frequent relapses after autologous transplantation in first (49% vs 14%) and second remission (64% vs 25%) leading to similar outcomes for these two transplantation choices. Transplantation in CR1 yielded similar 3-year survival rates for recipients of unrelated donor (51%) and autologous (44%) transplants, as did transplantation in CR2 (40% vs 32%, respectively). As with AML, adverse risk features appears to favor allogeneic transplantation in first remission. A large Phase III study involving 1500 patients with a new diagnosis of ALL comparing specific outcomes for autologous or related-donor allogeneic HSC transplantation or nontransplant consolidation/maintenance therapy has not yet been reported in final analysis, although the study confirmed previously reported risk groups for patients with this diagnosis.89 Phase II reports from investigators from the University of Minnesota and Dana-Farber transplant programs reported 56

528

recipients of 214 adults alive and without disease at a median of 25 months after autologous transplantation.90 The randomized addition of posttransplant interleukin-2 therapy after autologous transplantation did not improve the DFS. A similar matchedpair study involving children transplanted in second remission reported a 9-year probability of DFS of 26% for patients undergoing autologous transplantation compared to 32% for similar patients treated with chemotherapy alone.91 In contrast, others reported a 1-year DFS of 50% for adult patients transplanted in first remission and 27% for patients in second or later remission using marrow with or without ex-vivo purging.92 As with AML, autologous HSC transplantation appears effective for a subpopulation of patients with ALL, particularly those without adverse risk cytogenetic features and (for patients in second remission) who experienced longer durations of first remission.

Chronic Myelogenous Leukemia Limited numbers of patients with chronic myelogenous leukemia (CML) in chronic phase or with more advanced disease have been treated with autologous HSC transplantation.93 In a large, single-center study of 73 patients, the survival of patients with chronic-phase disease who underwent transplantation did not differ from that of patients treated with interferon. Of patients with more advanced disease, 58% achieved a complete hematologic remission but only 10% achieved a complete cytogenetic response, and the median survival for this group of patients was only 5 months. However, other studies have reported longer durations of survival and remission for patients who receive Philadelphia-negative grafts,94 leading to the hypothesis that products collected from patients in molecular remissions after treatment with imatinib may be useful in dose-intense therapy for patients with subsequent progression of disease.95 A metaanalysis of a number of incomplete clinical trials comparing autologous transplantation to nontransplant therapy (principally, interferon-alpha) found no evidence of a difference in survival or statistically significant differences between treatment groups in best hematologic or cytogenetic response achieved in the first year.96 Furthermore, this analysis was unable to determine whether autologous HSC transplantation with predominantly Philadelphia-negative cells early on in the disease resulted in a better outcome. Autologous HSC transplantation for this disease is, in general, limited to clinical research protocols.

Chronic Lymphocytic Leukemia Aggressive treatment is less likely to be used in CLL because of its long natural history, indolent nature, and the advanced age of most patients. Although the disease responds to chemotherapy and radiotherapy initially, relapse is inevitable. The extensive infiltration of marrow by malignant lymphocytes has served to focus transplantation trials on allogeneic transplantation or autologous transplantation with HSC products collected in remission or purged ex vivo. Few clinical studies have been reported. Investigators from the M.D. Anderson Cancer Center reported 6 of 11 patients treated with autologous marrow cells (seven purged

Chapter 34: HPCs: Autologous Transplantation

ex vivo) surviving in remission up to 29 months after transplantation.97 Investigators at the Dana-Farber Cancer Institute reported a high CR response rate for 12 patients who received autologous marrow that had been purged with multiple monoclonal antibodies,98 but a subsequent report of a total of 137 patients found no plateau in the survival probability, indicating that late relapses continue to occur. 99 Currently, autologous HSC transplantation in the treatment of CLL is limited to clinical research protocols.

on Phase III studies that demonstrated improved response rates and better survival for recipients of this treatment (Table 34-3). Dose-intense melphalan therapy was first noted to have a dramatic response in nine patients with refractory myeloma in 1983, including five patients who achieved a complete biochemical and marrow remission.101 Hematopoietic toxicity was initially dose-limiting for dose-intense melphalan, but this could be ameliorated by infusion of previously collected HSCs. The ability to administer high doses of melphalan with or without other drugs or TBI was confirmed in a series of Phase I and II studies. A matched-pair analysis by the University of Arkansas group and Southwest Oncology Group comparing dose-intense with standard-dose therapy demonstrated the benefit of autologous HSC transplantation for the treatment of multiple myeloma with a marked improvement in CR rate (22%) when compared

Multiple Myeloma Multiple myeloma, a malignancy of plasma cells, is now the most common indication for autologous stem cell transplantation in North America (Fig 34-1). Autologous stem cell transplantation with dose-intense melphalan is now considered a standard component of the initial therapy of newly diagnosed patients based

5,500 5,000

Allogeneic (total N  7,880)

4,500

Autologous (total N  10,840)

4,000 Transplants

3,500 3,000 2,500 2,000 1,500 1,000 500 0 Multiple NHL myeloma

AML Hodgkin disease

ALL

MDS/ MPD

CML Aplastic Other anemia leuk

Other cancer

Nonmalig disease

Figure 34-1. Indications for hematopoietic stem cell transplantation in North America in 2005.

Table 34-3. Risk Factors for Survival After Autologous Transplantation for Multiple Myeloma Event-Free Survival*

Overall Survival

Variable

RR

p

Variable

RR

p

No change in chromosome 13

0.5

0.0001

No change in chromosome 13

0.4

0.0001

B2M 2.5 mg/L

0.7

0.0001

B2M 2.5 mg/L

0.6

0.0001

12 months therapy

0.7

0.0001

12 months therapy

0.7

0.0001

Sensitive disease

0.8

0.0001

CRP 4.0 mg/L

0.7

0.0002

Any 2nd transplant

0.8

0.0004

Sensitive disease

0.8

0.0002

Non-IgA

0.7

0.002

Non-IgA

0.7

0.002

Any CR

0.8

0.002

Any 2nd transplant

0.04

0.0001

* Used with permission from Desikan et al.100 B2M  immunoglobulin A; CRP  C-reactive protein; CR  complete remission.

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Section II: Part III

Table 34-4. Randomized Trials Comparing Conventional Chemotherapy vs High-Dose Therapy Study

n

Age

Median F/U (months)

CR Rate (%)

Median EFS (months)

Median OS (months)

CC

HDT

p

CC

HDT

p

CC

HDT

p

22

0.001

18

28

0.01

44

57

0.03

200

65

84

5

MAG91

190

55-65

56

NE

NE

19

25

0.05

45

42

NS

PETHEMA106

164

65

42

11

30

0.002

34

42

NS

67

65

NS

Italian MMSG107

195

50-70

24

7

26

0.001

16

28

0.0036

43

58

0.0008

516

70

76

15

17

NS

21

25

0.05

53

58

NS

407

65

42

8

44

0.001

19.6

31.6

0.0001

42

54

0.001

IFM903 105

USIG

103

MRC7104

CC  conventional chemotherapy; CR  complete remission; EFS  event-free survival; F/U  follow-up; HDT  high-dose therapy; OS  overall survival; NE  not evaluated; NS  difference not statistically significant.

to currently available low-dose regimens (5%).102 Event-free (49 vs 27 months) and overall survivals (62 vs 48 months) were also improved for patients treated with dose-intense therapy as part of their initial treatments. Several randomized studies comparing dose-intense therapy with autologous stem cell support to standard therapy regimens have been conducted (Table 34-4).103 The French Myeloma Intergroup reported a large-scale randomized trial (IFM 90) that randomly assigned 200 patients to either conventional dose VMCP/VBAP polychemotherapy regimen or autologous marrow transplantation with melphalan and TBI after four cycles of induction treatment.3 The findings were strikingly in favor of the dose-intense approach with improvements in complete response rates (22% vs 5%) and median probabilities of EFS (27 months vs 18 months) and OS (60 months vs 37 months). The British Medical Research Council (MRC) Myeloma VII trial of 407 patients reported similar results for a comparison of six cycles of BCNU, doxorubicin, cyclophosphamide, and melphalan to at least three cycles of doxorubicin, vincristine, methylprednisolone, and cyclophosphamide followed by melphalan at 200 mg/m2 and autologous stem cell transplant.104 The French group Myelome Autogreffe (MAG) achieved results comparable to the IFM 90 trial; however, there was no significant difference in OS as a result of unexpected prolonged survivals in the chemotherapy arm.105 The Spanish PETHEMA group did not find a statistically significant difference in EFS and OS between chemotherapy and dose-intensive therapy.106 However, only patients who responded (CR: 15%; partial response: 68%; minor response: 17%) to conventional dose chemotherapy were randomly assigned to treatment groups and patients in the chemotherapy arm were allowed to cross over into the dose-intensive therapy group. The Italian Multiple Myeloma Study Group compared melphalan-prednisone to two courses of melphalan at 100 mg/ m2 with autologous stem cell support.107 They found significant improvement in CR and EFS in the transplant study arm. The MRC trial comparing high-dose therapy to conventional therapy deserves special consideration.104 It is the most recent of the randomized trials and was carried out in the era of more advanced

530

supportive care and the availability of newer therapeutic agents. It still showed a statistically significant advantage for high-dose therapy in terms of CR, EFS, and OS. Moreover, it did not show any increase in treatment-related mortality relative to conventional therapy. These randomized trials all showed a trend to significantly improved results for patients treated with dose-intense therapy. Adding the support of numerous Phase II trials, the data clearly indicate that dose-intense therapy with autologous HSC transplantation should be offered to appropriate patients as part of their initial treatment. The median EFS is remarkably constant (25-31 months). It is more difficult to analyze OS results because that depends partly on salvage therapy. However, median OS was significantly longer in the three studies where differences in EFS were more marked. In all studies, procedure-related death rate was 5% and not greater than that observed with conventional chemotherapy. With HSC support, mucositis becomes the dose-limiting effect of dose-intense melphalan. In an attempt to increase the total amount of melphalan administered, the Arkansas transplant program explored the feasibility of two consecutive autologous transplants given at intervals of only a few months, doubling the total dose of melphalan administered.108 The investigators described 42 patients who received two consecutive autologous PBSC transplants using melphalan 200 mg/m2. In patients who were capable of receiving two transplants, the CR rate increased from 24% after the first transplant to 43% after receipt of the second transplant. This experience has now been extended in over 1000 patients with overall CR rates of 40% and projected 5-year EFS and OS of 25% and 40%, respectively.100 The French Intergroup randomly assigned 399 newly diagnosed myeloma patients under age 60 to single vs tandem transplants.109 They found similar CR rates between the two groups (42% single transplant vs 50% tandem transplant), but superior 7-year EFS (10% vs 20%, p  0.03) and OS (21% vs 42%, p  0.01) for the tandem transplant group. For patients who did not achieve a very good partial response (less than 90% reduction in serum or urine paraprotein) after one transplant, the improvement in 7-year OS was more dramatic (11% vs 43%, p  0.01). These

Chapter 34: HPCs: Autologous Transplantation

results support tandem autologous transplants for patients with a diagnosis of multiple myeloma who do not show disease progression after the first transplant. The success of autologous transplantation led to several analyses of the risk factors predictive for the survival of patients after initial transplantation. A retrospective study of 344 patients with multiple myeloma treated with highdose chemotherapy and autologous stem cell transplantation showed a 5-year OS of 72% for patients who achieved a complete response, which is defined as absent monoclonal protein by both serum protein electrophoresis (SPEP) and immunofixation (IFE). This is compared to 48% for patients who achieved a near complete remission (monoclonal protein absent on SPEP but still detectable by IFE). The EFS was also superior in the CR group (35%) in comparison to the non-CR group of patients (21%).110 Investigators from the Arkansas transplant program reported the long-term outcomes for 515 consecutive patients intended to receive melphalan-based tandem transplants with follow-up of at least 5 years.111 One quarter of the patients had EFS of 5 years with no further relapses seen after 7 years (46 patients on the plateau). Factors associated with long-term EFS included initial transplantation within 1 year of diagnosis, absence of chromosome 11 or 13 abnormalities, and a lower β2-microglobulin at initial diagnosis. This group subsequently reported that of 135 patients enrolled in a study of tandem autologous HSC transplantation, patients with normal cytogenetics had median EPS and OS of 43 and 50 months or more, respectively.112 The EFS and OS for patients with abnormalities of chromosome 13 were 21 and 29 months; for patients with abnormalities involving 11q, 20 and 21 months; and for patients with abnormalities of both 13 and 11q, 11 and 12 months. The absence of chromosomal abnormalities was the single strongest prognostic factor associated with prolonged survival in this study. Other factors that are considered poor prognosticators are fluorescence in situ hybridization (FISH)defined chromosome 13 deletions113; hyperdiploidy114; the detection of abnormal metaphases100; elevations in C-reactive protein, lactate dehydrogenase, β2-microglobulin, and creatinine; and low levels of hemoglobin, albumin, and platelets.115 The role of maintenance therapy after transplantation remains open to question. A recently published study assigned 597 patients to three arms of (A) no maintenance therapy, (B) pamidronate therapy given monthly, or (C) pamidronate with thalidomide starting 2 months after a second autologous HSC transplantation.116 Those who received pamidronate and thalidomide (Arm C) had the best CR or very good partial response when compared to the Arm A and Arm B patients (67% vs 55% and 57%, respectively). The 3-year probability of relapse-free survival was also higher in Arm C vs Arm A and B (51% vs 38% and 39%, respectively); 87% compared to 77% and 74%, respectively. Of note, the thalidomide therapy showed no benefit in patients with chromosome 13 deletion or those who achieved least a very good partial response to the dose-intense therapy. Additional studies of this question are in progress.

Amyloidosis Primary amyloidosis is a plasma cell dyscrasia in which clonal plasma cells in the marrow produce a monoclonal immunoglobulin protein. These M-protein light chains or light-chain fragments form insoluble fibrils that are deposited into the extracellular matrix of a variety of tissues, resulting in severe multi-organ dysfunction and poor patient survival. Randomized studies have shown that melphalan-based therapy can prolong the life of patients with primary amyloidosis.117 Dose-intense therapy with melphalan and autologous PBSC transplantation can induce a complete hematologic remission, defined as the absence of detectable plasma-free light-chain protein.118 Responders who achieve a complete disappearance of light-chain secretion are more likely to achieve reversal of underlying organ dysfunction.119 Patients frequently present with extensive systemic disease, rendering this disease difficult to treat. Regimen-related mortality is close to 20% of patients in some studies.120 Multi-organ disease (particularly cardiac dysfunction) predicts a poor transplant outcome. In one study, the EFS was only 60% for patients with cardiac involvement but 100% for patients with renal involvement and no evidence of cardiac dysfunction.118 Single center studies, particularly with adjustment of melphalan dose for patients with higher risk of toxicity, report a much lower mortality incidence. This suggests that the use of dose-intense therapy should be limited to centers with greater experience in the treatment of this disease.121 A randomized study conducted in Europe did not find improved survival for patients treated with PBSC transplantation, but the transplant arm was characterized by a high mortality rate that could obscure the benefit of dose-intense therapy for selected patients.122 As with the treatment of multiple myeloma, one group found that tandem cycles of dose-intense melphalan with autologous PBSC support improved the response rate for patients with systemic amyloidosis.123 Thalidomide and lenolidamide are also effective therapies in the treatment of amyloidosis with a small proportion of patients achieving a complete hematologic response. These agents may be useful in the pre- or posttransplant management of patients with amyloidosis.124,125

Lymphoma The lymphomas represent a diverse group of hematologic malignancies arising from lymphoid tissues. They vary from the slowest to some of the fastest growing tumors. They also range from highly curable to essentially incurable. The lymphomas are highly responsive to chemotherapy and radiation in most cases. These tumors exhibit a strong dose-response relationship, and the benefit of high-dose treatment with autologous stem cell rescue is well established for some categories of this disease. Several high-dose chemotherapy regimens have been developed for the treatment of the lymphomas. Radiation-based regimens including cyclophosphamide with TBI and etoposide plus TBI have significant activity but frequently are difficult to administer in patients already exposed to radiotherapy. Popular

531

Section II: Part III

chemotherapy regimens include cyclophosphamide, BCNU, and etoposide (CBV) and BCNU, etoposide, cytarabine, and melphalan (BEAM). However, no single chemotherapeutic regimen has emerged as a superior treatment.

Hodgkin Disease Many patients with Hodgkin disease will achieve durable remissions with nontransplant chemotherapy with or without radiation therapy, and algorithms for staging and treatment of this disease are well defined. Dose-intense therapy with autologous transplantation should be considered for those patients who do not achieve a remission or who relapse after initial therapy and will succumb without aggressive therapy.4,126 The inclusion of dose-intense therapy as part of the initial therapy of patients with adverse-risk features is not supported in clinical studies.127 For patients who suffer a relapse after achieving CR, the prognosis with conventional salvage therapy is directly related to the duration of the initial remission. The outcome for patients whose remissions lasted less than 1 year is dismal with standarddose second-line treatments, and most experts concur that these patients are best treated with high-dose chemotherapy with stem cell rescue. Approximately 40% to 50% of patients with Hodgkin disease who suffer a relapse within 1 year will achieve durable remissions after autologous HSC transplantation.128-130 For patients whose first remission lasts more than 1 year, both conventional-dose chemotherapy and high-dose autologous stem cell therapy provide the possibility of overall survival, although HSC transplantation favors PFS.131 Two randomized trials comparing high-dose chemotherapy with conventional salvage treatment have been performed.132,133 These studies suggest similar overall long-term survival rates but possibly a better DFS rate for the transplant arms. The British national lymphoma trial compared high-dose BEAM-with-transplant with mini-BEAM salvage therapy. Accrual was terminated prematurely because patients refused randomization and requested the high-dose therapy. Patients who underwent autologous transplantation had statistically greater EFS and PFS rates. Overall survival rates, however, were the same in both groups in this very small trial. A second European Group for Blood and Marrow Transplantation (EBMT) trial comparing standard-dose with high-dose treatment revealed improved time to treatment failure with the autologous arm but, again, overall survival in this slightly larger trial was not yet improved. Numerous risk factors are now identified that predict lower rates if disease control after HSC transplantation is achieved in the treatment of Hodgkin disease. These risk factors include chemotherapy-resistant disease, more than minimal residual disease at the time of transplant, B symptoms at relapse, poor performance status, relapse within a prior radiation field, and high International Prognostic Factor Project score.134,135 Patients with refractory Hodgkin disease may achieve durable complete remissions with high-dose chemotherapy and stem cell rescue. Numerous series, including that from the Autologous Blood and Marrow Transplant Registry [ABMTR—now the Center for International Blood and Marrow Transplant Research

532

(CIBMTR)] demonstrate that high-dose treatment can overcome drug resistance in Hodgkin disease. In the ABMTR analysis, patients were considered to have primary refractory Hodgkin disease if they never achieved CR. Following transplantation, the probability of 3-year PFS was 38% with an OS rate of 50%.136 Results from an EBMT Registry analysis were similar to that in the American series. The European group reported an actuarial 5-year DFS rate of 32% and an OS rate of 36% for patients with refractory Hodgkin disease.126

Low-Grade Non-Hodgkin Lymphoma Low-grade NHLs, in general, exhibit variable and prolonged natural courses, with many patients not requiring treatment until symptoms or organ toxicity appear. Therefore, most of the experience with HSC transplantation has been in patients after initial relapse rather than at the time of initial diagnosis, avoiding the potential for morbidity and mortality of dose-intense therapy. Multiple studies have demonstrated a greater response after dose-intense chemotherapy with autologous HSC transplantation compared to combination chemotherapy rescue regimens in the treatment of advanced or relapsed follicular lymphoma. Patients enrolled in a European Phase III randomized trial received dose-intense therapy and marrow transplantation (with or without ex-vivo purging). They benefited from a longer PFS compared to patients who were randomly assigned to the more standard-dose chemotherapy arm (55-58% vs 26%) and a significantly longer OS at 4 years (71-77% vs 46%).137 The benefit of dose-intense therapy for newly diagnosed patients is less obvious. A German study enrolled 240 newly diagnosed patients with advanced follicular lymphoma in a Phase III randomized trial comparing myeloablative therapy followed by autologous HSC transplantation to combination chemotherapy with interferon.138 At a median follow-up of 4.2 years, the myeloablative therapy arm was found to have a much longer 5-year PFS (65% vs 33%). However, this treatment arm also had higher acute toxicity associated with it and a higher 5-year risk of developing other hematologic malignancies such as treatment-related myelodysplasia (3.8% vs 0%). In contrast, 401 newly diagnosed patients with advanced follicular lymphoma were also studied in the GELA randomized study that compared dose-intense chemotherapy and TBI with autologous HSC transplantation to combination chemotherapy with interferon and found no significant difference in EFS or OS between the two arms after a median follow-up of 7.5 years.139 Although response rates are high, a continuing pattern of relapse has been observed with autologous transplant. One group reported a 68% CR rate for a single-center series of 100 patients with relapsed disease but, at a median of 4 years, a probability of failure-free survival of 44% with no plateau to the curve depicting probability of relapse.140 The Dana-Farber transplant program reported a series of 86 patients with advanced follicular lymphoma who underwent autologous marrow transplantation as part of initial therapy. Their publication raised the question about the necessity for and efficacy of tumor cell purging.141

Chapter 34: HPCs: Autologous Transplantation

Patients who received marrow products that were free of disease (based on a sensitive PCR detection technique for the t14;18 abnormality commonly found in follicular non-Hodgkin lymphoma) experienced a much lower rate of relapse than similar patients whose products contained detectable tumor after purging. However, no plateau to the curve depicting probability of relapse was reported. Patients who undergo allogeneic transplantation will experience a higher probability of transplant-related mortality but a lower risk of relapse after transplantation.142 The difference in relapse rates could result either from reintroduction of lymphoma cells in the HSC product or, more likely, from the lack of the powerful and beneficial graft-vs-disease effect of allogeneic HSC transplantation. Small numbers of patients have undergone autologous HSC transplantation for aggressive non-Hodgkin lymphoma after transformation from indolent non-Hodgkin lymphoma,143,144 with several reports describing improved survival for patients treated with dose-intense therapy compared to standard combination chemotherapy regimens. In patients under the age of 60 with chemotherapy-sensitive disease, the 4- to 5-year DFS and OS were reasonable (30%-52% and 37%-63%, respectively),144 suggesting that this therapy should be considered for this group of patients.

Aggressive Non-Hodgkin Lymphoma The standard of care for patients with B-cell non-Hodgkin lymphoma in first “chemotherapy-sensitive” relapse is HSC transplantation.5,145-147 The success of this therapy reflects the extent and the responsiveness of the disease to chemotherapy at the time of transplantation.148,149 Relapse is the major cause of failure of autologous transplantation. The role of high-dose chemotherapy with stem cell rescue in patients with chemotherapy-sensitive aggressive (intermediate or high-grade) lymphomas in relapse has become well established. The Parma trial randomly assigned 109 patients in first chemotherapy-sensitive relapse after two cycles of salvage chemotherapy to either high-dose therapy or four additional cycles of conventional-dose treatment.5 The EFS at 5 years was superior for the group undergoing high-dose therapy compared with the group receiving standard-dose treatment (46% vs 12%, p  0.001). The same superiority for transplant was true of 5-year OS (53% vs 32%, p  0.038). Furthermore, it is notable that in the Parma trial no patients assigned to the conventional-dose salvage therapy could be rescued at the time of second relapse with a delayed transplant. Therefore, excessive pretransplant “debulking” therapy with or without delays in transplant timing should be avoided. This randomized trial confirmed previous Phase II studies and conclusively demonstrated that transplantation therapy represents the treatment of choice for most patients with chemotherapysensitive first-relapsed aggressive non-Hodgkin lymphoma. Hematopoietic stem cell transplant may also be indicated for patients who respond slowly to initial therapy, have high-risk disease, are resistant to initial therapy, and are in relapse with chemotherapy-insensitive disease. The appropriate approach

for patients who achieve less than a complete response or who exhibit a slow response to conventional induction therapy is not yet clear. In one small trial of 69 slow responders to CHOP chemotherapy (cyclophosphamide, doxorubicin, and vincristine along with prednisone), the use of early transplant compared to five additional cycles of CHOP was not associated with an overall improvement in survival or EFS (p  0.10).150 The International Prognostic Index (IPI) identifies patients with aggressive non-Hodgkin lymphoma who have a high likelihood of relapse and poor overall survival with conventional first-line therapy.151 The French LNH-87 trial examined the role of consolidation transplantation for patients in CR1 after standard-dose treatment.146,152 The initial analysis of this trial demonstrated no advantage gained by the addition of intensive consolidation. However, subsequent analysis that included only those patients with intermediate or high risk by the more restrictive criteria of the IPI, revealed a superior DFS for those patients undergoing high-dose transplantation. Similarly, the Italian Non-Hodgkin Lymphoma Study Group showed no benefit from high-dose therapy with stem cell rescue in patients judged to be at high risk by virtue of tumor bulk or advanced-stage disease.153 However, a striking advantage in DFS was noted when the 70 patients who qualified as high-intermediate or high risk based on the IPI were analyzed. In this subgroup, transplantation yielded a superior 6-year DFS rate (87% vs 48%, p  0.008). Autologous transplantation therapy is likely to be beneficial in patients with high-risk disease as measured by IPI. Analysis of ABMTR data showed that among 184 patients who had never achieved a complete remission with conventional therapy (primarily induction failures), 44% could achieve one after autologous marrow or PBSC transplantation.154 The probability of PFS 5 years after transplantation was 31%. Chemotherapy resistance, administration of multiple cycles of chemotherapy before transplantation, poor performance status at time of transplantation, older age, and the lack of use of consolidative radiotherapy before or after transplantation predicted poor outcome. Patients with disease that is unresponsive to chemotherapy at the time of relapse have a low (about 15% to 20%) chance of achieving a long-term remission with high-dose therapy and stem cell rescue. Mantle cell lymphoma is known for its unremitting clinical course when treated conventionally and has proven relatively resistant to high-dose treatment with autologous HSC support, especially when used as a salvage therapy.155 Incorporating high-dose therapy into the initial treatment plan, however, may be more likely to provide durable remissions. At M.D. Anderson Cancer Center the results of using an intensive induction regimen followed by autologous transplantation have been encouraging.156 Among previously untreated patients, the EFS rate at 3 years was 72%. Mantle cell NHL is very responsive to the graft-vs-disease effect of allogeneic transplantation, and this treatment approach should be given priority over autologous HSC transplantation.

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Burkitt lymphoma, Burkitt-like lymphomas, and lymphoblastic lymphomas are eminently curable in most children. However, these high-grade non-Hodgkin lymphomas are associated with relatively poor long-term survival rates in adults. Data from the EBMT Registry suggests that disease status at the time of transplant is the most important predictor of outcome in patients with high-grade disease.149 For Burkitt and Burkitt-like lymphoma, the 3-year actuarial OS rate was 72% for patients who received a transplant during CR1, compared with 37% in those with chemotherapy-sensitive relapse, and 7% in patients with disease that is unresponsive to chemotherapy. For patients with lymphoblastic lymphoma, the 6-year actuarial survival rate ranged from 63% in patients who were in first CR1 to 15% in those who had resistant disease. Patients in CR2 had an intermediate survival rate of 31% at 6 years. These results with transplantation are superior to conventional-dose salvage therapy.

Waldenström Macroglobulinemia Waldenström macroglobulinemia is a rare lymphoproliferative disorder characterized by the production of IgM paraprotein. Waldenström macroglobulinemia, despite the paraprotein production, is distinct in its natural history and response compared to multiple myeloma, with alkylating agents, nucleoside analogues, and rituximab all being effective in the initial treatment of this disease. Only limited numbers of patients who have undergone autologous (or allogeneic) transplantation are described in single center or registry reports but, despite this limitation, a recent workshop on the treatment of patients with this disease recommended collection of autologous HSC before extensive therapy that would limit the ability to collect adequate products.157 In a Phase I/II study of autologous transplantation, seven patients (untreated or after first-line therapy) with symptomatic disease underwent two or three cycles of cytoreductive chemotherapy with collection and purging of PBSC followed by autologous transplantation after conditioning with TBI and high-dose cyclophosphamide.158 Engraftment was prompt, without any procedure-related deaths. A strong reduction or normalization of the marrow infiltration and serum IgM levels occurred in all evaluable patients, but immunofixation electrophoresis revealed persistent paraproteinemia in five. A registry report of 10 patients who underwent autologous transplantation reported a PFS at 3 years of 65% with an OS of 70%.159

Solid Tumors Autologous transplantation for the treatment of solid tumors is less established, primarily because most malignancies do not exhibit the strong dose-response to myelosuppressive chemotherapeutic agents. Exceptions are germ cell tumors and some pediatric malignancies such as neuroblastoma.

Breast Cancer Breast cancer was at one point the most frequent indication for autologous HSC transplantation.160 However, this treatment approach is no longer considered appropriate. The experience of

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Bonadonna and others showing a dose response for this malignancy led investigators in the early 1990s to explore the use of dose-intense chemotherapy regimens for women with micrometastases involving 10 or more axillary lymph nodes and for women with inflammatory breast cancer. Peters et al161 treated 102 women with high-risk (10 axillary lymph nodes positive for disease) Stage IIA, IIB, IIIA, or IIIB cancer with four cycles of standard-dose CAF (cyclophosphamide, doxorubicin, fluoroucil) chemotherapy followed by high-dose STAMP-1 (cyclophosphamide, BCNU, and cisplatin) chemotherapy with autologous stem cell rescue. The actuarial EFS for the study population at 2.5 years was 72%. Comparison to three historic or concurrent Cancer and Leukemia Group B adjuvant chemotherapy trials selected for similar patients showed an EFS at 2.5 years to be between 38% and 52%. However, several randomized studies did not demonstrate a survival advantage for patients treated with dose-intense therapy compared to those treated with more standard-dose treatments.162-165 Dose-intense therapy with HSC transplantation also does not appear to provide a survival advantage for women with metastatic disease.166

Germ Cell Tumors Germ cell cancers are relatively uncommon diseases accounting for about 1% of all malignancies in primarily adolescent and young men. Germ cell tumors, highly curable with nontransplant therapies, are classified as seminomatous or nonseminomatous tumors. Despite a propensity for metastatic spread, germ cell cancers are one of the most highly curable human malignancies. Over 60% of patients with high-risk disseminated disease will achieve durable responses after treatment with four cycles of cisplatin, etoposide, and bleomycin. Patients with refractory disease or who relapse after initial treatment can be effectively rescued with one or two cycles of dose-intense therapy. Forty cisplatin-refractory patients were entered into a large multi-center Phase II (ECOG-sponsored) trial of carboplatinum and etoposide, of whom 22 (58%) were able to proceed to a second cycle.167 Five patients (13%) died of treatment-related causes including infection, hemorrhage, and hepatic veno-occlusive disease. All treatment-related deaths occurred during the first course of therapy. Nine patients (24%) achieved a complete response (including two patients after surgical resection of residual disease). Another eight patients achieved a partial response for an overall response rate of 45%. Three of the complete responses occurred after the first marrow transplant, and for four patients the partial response converted to complete response after the second marrow transplant. Five of the nine patients were alive and free of disease at a minimum follow-up of 18 months. A striking finding of this study was the poor outcome in patients with nonseminomatous primary mediastinal germ cell tumors. Eleven patients with this diagnosis were enrolled in this study and none obtained a durable complete remission. The initial ECOG experience with multiple cycles of highdose therapy has been replicated at single centers. The Memorial Sloan Kettering group treated 58 patients with refractory germ

Chapter 34: HPCs: Autologous Transplantation

cell tumors with two cycles of high-dose carboplatin, etoposide, and cisplatin. Forty percent achieved a complete response, with a 2-year survival rate of 31%. Patients with pretreatment beta human chorionic gonadotropin (β-hCG) values below 100 and those without retroperitoneal masses fared better.168 In contrast, this same group reported that the inclusion of dose-intense therapy did not improve survival for newly diagnosed patients with poor-risk features, although there was a trend for better survival for patients not showing a rapid response to initial treatment who then received dose-intensification with HSC support.169 The Indiana University group reported 184 patients with seminomatous and nonseminomatous germ cell tumors who had progressed following cisplatin-based regimens.170 Patients received one or two cycles of conventional-dose salvage, followed by one (n11) or two (n173) cycles of high-dose carboplatin and etoposide with stem cell support. At a median of 48 months of follow-up, 116 patients remain progression free, including patients with cancer refractory to standard doses of cisplatinum. This very aggressive schedule resulted in one of the highest response rates noted to date.

Ovarian Carcinoma Autologous transplantation for the treatment of epithelial (nongerm-cell) ovarian carcinoma remains under active investigation, and promising results have been reported for some groups of women. A review of registry data for 421 patients undergoing transplantation identified a better outcome for patients with platinum-sensitive, low-bulk disease with the authors recommending prospective clinical studies.171 However, one reported Phase III study that enrolled 149 previously untreated women with epithelial ovarian cancer found no differences in EFS or OS, indicating that further studies are needed.172

Other Diseases The improved safety of autologous PBSC transplantation encourages studies of dose-intense therapy in the treatment of nonmalignant diseases, including autoimmune disorders such as multiple sclerosis, scleroderma, systemic lupus erythematosus (SLE), Crohn disease, and refractory rheumatoid arthritis.173 The hypothesis of currently ongoing studies is that dose-intense lymphoablative conditioning regimens will achieve prolonged remission or cure of these diseases by destruction of the clone of cells responsible for the disease. This hypothesis is supported by reports of remission of autoimmune diseases (eg, rheumatoid arthritis, SLE) that existed as a comorbid condition for patients undergoing autologous or allogeneic transplantation,174,175 although this has not been a universal finding.176 The use of intensive chemoradiotherapy with HSC rescue offers an opportunity to deliver maximally tolerated immunosuppression. Factors that may influence the relative efficacy of this approach include the number and relative resistance to ablation of the responsible lymphoid effector populations and the nature and persistence of the relevant antigen against which the immune response is raised. Although lymphoid effectors are not well defined for

these diseases, evidence points to the involvement of T and B lymphocytes. High-dose therapy followed by autologous HSC transplantation may allow the immune system to “reset” with control of the autoreactive lymphocytes. As with the treatment of malignant diseases, depletion of the unwanted lymphoid cell population from the HSC inoculum (purging) might be necessary for optimal clinical outcome. These nonmalignant diseases are debilitating, and appropriate patient selection is necessary so that the risks (morbidity and mortality) are appropriately balanced against the benefits (possibly, lack of progression instead of reversal of damage already incurred) of this therapy. Multiple centers have now published their experiences in the treatment of small numbers of patients indicating that this treatment may be effective in controlling autoimmune diseases in certain patient populations. Burt et al177 recently reported 50 patients who received a nonmyeloablative conditioning regimen of high-dose cyclophosphamide and equine antithymocyte globulin with autologous PBSC support in the treatment of SLE. The treatment-related mortality was 4% (2/50; one death occurring after mobilization of PBSC and one after transplantation), but the probability of 5-year OS was 84%, and the probability of DFS was 50%. These patients also demonstrated stabilization of renal and pulmonary functions and significant improvement or stabilization in markers of disease activity. Similarly, 73 patients were identified from registry data as having received autologous HSC transplantation for the treatment of rheumatoid arthritis, with 49 patients (67%) achieving at least a 50% response at some point after transplantation. There was a significant reduction in the level of disability. Most patients restarted other therapies within 6 months for persistent or recurrent disease activity. This provided disease control in about half the cases, suggesting that the transplant may have ameliorated the disease.178 These publications set the stage for larger Phase II and III clinical studies to determine proper patient selection and choice of treatment strategies.

Summary Autologous HSC transplantation with dose-intense therapy is now a standard treatment for many diseases. Improved safety of this therapy has increased acceptance by patients and allowed its extension to older patients and those with comorbid diseases. The major failing is the difficulty in curing patients with advanced or refractory diseases. Older patients face a higher risk of relapse that appears to be a result of differing sensitivity of their diseases to dose-intense therapy. This suggests the need for a different approach based on age. Posttransplant immunologic approaches now being studied may, however, be able to convert the minimal disease remaining after transplantation into a cure. Continued effort will determine if modifications of pretransplant conditioning regimens, choice of HSC source, ex-vivo processing, or supplementation of the cell inoculum with other myeloid or lymphoid cells affects engraftment kinetics or immunologic

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reconstitution, and thereby enhances the therapeutic value of autologous transplantation for patients with malignant and nonmalignant diseases.

Disclaimer H. Rashidi has disclosed no conflicts of interest. S. Rowley has disclosed a financial relationship with Johnson and Johnson.

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Society of Blood and Marrow Transplantation. Biol Blood Marrow Transplant 2006;12:138-51. Nagler A, Ackerstein A, Or R, et al. Immunotherapy with recombinant human interleukin-2 and recombinant interferon-α in lymphoma patients postautologous marrow stem cell transplantation. Blood 1997;89:3951-3. Magni M, DeNicola M, Devizzi L, et al. Successful in vivo purging of CD34-containing peripheral blood harvests in mantle cell and indolent lymphoma: Evidence for a role of both chemotherapy and rituximab infusion. Blood 2000;96:864-9. Timmerman JM, Levy R. Dendritic cell vaccines for cancer immunotherapy. Annu Rev Med 1999;50:507-29. Cassileth PA, Lynch E, Hines JD, et al. Varying intensity of postremission therapy in acute myeloid leukemia. Blood 1992;79:1924-30. Cassileth PA, Harrington DP, Appelbaum FR, et al. Chemotherapy compared with autologous or allogeneic bone marrow transplantation in the management of acute myeloid leukemia in first remission. N Engl J Med 1998;339:1649-56. Burnett AK, Goldstone AH, Stevens RM, et al. Randomised comparison of addition of autologous bone-marrow transplantation to intensive chemotherapy for acute myeloid leukaemia in first remission: Results of MRC AML 10 trial. Lancet 1998;351:700-8. Harousseau JL, Cahn JY, Pignon B, et al. Comparison of autologous bone marrow transplantation and intensive chemotherapy as postremission therapy in adult acute myeloid leukemia. Blood 1997;90:2978-86. Ravindranath Y, Yeager AM, Chang MN, et al. Autologous bone marrow transplantation versus intensive consolidation chemotherapy for acute myeloid leukemia in childhood. N Engl J Med 1996;334: 1428-34. Zittoun RA, Mandelli F, Willemze R, et al. Autologous or allogeneic bone marrow transplantation compared with intensive chemotherapy in acute myelogenous leukemia. N Engl J Med 1995;332:217-23. Slovak ML, Kopecky KJ, Cassileth PA, et al. Karyotypic analysis predicts outcome of preremission and postremission therapy in adult acute myeloid leukemia: A Southwest Oncology Group/Eastern Cooperative Oncology Group Study. Blood 2000;96:4075-83. Yeager AM, Kaizer H, Santos GW, et al. Autologous bone marrow transplantation in patients with acute nonlymphocytic leukemia, using ex vivo marrow treatment with 4-hydroperoxycyclophosphamide. N Engl J Med 1986;315:141-7. Lenarsky C, Weinberg K, Petersen J, et al. Autologous bone marrow transplantation with 4-hydroperoxycyclophosphamide purged marrow for children with acute non-lymphoblastic leukemia in second remission. Bone Marrow Transplant 1990;6:425-9. Chantry AD, Snowden JA, Craddock C, et al. Long-term outcomes of myeloablation and autologous transplantation of relapsed acute myeloid leukemia in second remission: A British Society of Blood and Marrow Transplantation registry study. Biol Blood Marrow Transplant 2006;12:1310-7. Gorin NC, Aegerter P, Auvert B, et al. Autologous bone marrow transplantation for acute myelocytic leukemia in first remission: A European survey of the role of marrow purging. Blood 1990;75:1606-14. Rowley SD, Jones RJ, Piantadosi S, et al. Efficacy of ex vivo purging for autologous bone marrow transplantation in treatment of acute nonlymphoblastic leukemia. Blood 1989;74:501-6. Miller CB, Zehnbauer BA, Piantadosi S. Correlation of occult clonogenic leukemia drug sensitivity with relapse after autologous bone marrow transplantation. Blood 1991;78:1125-31.

84. Copelan EA, McGuire EA. The biology and treatment of acute lymphoblastic leukemia in adults. Blood 1995;85:1151-68. 85. Weisdorf DJ, Billett AL, Hannan P, et al. Autologous versus unrelated donor allogeneic marrow transplantation for acute lymphoblastic leukemia. Blood 1997;90:2962-8. 86. Attal M, Blaise D, Marit G, et al. Consolidation treatment of adult acute lymphoblastic leukemia: A prospective, randomized trial comparing allogeneic versus autologous bone marrow transplantation and testing the impact of recombinant interleukin-2 after autologous bone marrow transplantation. BGMT Group. Blood 1995;86:1619-28. 87. Hunault M, Harousseau JL, Delain M, et al. Better outcome of adult acute lymphoblastic leukemia after early genoidentical allogeneic bone marrow transplantation (BMT) than after late highdose therapy and autologous BMT: A GOELAMS trial. Blood 2004;104:3028-37. 88. Weisdorf D, Bishop M, Dharan B, et al. Autologous versus allogeneic unrelated donor transplantation for acute lymphoblastic leukemia: Comparative toxicity and outcomes. Biol Blood Marrow Transplant 2002;8:213-20. 89. Rowe JM, Buck G, Burnett AK, et al. Induction therapy for adults with acute lymphoblastic leukemia: Results of more than 1500 patients from the international ALL trial: MRC UKALL XII/ECOG E2993. Blood 2005;106:3760-7. 90. Weisdorf DJ, Billett AL, Hannan P, et al. Autologous versus unrelated donor allogeneic marrow transplantation for acute lymphoblastic leukemia. Blood 1997;90:2962-8. 91. Borgmann A, Schmid H, Hartmann R, et al. Autologous bonemarrow transplants compared with chemotherapy for children with acute lymphoblastic leukaemia in a second remission: A matchedpair analysis. The Berlin-Frankfurt-Munster Study Group. Lancet 1995;346:873-6. 92. Doney K, Buckner CD, Fisher L, et al. Autologous bone marrow transplantation for acute lymphoblastic leukemia. Bone Marrow Transplant 1993;12:315-21. 93. Khouri IF, Kantarjian HM Talpaz M, et al. Results with high-dose chemotherapy and unpurged autologous stem cell transplantation in 73 patients with chronic myelogenous leukemia: The MD Anderson experience. Bone Marrow Transplant 1996;17:775-9. 94. Koziner B, Dengra C, Lucero G, et al. Autologous stem cell transplantation for patients with chronic myeloid leukemia. The Argentine Group of Bone Marrow Transplantation (GATMO) experience. Cancer 2002;95:2339-45. 95. Drummond MW, Marin D, Clark RE, et al. Mobilization of Ph chromosome-negative peripheral blood stem cells in chronic myeloid leukaemia patients with imatinib mesylate-induced complete cytogenetic remission. Br J Haematol 2003;123:479-83. 96. CML Autograft Trials Collaboration. Autologous stem cell transplantation in chronic myeloid leukaemia: A meta-analysis of six randomized trials. Cancer Treat Rev 2007;33:39-47. 97. Khouri IF, Keating MJ, Vriesendorp HM, et al. Autologous and allogeneic bone marrow transplantation for chronic lymphocytic leukemia: Preliminary results. J Clin Oncol 1994;12:748-58. 98. Rabinowe SN, Soiffer RJ, Gribben JG, et al. Autologous and allogeneic bone marrow transplantation for poor prognosis patients with B-cell chronic lymphocytic leukemia. Blood 1993;82:1366-76. 99. Gribben JG, Zahrieh D, Stephans K, et al. Autologous and allogeneic stem cell transplantations for poor-risk chronic lymphocytic leukemia. Blood 2005;106:4389-96.

Chapter 34: HPCs: Autologous Transplantation

100. Desikan R, Barlogie B, Sawyer J, et al. Results of high dose therapy for 1000 patients with multiple myeloma: Durable complete remission and superior survival in the absence of chromosome 13 abnormalities. Blood 2000;95:4008-10. 101. McElwain TJ, Powles RL. High-dose intravenous melphalan for plasma-cell leukaemia and myeloma. Lancet 1983;2:822-4. 102. Barlogie B, Jagannath S, Vesole DH, et al. Superiority of tandem autologous transplantation over standard therapy for previously untreated multiple myeloma. Blood 1997;89:789-93. 103. Barlogie G, Kyle R, Anderson K, et al. Standard chemotherapy compared with high-dose chemoradiotherapy for multiple myeloma: Final results of phase III US Intergroup Trial S9321. J Clin Oncol 2006;24:929-36. 104. Child JA, Morgan GJ, Davies FE, et al. High-dose chemotherapy with hematopoietic stem-cell rescue for multiple myeloma. N Engl J Med 2003;348:1875-83. 105. Fermand JP, Ravaud P, Katsahian S, et al. High-dose therapy and autologous blood stem-cell transplantation compared with conventional treatment in myeloma patients aged 55 to 65 years: Long-term results of a randomized control trial from the Group Myelome-Autogreffe. J Clin Oncol 2005;23:9227-33. 106. Blade J, Sureda A, Rivera JM, et al. High-dose therapy intensification compared with continued standard chemotherapy in multiple myeloma patients responding to the initial chemotherapy: Longterm results from a prospective randomized trial from the Spanish cooperative group PETHEMA. Blood 2005;106:3755-9. 107. Palumbo A, Bringhen S, Petrucci MT, et al. Intermediatedose melphalan improves survival of myeloma patients aged 50 to 70: Results of a randomized controlled trial. Blood 2004;104: 3052-7. 108. Vesole D, Barlogie B, Jagannath S, et al. High dose chemotherapy for refractory multiple myeloma: Improved prognosis with better supportive care and double transplants. Blood 1994:84:950-6. 109. Attal M, Harousseau JL, Facon T, et al. Single versus double autologous stem-cell transplantation for multiple myeloma. N Engl J Med 2003;349:2495-502. 110. Lahuerta JJ, Martinez-Lopez J, Serna JD, et al. Remission status defined by immunofixation vs. electrophoresis after autologous transplantation has a major impact on the outcome of multiple myeloma patients. Br J Haematol 2000;109:438-46. 111. Tricot G, Spencer T, Sawyer J, et al. Predicting long-term (5 years) event-free survival in multiple myeloma patients following planned tandem autotransplants. Br J Haematol 2002;116:211-17. 112. Tricot G, Barlogie B, Jagannath S, et al. Poor prognosis in multiple myeloma is associated only with partial or complete deletions of chromosome 13 or abnormalities involving 11q and not with other karyotype abnormalities. Blood 1995;86:4250-6. 113. Facon T, Avet-Loiseau H, Guillerm G. Intergroupe Francophone du Myelome. Chromosome 13 abnormalities identified by FISH analysis and serum beta2-microglobulin produce a powerful myeloma staging system for patients receiving high-dose therapy. Blood 2001;97:1566-71. 114. Smadja NV, Bastard C, Brigaudeau C, et al. Groupe Francais de Cytogenetique Hematologique. Hypodiploidy is a major prognostic factor in multiple myeloma. Blood 2001;98:2229-38. 115. Hahn R, Wingard J, Anderson K, et al. The role of cytotoxic therapy with hematopoietic stem cell transplantation in the therapy of multiple myeloma: an evidence-based review. Biol Blood Marrow Transplant 2003;9:4-37.

116. Attal M, Harousseau JL, Leyvraz S, et al. Maintenance therapy with thalidomide improves survival in patients with multiple myeloma. Blood 2006;108:3289-94. 117. Kyle RA, Gertz MA, Greipp PR, et al. A trial of three regimens for primary amyloidosis: Colchicine alone, melphalan and prednisone, and melphalan, prednisone, and colchicine. N Engl J Med 1997;336:1202-7. 118. Comenzo RL, Vosburgh E, Simms RW, et al. Dose-intensive melphalan with blood stem-cell support for the treatment of AL (amyloid light-chain) amyloidosis: Survival and responses in 25 patients. Blood 1998;91:3662-70. 119. Dember LM, Sanchorawala V, Seldin DC. Effect of dose-intensive intravenous melphalan and autologous blood stem-cell transplantation on AL amyloidosis-associated renal disease. Ann Intern Med 2001;134:746-53. 120. Vesole DH, Pérez WS, Akasheh M, et al. High-dose therapy and autologous hematopoietic stem cell transplantation for patients with primary systemic amyloidosis: A Center for International Blood and Marrow Transplant Research Study. Mayo Clin Proc 2006;81:880-8. 121. Cohen AD, Zhou P, Chou J, et al. Risk-adapted autologous stem cell transplantation with adjuvant dexamethasone / thalidomide for systemic light-chain amyloidosis: Results of a phase II trial. Br J Haematol 2007;139:224-33. 122. Jaccard A, Moreau P, Leblond V, et al. High-dose melphalan versus melphalan plus dexamethasone for AL amyloidosis. N Engl J Med 2007;357:1083-93. 123. Sanchorawala V, Wright DG, Quillen K, et al. Tandem cycles of high-dose melphalan and autologous stem cell transplantation increases the response rate in AL amyloidosis. Bone Marrow Transplant 2007;40:557-62. 124. Palladini G, Perfetti V, Perlini S, et al. The combination of thalidomide and intermediate-dose dexamethasone is an effective but toxic treatment for patients with primary amyloidosis (AL). Blood 2005;105:2949-51. 125. Sanchorawala V, Wright DG, Rosenzweig M, et al. Lenalidomide and dexamethasone in the treatment of AL amyloidosis: Results of a phase 2 trial. Blood 2007;109:492-6. 126. Sweetenham JW, Carella AM, Taghipour G, et al. High-dose therapy and autologous stem-cell transplantation for adult patients with Hodgkin’s disease who do not enter remission after induction chemotherapy: Results in 175 patients reported to the European Group for Blood and Marrow Transplantation. Lymphoma Working Party. J Clin Oncol 1999;17:3101-9. 127. Federico M, Bellei M, Brice P, et al. High-dose therapy and autologous stem-cell transplantation versus conventional therapy for patients with advanced Hodgkin’s lymphoma responding to frontline therapy. J Clin Oncol 2003;21:2320-5. 128. Chopra R, McMillan AK, Linch DC, et al. The place of high-dose BEAM therapy and autologous bone marrow transplantation in poor-risk Hodgkin’s disease. A single-center eight-year study of 155 patients. Blood 1993;81:1137-45. 129. Bierman PJ, Anderson JR, Freeman MB, et al. High-dose chemotherapy followed by autologous hematopoietic rescue for Hodgkin’s disease patients following first relapse after chemotherapy. Ann Oncol 1996;7:151-6. 130. Yuen AR, Rosenberg SA, Hoppe RT, et al. Comparison between conventional salvage therapy and high-dose therapy with autografting for recurrent or refractory Hodgkin’s disease. Blood 1997;89:814-22.

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131. Bonfante V, Santoro A, Viviani S, et al. Outcome of patients with Hodgkin’s disease failing after primary MOPP-ABVD. J Clin Oncol 1997;15:528-34. 132. Linch DC, Winfield D, Goldstone AH, et al. Dose intensification with autologous bone-marrow transplantation in relapsed and resistant Hodgkin’s disease: Results of a BNLI randomised trial. Lancet 1993;341:1051-4. 133. Schmitz N, Sextro M, Pfistner D, et al. Aggressive conventional chemotherapy compared with high-dose chemotherapy with autologous haemopoietic stem-cell transplantation for relapsed chemosensitive Hodgkin’s disease: A randomised trial. Lancet 2002;359:2065-71. 134. Majhail NS, Weisdorf DJ, Defor TE, et al. Long-term results of autologous stem cell transplantation for primary refractory or relapsed Hodgkin’s lymphoma. Biol Blood Marrow Transplant 2006;12:1065-72. 135. Bierman PJ, Lynch JC, Bociek RG, et al. The International Prognostic Factors Project score for advanced Hodgkin’s disease is useful for predicting outcome of autologous hematopoietic stem cell transplantation. Ann Oncol 2002;13:1370-7. 136. Lazarus HM, Rowlings PA, Zhang MJ, et al. Autotransplants for Hodgkin’s disease in patients never achieving remission: A report from the Autologous Blood and Marrow Transplant Registry. J Clin Oncol 1999;17:534-45. 137. Schouten HC, Qian W, Kvaloy S, et al. High-dose therapy improves progression-free survival and survival in relapsed follicular nonHodgkin’s lymphoma: Results from the randomized European CUP trial. J Clin Oncol 2003;21:3918-27. 138. Lenz G, Dreyling M, Schiegnitz E, et al. Moderate increase of secondary hematologic malignancies after myeloablative radiochemotherapy and autologous stem-cell transplantation in patients with indolent lymphoma: Results of a prospective randomized trial of the German low grade lymphoma study group. J Clin Oncol 2004;22:4926-33. 139. Sebban C, Mounier N, Brousse N, et al. Standard chemotherapy with interferon compared with CHOP followed by high-dose therapy with autologous stem cell transplantation in untreated patients with advanced follicular lymphoma: The GELF-94 randomized study from the Groupe d’Etude des Lymphomes de l’Adulte (GELA). Blood 2006;108:2540-4. 140. Bierman PJ, Vose JM, Anderson JR, et al. High-dose therapy with autologous hematopoietic rescue for follicular low-grade nonHodgkin’s lymphoma. J Clin Oncol 1997;15:445-50. 141. Freedman AS, Gribben JG, Neuberg D, et al. High-dose therapy and autologous bone marrow transplantation in patients with follicular lymphoma during first remission. Blood 1996;88:2780-6. 142. Verdonck LF, Deffer AW, Lokhorst HM, et al. Allogeneic versus autologous bone marrow transplantation for refractory and recurrent low-grade non-Hodgkin’s lymphoma. Blood 1997;90:4501-5. 143. Williams CD, Harrison CN, Lister TA, et al. High-dose therapy and autologous stem-cell support for chemosensitive transformed lowgrade follicular non-Hodgkin’s lymphoma: A case-matched study from the European Bone Marrow Transplant Registry. J Clin Oncol 2001;19:727-35. 144. Freedman AS. Biology and management of histologic transformation of indolent lymphoma. Hematology Am Soc Hematol Educ Program 2005;314-20. 145. Gianni AM, Bregni M, Siena S, et al. High-dose chemotherapy and autologous bone marrow transplantation compared with MACOP-B in aggressive B-cell lymphoma. N Engl J Med 1997;336: 1290-7.

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146. Haioun C, Lepage E, Gisselbrecht C, et al. Benefit of autologous bone marrow transplantation over sequential chemotherapy in poor-risk aggressive non-Hodgkin’s lymphoma: Updated results of the prospective study LNH87-2. J Clin Oncol 1997;15:1131-7. 147. Guglielmi C, Gomez F, Philip T, et al. Time to relapse has prognostic value in patients with aggressive lymphoma enrolled onto the Parma trial. J Clin Oncol 1998;16:3264-9. 148. Vose JM, Anderson JR, Kessinger A, et al. High-dose chemotherapy and autologous hematopoietic stem-cell transplantation for aggressive non-Hodgkin’s lymphoma. J Clin Oncol 1993;11:1846-51. 149. Sweetenham JW, Pearce R, Taghipour G, et al. Adult Burkitt’s and Burkitt-like non-Hodgkin’s lymphoma—outcome for patients treated with high-dose therapy and autologous stem-cell transplantation in first remission or at relapse: Results from the European Group for Blood and Marrow Transplantation. J Clin Oncol 1996;14:2465-72. 150. Verdonck LF, van Putten WL, Hagenbeek A, et al. Comparison of CHOP chemotherapy with autologous bone marrow transplantation for slowly responding patients with aggressive non-Hodgkin’s lymphoma. N Engl J Med 1995;332:1045-51. 151. A predictive model for aggressive non-Hodgkin’s lymphoma. The International Non-Hodgkin’s Lymphoma Prognostic Factors Project. N Engl J Med 1993;329:987-94. 152. Haioun C, Lepage E, Gisselbrecht C, et al. Comparison of autologous bone marrow transplantation with sequential chemotherapy for intermediate-grade and high-grade non-Hodgkin’s lymphoma in first complete remission: A study of 464 patients. Groupe d’Etude des Lymphomes de l’Adulte. J Clin Oncol 1994;12:2543-51. 153. Santini G, Salvagno L, Leoni P, et al. VACOP-B versus VACOP-B plus autologous bone marrow transplantation for advanced diffuse non-Hodgkin’s lymphoma: Results of a prospective randomized trial by the non-Hodgkin’s Lymphoma Cooperative Study Group. J Clin Oncol 1998;16:2796-802. 154. Vose JM, Zhang MJ, Rowlings PA, et al. Autologous transplantation for diffuse aggressive non-Hodgkin’s lymphoma in patients never achieving remission: A report from the Autologous Blood and Marrow Transplant Registry. J Clin Oncol 2001;19:406-13. 155. Freedman AS, Neuberg D, Gribben JG, et al. High-dose chemoradiotherapy and anti-B-cell monoclonal antibody-purged autologous bone marrow transplantation in mantle-cell lymphoma: No evidence for long-term remission. J Clin Oncol 1998;16:13-18. 156. Khouri IF, Romaguera J, Kantarjian H, et al. Hyper-CVAD and high-dose methotrexate/cytarabine followed by stem-cell transplantation: An active regimen for aggressive mantle-cell lymphoma. J Clin Oncol 1998;16:3803-9. 157. Gertz MA, Anagnostopoulos A, Anderson K, et al. Treatment recommendations in Waldenstrom’s macroglobulinemia: Consensus panel recommendations from the Second International Workshop on Waldenstrom’s Macroglobulinemia. Semin Oncol 2003;30:121-6. 158. Dreger P, Glass B, Kuse R. Myeloablative radiochemotherapy followed by reinfusion of purged autologous stem cells for Waldenström’s macroglobulinaemia. Br J Haematol 1999;106: 115-18. 159. Anagnostopoulos A, Hari PN, Pérez WS, et al. Autologous or allogeneic stem cell transplantation in patients with Waldenstrom’s macroglobulinemia. Biol Blood Marrow Transplant 2006;12:845-54. 160. Antman KH, Rowlings PA, Vaughan WP, et al. High-dose chemotherapy with autologous hematopoietic stem-cell support for breast cancer in North America. J Clin Oncol 1997;15:1870-9.

Chapter 34: HPCs: Autologous Transplantation

161. Peters WP, Ross M, Vredenburgh JJ, et al. High-dose chemotherapy and autologous bone marrow support as consolidation after standard-dose adjuvant therapy for high-risk primary breast cancer. J Clin Oncol 1993;11:1132-43. 162. Tallman MS, Gray R, Robert NJ, et al. Conventional adjuvant chemotherapy with or without high-dose chemotherapy and autologous stem-cell transplantation in high-risk breast cancer. N Engl J Med 2003;349:17-26. 163. Zander AR, Kröger N, Schmoor C, et al. High-dose chemotherapy with autologous hematopoietic stem-cell support compared with standard-dose chemotherapy in breast cancer patients with 10 or more positive lymph nodes: First results of a randomized trial. J Clin Oncol 2004;22:2273-83. 164. Leonard RC, Lind M, Twelves C, et al. Conventional adjuvant chemotherapy versus single-cycle, autograft-supported, high-dose, late-intensification chemotherapy in high-risk breast cancer patients: A randomized trial. J Natl Cancer Inst 2004;96:1076-83. 165. Coombes RC, Howell A, Emson M, et al. High dose chemotherapy and autologous stem cell transplantation as adjuvant therapy for primary breast cancer patients with four or more lymph nodes involved: Long-term results of an international randomised trial. Ann Oncol 2005;16:726-34. 166. Stadtmauer EA, O’Neill A, Goldstein LJ, et al. Conventional-dose chemotherapy compared with high-dose chemotherapy plus autologous hematopoietic stem-cell transplantation for metastatic breast cancer. Philadelphia Bone Marrow Transplant Group. N Engl J Med 2000;342:1069-76. 167. Nichols CR, Andersen J, Lazarus HM, et al. High-dose carboplatin and etoposide with autologous bone marrow transplantation in refractory germ cell cancer: An Eastern Cooperative Oncology Group protocol. J Clin Oncol 1992;10:558-63. 168. Motzer RJ, Mazumdar M, Bosl GJ, et al. High-dose carboplatin, etoposide, and cyclophosphamide for patients with refractory germ cell tumors: Treatment results and prognostic factors for survival and toxicity. J Clin Oncol 1996;14:1098-105.

169. Motzer RJ, Nichols CJ, Margolin KA, et al. Phase III randomized trial of conventional-dose chemotherapy with or without high-dose chemotherapy and autologous hematopoietic stem-cell rescue as first-line treatment for patients with poor-prognosis metastatic germ cell tumors. J Clin Oncol 2007;25:247-56. 170. Einhorn LH, Williams SD, Chamness A, et al. High-dose chemotherapy and stem-cell rescue for metastatic germ-cell tumors. N Engl J Med 2007;357:340-8. 171. Stiff PJ, Veum-Stone J, Lazarus HM, et al. High-dose chemotherapy and autologous stem-cell transplantation for ovarian cancer: An autologous blood and marrow transplant registry report. Ann Intern Med 2000;133:504-15. 172. Möbus V, Wandt H, Frickhofen N, et al. Phase III trial of high-dose sequential chemotherapy with peripheral blood stem cell support compared with standard dose chemotherapy for first-line treatment of advanced ovarian cancer: Intergroup trial of the AGO-Ovar/AIO and EBMT. J Clin Oncol 2007;25:4187-93. 173. Kapoor S, Wilson AG, Sharrack B, et al. Haemopoietic stem cell transplantation—an evolving treatment for severe autoimmune and inflammatory diseases in rheumatology, neurology and gastroenterology. Hematology 2007;12:179-91. 174. Schachna L, Ryan PF, Schwarer AP. Malignancy-associated remission of systemic lupus erythematosus maintained by autologous peripheral blood stem cell transplantation. Arthritis Rheum 1998;41:2271-2. 175. Snowden JA, Kearney P, Kearney A, et al. Long-term outcome of autoimmune disease following allogeneic bone marrow transplantation. Arthritis Rheum 1998;41:453-9. 176. Euler HH, Marmont AM, Bacigalupo A, et al. Early recurrence or persistence of autoimmune diseases after unmanipulated autologous stem cell transplantation. Blood 1996;88:3621-5. 177. Burt RK, Traynor A, Statkute L, et al. Nonmyeloablative hematopoietic stem cell transplantation for systemic lupus erythematosus. JAMA 2006;295:527-35. 178. Snowden JA, Passweg J, Moore JJ, et al. Autologous hemopoietic stem cell transplantation in severe rheumatoid arthritis: A report from the EBMT and ABMTR. J Rheumatol 2004;31:482-8.

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Hematopoietic Progenitor Cells: Allogeneic Transplantation Amin M. Alousi1 & Sergio A. Giralt2 1 2

Assistant Professor, Stem Cell Transplantation, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA Professor, Stem Cell Transplantation and Lymphoma/Myeloma, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA

In 1957 Thomas and colleagues published the first reported attempts at marrow transplantation in six patients with advanced malignancies who were given marrow after receiving limited doses of chemo- or radiotherapy.1 For the next decade this therapy was limited to patients with terminal leukemia or severe marrow failure resulting from radiation exposure or disease. Although these first efforts provided groundwork for later animal and human studies, success was minimal with almost all of these early patients dying from complications of graft failure, graft-vs-host disease (GVHD), infections, or their primary disease. It was not until 1968 that the first successful allogeneic marrow transplant was reported, in a patient with severe combined immunodeficiency.2 As a result of the pioneering work of Thomas and colleagues, the number of allogeneic transplants began to increase dramatically over the next decades into the established specialty it is today.3,4 This chapter provides a brief overview of hematopoietic stem cell transplantation (HSCT) including the types of transplants, key procedural elements, donor sources, disease indications, and potential complications.

Hematopoietic Stem Cell Transplantation HSCT can be divided into three main types: 1) autologous transplantation, in which a patient serves as a self-donor, 2) syngeneic transplantation wherein the donor is a genetically identical twin, and 3) allogeneic transplantation wherein the donor is genetically different from the recipient. Marrow transplantation was initially conceived as a means for allowing hematopoietic recovery following the administration of dose-intensive chemo- and radiotherapy for treatment of malignancies. For autologous and syngeneic transplants, the therapeutic role remains hematopoietic cellular rescue following high-dose therapies. In comparison, for allogeneic Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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transplants, the infused cells result in an additional therapeutic potential arising from a powerful donor immune-mediated response against the host malignancy, a phenomenon referred to as graft-vs-tumor (GVT) effect. Numerous lines of evidence support the therapeutic potential in the GVT effect and are detailed in Table 35-1.5-10 Although a GVT benefit does not occur with autologous transplants, these types of grafts are associated with less risk because the infused cells will not be immunologically rejected or mediate an immunologic reaction. In comparison, allogeneic transplants have a greater rate of complications because of the potential for graft rejection, or more commonly, GVHD.

Procedure Fundamentals: Autologous Transplantation Autologous transplantation is a strategy in which high-dose therapy is administered to patients with a malignancy known to be dose-responsive. In order to receive an autologous HSCT, the patient must first undergo stem cell collection, followed by cryopreservation and storage of the marrow or peripheral blood progenitor cells (PBPCs). Once marrow or PBPCs are successfully collected, the patient can receive intensive myeloablative chemoor radiotherapy (or both), followed by infusion of the previously

Table 35-1. Evidence Substantiating a Graft-vs-Tumor Effect 1. Relapse is lower in allogeneic transplants when compared to autologous or syngeneic transplants. 2. T-lymphocyte-depleted transplants have a higher incidence of relapse when compared to T-lymphocyte-replete transplants. 3. Relapse rates are inversely associated with acute graft-vs-host disease (GVHD) incidence and severity. 4. Relapse rates inversely associated with incidence of chronic GVHD. 5. Delayed clearance of minimal residual disease following allogeneic transplantation (beyond the timeframe in which it could be attributed to cytotoxic therapy administered with the conditioning regimen). 6. Induction of remission following withdrawal of immunosuppression after transplantation in a minority of cases. 7. Induction of remission following infusion of donor lymphocytes.

Chapter 35: HPCs: Allogeneic Transplantation

cryopreserved cells to restore hematopoiesis. Autologous HSCT is discussed thoroughly in Chapter 34. Its brief description here is for ease of comparison with allogeneic transplantation.

Progenitor Cell Collection Ideally, the collection process should occur at a time when the marrow has normal cellularity and a limited number of malignant cells. The resting marrow contains both hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). HSCs are defined as clonal precursor cells that are capable of self-renewal as well as giving rise to a defined set of differentiated progeny. HPCs lack the ability for self-renewal but, similar to HSCs, have the ability to differentiate and proliferate.11,12 The ability of the stored product to engraft depends on the presence of both types of cells. This chapter collectively refers to these cells as HPCs, which can be identified by the cell expression of CD34 and Thy-1 and the absence of Lin and CD38 cell markers.11,13 Historically, HPCs capable of reconstituting hematopoiesis were collected through harvesting the marrow via multiple aspirations from sites such as the ilium. More commonly today, HPCs are collected from the peripheral blood by leukapheresis. In this procedure, the normally low levels of circulating HPCs are increased through the use of hematopoietic growth factors such as granulocyte colony-stimulating factor (G-CSF) alone or in combination with mobilizing doses of cytoreductive chemotherapy (termed chemomobilization).14,15 Chemomobilization strategies generally result in a higher yield of HPCs when compared to strategies using hematopoietic growth factors alone.16 PBPC collections have now largely replaced marrow harvesting procedures because of the ease and safety (sparing the need for general anesthesia) of the procedure. In addition, PBPC transplantation results in quicker recovery of granulocytes and platelets than does marrow transplantation.17 Quantification of CD34-expressing cells in the PBPCs is often performed to assess the probability of successful reconstitution of hematopoiesis upon infusion.18 Threshold levels resulting in timely neutrophil and platelet recovery range from 3 to 5 ⫻ 106 CD34⫹ cells/kg body weight.19

Preparative Regimens The combination of chemo-, radio-, and biologic therapies in preparation for HSCT is referred to as a preparative (or conditioning) regimen. The sole role of the preparative regimen in the autologous transplant setting is eradication of any residual cancer. High-dose therapy is employed to overcome tumor resistance. In addition, the combination of chemotherapies may provide a way to act on different targets within cancer cells.20 Creation of regimens is generally based on the principles of minimizing overlapping toxicity and mechanisms of resistance. Dose escalation achievable with the combination regimens is usually less than with each drug alone. Candidate drugs for incorporation into preparative regimens are those agents with a steep dose-response curve and myelosuppression as their doselimiting toxicity. However, dose escalation is not feasible when

extramedullary toxicity occurs at lower doses or at the same dose levels as myelosuppression. For this reason, alkylating agents are commonly employed. For instance, the dose of thiotepa can be escalated significantly with stem cell support.21,22 Conversely, adriamycin is seldom used because the mucositis and cardiac toxicity associated with dose escalation of this drug cannot be overcome by the use of HPCs. Numerous conditioning regimens have been evaluated for various diseases with choice of regimens often based on the preference and experience of the clinician. High-dose chemoradiation therapy for multiple myeloma and lymphoma, two of the most common indications for autologous transplantation, is reviewed later in this chapter. Recently, the addition of newer “targeted” therapies to conditioning regimens has been explored as a way of increasing efficacy and minimizing toxicity. Monoclonal antibodies and radioconjugates for B-cell lymphomas and certain leukemias are examples of newer therapies that have been added safely to conditioning regimens and may offer additional therapeutic benefit with little or no increase in toxicity.23,24 Autologous transplantation is a relatively safe procedure with minimal treatment-related mortality. There is no specific age cut-off for the procedure, and patients well into their seventh and eighth decades of life safely undergo the therapy.25,26 Specific disease indications and complications are discussed later in the chapter.

Allogeneic Transplantation The most obvious difference between autologous and allogeneic HSCT is the need for a donor in the latter. Historically, most allografts used marrow cells obtained from an HLA-matched sibling. However, many potential candidates for this therapy lack a matched sibling donor and transplant procedures using cells obtained from volunteer unrelated donors, mismatched family members, and cord blood are becoming more common. HLA compatibility is the strongest predictor for the occurrence of severe GVHD and is the single most important factor to consider in selecting an allogeneic donor.27 The major histocompatibility complex (MHC) is located at p21.3 on the short arm of chromosome 6, where several closely linked genes referred to as the HLA system are located.28 These genes have been subdivided into Class I loci, which includes HLA-A, -B, and -C, and Class II loci, which includes HLA-DR, -DRW, -DQ, and -DP. For HSCT, HLA-A, HLA-B, and HLA-DR are routinely evaluated.29,30 Matching at HLA-C has been confirmed to have an impact on outcome.27 In its strictest sense, HLA identity means that the donor and recipient are matched for the amino acid sequence encoded by all HLA loci. It is important to note that conventional typing techniques detect a limited number of HLA polymorphic sequences. Therefore, “HLA matched” may not be “HLA identical.” HLA genotypically identical siblings inherit the same MHC haplotypes and, therefore,

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are also identical for other non-HLA genes and polymorphisms that are carried on those haplotypes. This is unlike the case for unrelated donors and patients who, although matched for Class I and II alleles, may not necessarily be identical for non-HLA polymorphisms. However, to date studies have not been able to substantiate an effect on outcome for mismatching at any of the known microsatellite polymorphic regions tested.31 Conventional serologic typing is based on the complementdependent microlymphocytotoxicity test and uses selected HLA-specific alloantisera or monoclonal antibodies to identify HLA antigens.32 Mismatching between cross-reactive antigens is considered a minor mismatch, whereas mismatching between non-cross-reactive antigens is considered a major mismatch. For related patient-donor pairs, a single minor mismatch may be of little biological significance. Molecular typing relies on polymerase chain reaction (PCR) amplification of specific gene segments and can be performed 1) at a level corresponding to the specificities identified by serology (low resolution), 2) at a level where a limited number of alleles are possible (intermediate resolution), or 3) at a level where the specific allele is identified (high resolution). Sequence-based HLA typing is the most precise technique available. Improvement in typing techniques through high-resolution molecular typing has had a profound impact in reducing the risks of immunologic complications with unrelated allogeneic transplantation.33 This is highlighted by studies finding that just over 50% of donor-patient pairs typed by conventional serologic typing are found to be matched at the allele level when retrospectively typed using high-resolution techniques. This translated into higher probabilities of severe acute GVHD and transplant-related mortality, as well as lower survival in those retrospectively identified allele-mismatched patients.34 The fact that immunologic complications (GVHD) often develop even among HLA genotypically identical siblings suggests that this system is not the only mediator of immunologic complications following allogeneic transplantation. In HLA-matched transplants, GVHD is felt to result from mismatching of poorly defined minor histocompatibility antigens. The only easily identified minor histocompatibility antigen is the HY antigen.35 The chance of mismatching for these minor antigens should increase with increasing distance in the relationship between donor and recipient.36

Donor Factors Efforts to identify a donor is a lengthy process and must be carried out as soon as a patient has been referred, often before a definitive decision related to transplantation has been made. Usually all potential sibling donors are typed first and if no compatible donors are identified, an unrelated donor search is performed. Because of the shrinking size of households, the frequency of unrelated donor transplant is becoming more common and is currently estimated to account for one third of all allogeneic transplants.37 To help facilitate this process, registries containing HLA-typed potential adult donors were created. The largest of theses registries is the National Marrow Donor

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Program (NMDP), which was established in 1986 to facilitate donor enrollment as well as the donor search process. There are more than 7.5 million individuals listed on these registries with approximately 800,000 new volunteers added annually.38 Since the early 1990s the use of PBPCs has become increasingly common and now constitutes the most common HPC source. The rationale for using PBPCs is derived from studies in the autologous setting that demonstrated accelerated recovery of hematopoiesis when compared to traditional marrow transplantation.39-40 For donors, this has eliminated the risks and morbidity associated with general anesthesia and marrow harvest. Recombinant growth factors such as G-CSF are used to mobilize HPCs into the peripheral blood where they may be collected through one or more leukapheresis procedures. In addition to the benefits to the donor, PBPC transplantation offers a number of advantages to the recipient. G-CSF-mobilized PBPCs contain more HPCs than marrow grafts.41 This has shortened the duration of absolute neutropenia and thrombocytopenia in recipients by approximately 4 to 8 days.41,42 Donor T cells are found in much higher concentration in PBPC grafts and help to facilitate engraftment especially after less dose-intense preparative regimens. The presence of higher numbers of T cells initially raised the concern for greater frequency and severity of GVHD; however, many large studies have shown that the risk of severe acute GVHD using PBPC grafts does not exceed the risk seen in traditional marrow grafts.43,44 The incidence of chronic GVHD in patients receiving PBPC grafts has tended to be higher than those receiving marrow grafts. Thus, longer follow-up will be needed before it is safe to conclude that these two sources are comparable.45,46 Only a fraction of patients will have a fully matched unrelated donor identified despite the growing number of typed volunteers on worldwide registries. Populations of European ancestry have an approximately 50% chance of identifying a matched unrelated donor, whereas ethnic or mixed communities have as low as a 10% chance.47 This disparity is largely the result of the increased polymorphisms with respect to HLA seen in certain minority groups.48 For this reason, alternative donor transplants are an area of active research. Umbilical cord blood (UCB) offers another source of hematopoietic stem cells for use in allogeneic transplantation. UCB transplants have increased the available donor pool because of the reduced need for HLA compatibility between the donor and recipient. In addition, UCB has shortened the time it takes to secure a donor, because the cells are readily available and do not require delays in the collection process. One review found that 19% of patients who did not have a potential HLA-matched unrelated donor were able to find a UCB unit matched at four of six HLA antigens. This same analysis found that the median time from search to clearance of an unrelated donor was 49 days; securing UCB units took just 13.5 days.49 Because the number of cells required for successful engraftment is dependent on recipient size, a limiting factor for this form of transplant is the low number of HPCs in a unit of cord blood. Recent registry

Chapter 35: HPCs: Allogeneic Transplantation

Recipient Factors It is estimated that over 20,000 allogeneic transplant procedures have been performed worldwide for a variety of malignant and nonmalignant disorders.53 The International Bone Marrow Transplant Registry (IBMTR), an invaluable resource that collects data from more than 400 centers worldwide, allows transplant physicians to see current transplant trends, compare outcomes of different transplant strategies, and conduct retrospective and prospective studies in HSCT. According to data submitted to the IBMTR, the following trends have been noted: Currently the most common diagnoses for which allogeneic progenitor cell transplantation is being performed are acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), and

100

Transplants, %

80

ⱕ20 yrs 21-40 yrs 41-50 yrs 51-60 yrs ⬎60 yrs

60 40 20 0 1987-1992

1993-1998

1999-2004

Figure 35-1. The proportion of older recipients of allogeneic transplants for AML, ALL, CML [as reported to the Center for International Blood and Marrow Transplant Research (www.cibmtr.org)] is increasing.

Transplants

analysis50 evaluating outcomes for UCB transplantation in pediatric patients with leukemia found similar leukemia-free survival for one- or two-antigen mismatch UCB transplants when compared to HLA-matched unrelated transplants. This study found similar rates for acute GVHD between the mismatched UCB transplant and unrelated donor transplant recipients. Treatment-related mortality was higher in two-antigen mismatch UCB when compared to one-antigen UCB or matched unrelated donor recipients. In addition, patients who had a low-cell dose, one- or two-antigen mismatch UCB transplant had higher transplant-related mortality than those receiving a matched, unrelated donor transplant. Median time to neutrophil and platelet engraftment were higher following one- to two-antigen mismatched UCB transplants when compared to unrelated marrow recipients.50 Retrospective studies in adults have confirmed a low rate of acute GVHD when comparing one-antigen and two-antigen mismatch UCB transplants to one-antigen mismatch unrelated marrow recipients but most have demonstrated inferior outcomes when compared with matched unrelated donor recipients. These studies also confirmed a higher graft failure rate and treatment-related mortality for UCB transplants in adults when compared to matched unrelated donor transplants and lower leukemia-free survival.51 Nonetheless, UCB transplants are a feasible option for adults who do not have a related or unrelated, matched donor and strategies for improving engraftment are an active area of research. Haploidentical transplants offer another option when a matched sibling or unrelated donor cannot be identified. The advantage of these transplants is that parents, children, or halfmatched siblings can serve as a donor. These forms of transplants call for highly selecting the collected product for CD34⫹ HPCs in an effort to minimize the amount of T cells in the final infusion. In this way, the risk for GVHD (which would otherwise be universal with transplants from haploidentical donors) is reduced. In addition, the conditioning regimen often includes antibodies directing against T cells. While these efforts reduce the risk for GHVD, delayed immune reconstitution occurs, resulting in a high risk for infectious complications.47,52

2,000 1,800 1,600 1,400 1,200 1,000 800 600 400 200 0

Reduced intensity conditioning Traditional

1998

1999

2000

2001

2002

2003

2004*

*Data incomplete

Figure 35-2. The proportion of allogeneic transplants performed using reducedintensity conditioning is increasing [data incomplete for 2004; provided by the Center for International Blood and Marrow Transplant Research (www.cibmtr.org)].

myelodysplastic syndromes (MDS) (in decreasing order). The indications for transplantation have changed over time; eg, CML is no longer the most commonly transplanted hematologic malignancy because of the advent of imatinib. Likewise, the average age of transplant recipients has increased over time. The proportion of patients receiving allografts over the age of 50 years is approaching 15%. Paralleling the increasing age of recipients has been the use of less-intense conditioning regimens (Figs 35-1 and 35-2).37 Numerous recipient factors determine the outcome following transplantation and include the disease stage at transplantation, recipient age, performance status, and presence of comorbidities. Apart from poor performance status, there is no one factor that precludes allografting in an individual patient. Instead, the decision to undertake transplantation depends on a careful assessment and discussion of the risks and benefits of the procedure when contrasted with the natural history of the disease. All potential transplant recipients undergo an extensive pretransplant evaluation that usually includes a complete history and physical examination in addition to evaluation of cardiac, pulmonary, hepatic, and renal function.

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Preparative Regimens In allogeneic transplantation, the preparative regimen serves two main purposes: eradicating the underlying malignancy and inducing a state of immune tolerance to allow for the donor cells to engraft and expand. Generally speaking, efforts to decrease relapse through intensification of the preparative regimen have come at the expense of increased treatment-related mortality. As in autologous transplantation, there is no one standard preparative regimen in the setting of allogeneic transplantation. Allogeneic transplant conditioning regimens can generally be divided into chemotherapy-based protocols or total body irradiation (TBI)-based protocols. TBI provides both immunosuppressive and myeloablative effects. The use of fractionated regimens and organ-shielding has lowered the toxicity.54-57 Most modern regimens deliver a total dose of 1000 cGy to 1500 cGy using a variety of fractionation schedules. Although there is some evidence that further escalation of the total dose of TBI is more effective at preventing relapse, these benefits have been offset by increased nonrelapse mortality.58,59 TBI is most often combined with high-dose cyclophosphamide (Cy), a potent immunosuppressive agent.60 Other chemotherapy regimens with demonstrated efficacy in conjunction with TBI included etoposide either alone or together with Cy. Efficacy appears similar between these regimens with the combination of etoposide and Cy showing increased toxicity in patients with advanced disease.61,62 TBI-based regimens are still commonly used in all disease states; however, it remains the most favored transplant regimen for patients with ALL (often combined with etoposide).63 Although efficacious, TBI is associated with a number of short- and long-term complications including secondary malignancies, cataracts, and endocrine dysfunction.64 The creation of non-TBI-containing regimens developed out of a wish to decrease the above-mentioned long-term toxicities as well as expand this therapy to centers without dedicated radiotherapists. The most extensively studied regimen was one created by Santos in the early 1980s65 and later modified by Tutschka.66 Santos’ original regimen replaced TBI with busulfan (Bu) at a dose of 16 mg/kg divided orally every 6 hours over 4 days combined with Cy 200 mg/kg divided in four daily doses (BuCy4). This regimen was found to be too toxic with a high rate of early transplant-related mortality and was later modified to the BuCy2 regimen with a decrease in the dose of Cy to 60 mg/kg for 2 days. This regimen remains in wide use today for various malignancies in both allogeneic and autologous transplantation. The safety and efficacy of the BuCy2 regimen when compared to TBI-Cy has been extensively evaluated in multiple, randomized studies.67-71 Most of these studies showed comparable outcomes for these two regimens; however, a few demonstrated superiority of the TBI-containing regimens especially for those AML and ALL patients with advanced disease. With these exceptions there currently are no data that confirm the superiority of one specific regimen over another. Recent modifications to the BuCy2 regimen with pharmacokinetic (PK)-guided dosing of Bu and/or an intravenous (IV) formulation probably eliminate any small

546

advantage in the TBI-Cy regimen that has thus far been inconsistently demonstrated. The dose-limiting side effect of oral Bu is hepatoxicity with veno-occlusive disease (VOD) of the liver a major limitation of the BuCy2 regimen. It has been well documented that oral dosing of Bu results in frequent variability in inter- and intrapatient steady-state drug concentrations.72-76 Elevated levels of Bu in the blood have been shown to result in higher incidences of VOD of the liver,77 while low doses may be associated with relapse (in CML)78 and nonengraftment.79-82 It has been hypothesized that by reducing this variability, results with Bu-containing regimens might be improved. PK-guided oral dosing followed by administration of an IV formulation of the drug has been evaluated. The need for PK-guided Bu dosing is strongest in pediatric transplantation where the variability of oral dosing is most pronounced. This is especially true for patients less than 4 years of age who have increased clearance of the drug when administered orally because of a higher conjugation rate with glutathione in the hepatocyte.83 Bolinger et al84 performed one of the earliest trials evaluating targeted-dosed Bu in pediatric patients undergoing allogeneic transplantation. In this study, adjustment from standard dosing was required in a majority of the patients to achieve the target steadystate concentration. By so doing, 30 of 32 (94%) patients successfully achieved the target concentration. This strategy improved the rate of engraftment when compared to a historical group of patients who received standard-dosed Bu. Similar results were shown by Bleyzac et al85 who were also able to document lower incidences of VOD of the liver when compared to historical controls. PK-guided strategies have also been shown to be feasible and result in low treatmentrelated mortality in adult patients receiving oral Bu regimens.86,87 In 1996, a patent was filed for an IV formulation of Bu. In 2000, researchers at M.D. Anderson Cancer Center performed a safety and feasibility study of the IV formulation and determined the bioequivalent dose.88 Phase II studies performed with the IV formulation have determined a lower incidence of VOD of the liver, death from VOD, and treatment-related mortality when retrospectively compared to patients treated during the same period who received the standard oral BuCy2 regimen.89-93 However, questions related to whether IV Bu is superior to oral Bu, whether PK-guided dosing of the IV formulation is needed, and whether oral PK-guided doses are equivalent to the IV formulation remain unanswered in the absence of randomized studies. Additional modifications to the BuCy regimen have stemmed from reports determining that Cy and its metabolites contribute not only to the development of hemorrhagic cystitis after transplantation but also to the liver toxicity of the preparative regimen.94 Therefore, fludarabine has replaced Cy in many modern myeloablative regimens. Initial studies with the combination of fludarabine and Bu have produced high engraftment rates with low levels of toxicity.95,96

Reduced-Intensity Conditioning Regimens Traditionally, allogeneic transplantation has been limited to younger patients because of toxicity related to the intensive

Chapter 35: HPCs: Allogeneic Transplantation

Table 35-2. Examples of Reduced-Intensity Conditioning Regimens 1. Total body irradiation at doses ⭐500 cGy 2. Total busulfan dose ⭐9 mg/kg 3. Total melphalan dose ⭐140 mg/m2 4. Regimen includes a purine analog—fludarabine, cladribine, or pentostatin

and chemotherapy-induced cystitis,101 respectively. The risk for serious organ toxicities and infections are detailed below. Generally accepted mortality rates associated with autologous transplants are approximately 5%, whereas allogeneic transplant recipients have a 10% to 50% risk for transplant-related death depending on host factors, including age, comorbidities, disease stage, baseline toxicities, and donor factors (namely, matching).

Table 35-3. Most Commonly Used Reduced-Intensity Conditioning Regimens 1. Low-dose total body irradiation (200 cGy) ⫾ fludarabine 2. Busulfan ⫹ fludarabine ⫾ others 3. Fludarabine ⫹ cyclophosphamide ⫾ others 4. Fludarabine ⫹ melphalan ⫾ others 5. Fludarabine ⫾ others

conditioning regimens. Unfortunately, the cancers for which this therapy is most commonly prescribed occur in older adults. Reduced-intensity conditioning regimens employ lower doses of chemo- or radiotherapy to allow for engraftment and rely on the GVT effect to prevent relapse. As a result, patients who traditionally were felt to be too old for allogeneic transplant or who suffer comorbidities that previously would have precluded them can now safely undergo this therapy. There is no one standard reduced-intensity regimen, with commonly used regimens differing in the degree of intensity administered. Some regimens employ the same agents used in full-intensity regimens, but at reduced doses. On the other end of the spectrum are regimens that provide minimal cytotoxic agents and instead rely on immunosuppressive treatments to allow donor engraftment. Agreed-upon definitions of reduced-intensity regimens as well as commonly employed agents used are listed in Tables 35-297 and 35-3.98

Myelosuppression Shortly after infusion, HPCs migrate to sites in the lungs, liver, spleen, and marrow. For most patients, some degree of marrow cellularity can be demonstrated within 14 days of transplantation. Neutrophil and platelet engraftment is defined as being the first of three consecutive days in which the absolute neutrophil count is ⬎500/µL and the platelet count is ⬎20,000/µL (without transfusion support), respectively. This generally occurs 14 to 24 days after stem cell infusion, with longer time to engraftment in cord blood transplants. Before engraftment, patients require aggressive hematologic support with transfusion of platelets and red cells. Blood components must be irradiated to minimize the possibility of graft-vs-host reactions mediated by blood donor T cells.102 For patients experiencing prolonged cytopenias, growth factors may be used to shorten the duration of aplasia without increasing the risk of GVHD or relapse.103,104 In the allogeneic setting, engraftment of donor cells can be documented by use of molecular tests such as DNA restriction fragment length polymorphisms, or by PCR of microsatellite regions demonstrating a donor pattern. Following successful transplantation, cells of the recipient’s reconstituted hematologic and immunologic systems are derived primarily from the donor’s blood or marrow, although in some cases mixed chimerism occurs in which both donor- and recipient-derived cells are present in the recipient’s circulation and marrow.105

Graft Failure

Potential Complications of Dose-Intensive Therapy Followed by HSCT The potential risks resulting from high-dose chemoradiotherapy and blood and marrow transplantation are numerous and include toxicity from the preparative regimen, infections resulting from granulocytopenia or posttransplant immunodeficiency, hemorrhage caused by thrombocytopenia and tissue toxicity, and, in the case of allogeneic transplants, transplant rejection and GVHD. The creation of better supportive medications has minimized many of the acute toxicities associated with high-dose chemoradiotherapy regimens. This includes the incorporation of selective type three 5-hydroxytryptamine (5-HT3) receptor antagonists, which has decreased the incidence of severe treatment-related nausea.99 The addition of palifermin (recombinant human keratinocyte growth factor) and mesna (2-mercaptoethane sodium sulfonate) have been shown to reduce the incidence of severe radiation-induced mucositis100

The failure to recover hematologic function or the loss of marrow function after initial reconstitution constitutes graft failure. Graft failure is an unusual event for autologous transplant recipients but can occur in 5% to 11% of HLA-identical allogeneic recipients.106 Graft failure generally takes place within 60 days of transplantation although late graft failure has been known to occur.107 Several factors are known to increase the risk of graft failure in allogeneic transplantation, including a low nucleated cell count infused, increasing HLA disparity between donor and host, T-cell depleted grafts, and inadequate immunosuppression of the host. Historical graft failure rates were as high as 35% in patients undergoing transplantation for aplastic anemia, which was felt to be caused in large part by alloimmunization from prior transfusions. This has now been reduced to ⬍10% for patients with aplastic anemia through the minimizing of pretransplant transfusions and administration of leukocyte-reduced, irradiated blood components.108 Evidence now exists implicating host T cells as the mediators of an active host immune response against minor alloantigens expressed by the donor cells. On the

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basis of this understanding, several strategies to further eliminate or inactivate host T cells have evolved, including the use of additional or increased intensity immunosuppressive agents such as fludarabine or the elimination of host T cells through the use of antithymocyte globulin (ATG) or anti-CD52.109-111 Treatment of graft failure depends on whether donor cells can be detected in the patient’s marrow. If present, a repeat infusion of donor cells can be attempted with or without further conditioning. When no donor hematopoietic cells are detected, a second conditioning regimen may be administered followed by donor stem cells; however, outcomes in this setting are extremely poor.112

Graft-vs-Host Disease GVHD represents one of the most frequent complications of allogeneic transplantation and has been the major barrier to wide-scale application of this therapy. GVHD results from the recognition of host tissues as foreign by donor immunocompetent cells. The incidence increases with greater HLA disparity between the donor and host. A fundamental problem for allogeneic transplantation is the close association between this complication and the derived benefit resulting from a GVT effect. Both GVHD and GVT effect are mediated by donor T cells with both being reduced when T cells are depleted from the infused graft (referred to as T-cell-depleted transplants). Identifying and separating the target antigens resulting in GVHD and GVT effect are active areas of research that may one day result in maximizing the therapeutic potential of this modality while eliminating this frequent obstacle.113 Two forms of GVHD are commonly distinguished: acute and chronic. Historically, acute GVHD was defined as occurring within the first 100 days of transplant and chronic GVHD was defined as occurring after the 100-day mark. It is now clear that this time point was an artificial one and clinical manifestation and histologic findings are now the sole factors used in defining these distinct entities.114

The incidence of acute GVHD varies from 20% to 70%, depending upon the intensity of the conditioning regimen, the extent of histocompatibility differences with the donor, the age of the recipient, and the stage of primary disease.115-120 Acute GVHD is graded clinically using a standardized system that takes into account clinical changes in the primarily affected organs: skin, liver, and gastrointestinal tract. This grading system allows for quantitative estimates of disease severity and response to therapy with scoring consisting of Grades I to IV (Table 35-4).121 These clinical findings are usually confirmed by pathologic changes in the affected organs, although the pathologic findings do not change the grading or staging of the disease. Corticosteroids remain the standard initial treatment, but even with prompt initiation of such therapy, the treatment is suboptimal. Fewer than 50% of patients with acute GVHD have a durable response after initial therapy; most patients require secondary treatment.122,123 Attempts to improve the complete response rate by adding additional therapies to corticosteroids or by using higher doses have been unsuccessful.124,125 Moreover, the outcome for patients with steroid-refractory GVHD is poor, with a mortality rate of 70%; at this time, no therapy has been shown to affect survival.126 The causes of death among patients with advanced GVHD include organ failure and infections related to poor immune reconstitution. For these reasons, improving prophylaxis has been the preferred strategy. The prophylactic use of cyclosporine (CSA) and methotrexate (MTX) are effective prophylaxis for acute GVHD and have been shown to improve survival.115 Corticosteroids have also been successfully used in combination with CSA for GVHD prophylaxis; however, the addition of prednisone to CSA and MTX has been shown not to add additional benefit.127 CSA is a cyclic polypeptide that prevents T-cell activation by inhibiting interleukin-2 production and expression. Although effective as GVHD prophylaxis, CSA has significant toxicities including hypertension, nephrotoxicity, hypomagnesemia, risks

Table 35-4. Organ Stages of Acute GVHD Organ Stage

Organ Skin (body surface area)

Liver (mg/dL bilirubin)

Gastrointestinal Tract (24-hour stool output)

0

No rash

⬍2.0

⬍500 mL or ⬍280 mL/m2

I

Rash ⬍25% or 25% to ⬍50%

⬍2.0

⬍500 mL or ⬍280 mL/m2

II

⭓50% or generalized erythroderma

2.1 to 3.0

501 to 1000 mL or 280 to 555 mL/m2 or persistent nausea and/or vomiting

3.1 to 6.0 or 6.1 to 15.0

1001 to 1500 mL or 556 to 833 mL/m2 or ⬎1501 mL or 833 mL/m2

⬎15.1

⬎1501 mL or 833 mL/m2 with severe abdominal pain and ileus

III

IV

548

Generalized erythroderma with bullous formation and desquamation

Chapter 35: HPCs: Allogeneic Transplantation

for seizures, hypertrichosis, gingival hyperplasia, tremors, and anorexia.128 Typically CSA is initiated intravenously 1 to 2 days before stem cell infusion and converted to oral dosing when possible. The risk of acute GVHD increases when cyclosporine concentrations in blood drop below a target level.129 Tacrolimus is a macrolide lactone that closely resembles CSA in mechanism of action, spectrum of toxicities, and pharmacologic interactions. In a randomized study,119 the prophylactic regimen of tacrolimus and MTX was demonstrated to reduce Grade II-IV acute GVHD when compared to CSA and MTX. However, there was a higher regimen-related death rate in patients with advanced disease who received tacrolimus in this study, possibly related to the use of tacrolimus at levels beyond those currently recommended. The implications of this finding are unclear and generally CSA and tacrolimus are viewed as equivalent. The addition of MTX has the potential for increased severity of regimenrelated mucositis and delays in engraftment. At M.D. Anderson Cancer Center, a modification of the tacrolimus-MTX regimen using “mini-dose MTX” was found to be as effective as full dose therapy with less toxicity (Fig 35-3).130 ATG has also been used effectively in the prophylactic setting.131 In patients with aplastic anemia who underwent transplantation using a Cy and ATG conditioning regimen, low rates of acute GVHD (15%) and chronic GVHD (34%) were reported and a significant survival benefit at 3 years was identified for patients receiving this regimen when compared with historical controls who received Cy alone (92% vs 72%).109 In addition to the immunosuppressive agents above, T-cell depletion before marrow infusion has been shown to effectively reduce the incidence and severity of GVHD; however, it also results in increased graft rejection, infectious complications, and relapse rates in a disease-specific manner.8 Selective T-cell subset depletion or “add-back” of allo-depleted T cells are future strategies that may be effective for reducing the risk of acute GVHD while preserving the GVT effect.52,132 Chronic GVHD shares many features with various autoimmune disorders, including autoantibodies and disease

manifestations that are similar to such disorders as the sicca syndrome and scleroderma. The manifestations of chronic GVHD are extensive with nearly every organ potentially affected.114 The consequences for affected patients can be profound, with its presence or absence being the most significant determinant of longterm outcome and quality of life following allogeneic HSCT.133 Approximately 60% to 70% of patients who receive an allogeneic transplant are affected to some degree by chronic GVHD.134 Despite progress in other areas of this field, our understanding of this entity is limited; therefore, advancements in the prevention and treatment of chronic GVHD have been sparse.

Organ Toxicity Veno-Occlusive Disease of the Liver VOD, also referred to as sinusoidal obstruction syndrome, is a complication of both allogeneic and autologous transplant as well as occasionally chemotherapy regimens in the nontransplant setting. The process occurs as a result of direct injury to the hepatic venous endothelium. The syndrome is characterized by the constellation of signs and symptoms consisting of tender hepatomegaly, jaundice, weight gain, and ascites. The incidence of this syndrome has varied among reports and has ranged from 5% to 50% of all allogeneic and autologous transplants.135,136 These studies suggest VOD is life-threatening in roughly one-quarter of cases. Risk factors include preexisting liver disease, high-intensity conditioning regimens, prior chemotherapy regimens, and mismatched or unrelated donor transplants.136 Preliminary reports have found defibrotide (a polydeoxyribonucleotide) to be a promising treatment for patients suffering from severe VOD.137 Lung Myriad pulmonary complications can occur in patients following autologous and allogeneic transplantation. Most of these complications are a result of acute or delayed complications of the conditioning regimen and previous treatments. It is important to distinguish these entities from an infectious etiology. Direct toxicity can result from the conditioning regimen including carmustine-induced

Start intravenous tacrolimus or CSA Stem cell infusion

Mini-dose MTX 5 mg/m2 Days ⫹1,3,6 and 11

Day ⫺5 ⫺4 ⫺3 ⫺2 ⫺1 0 ⫹1 ⫹2 ⫹3 ⫹4 ⫹5 ⫹6 ⫹7 ⫹8 ⫹9 ⫹10 ⫹11

Figure 35-3. Standard GVHD prophylaxis at M.D. Anderson Cancer Center. CSA ⫽ cyclosporin A; ATG ⫽ antithymocyte globulin; GVHD ⫽ graft-vs-host disease.

*ATG for unrelated donors or mismatch-related donor recipients

Wean tacrolimus/CSA @ 3-4 months post-transplant in sibling or 6 months in unrelated donor recipients if no GVHD

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pulmonary toxicity and radiation-induced lung injury. Diffuse alveolar hemorrhage (DAH) may occur in the immediate posttransplant period during pancytopenia. One study found a strong association between DAH and previous thoracic irradiation, whereas no association was found between the presence of thrombocytopenia, coagulopathy, or neutropenia.138 Idiopathic pneumonia syndrome may occur following allogeneic transplant. It is described as a diffuse, non-infectious pneumonia that in animal models appears to be an immune-mediated process in part induced by the toxicity of the conditioning regimen.139,140 Bronchiolitis obliterans is a pulmonary manifestation of chronic GVHD.114

Infections Patients undergoing autologous and allogeneic transplants are at risk for a variety of infections as a result of the myelosuppression induced by the chemo- and radiotherapies employed during conditioning, delayed immune reconstitution following myeloablative therapy, and, in the case of allogeneic transplantation, immunosuppression used to prevent and treat GVHD as well as GVHD itself. Risk for infection is generally divided into three phases: Phase I, the period following preparative regimen while awaiting engraftment; Phase II, following engraftment and up to Day 100; and Phase III, from Day 100 to months and year(s) following transplantation. Infectious risks for each of the three periods varies, respectively, depending on the degree of tissue toxicity and length of neutropenia following the preparative period, the presence of acute GVHD, and the presence of chronic GVHD. Based on the high risk and associated morbidity resulting from bacterial, fungal, and viral pathogens, several prophylactic and surveillance strategies have been developed for patients undergoing transplantation. Bacterial, candidial, and herpes simplex virus (HSV) infections pose the greatest risk for patients during the preengraftment period. In addition to conditioning-induced myelosuppression, patients often have impaired primary immunity as a result of indwelling catheters and tissue toxicity (mucositis, gastroenteritis, etc). Bacterial prophylaxis with a quinolone antibiotic is often employed in an effort to suppress intestinal flora and prevent serious gram-negative infections141,142; however, this is controversial and currently not recommended in guidelines from the Infectious Disease Society of America (IDSA)/Centers for Disease Control and Prevention (CDC).143 The prompt administration of broadspectrum antibiotics following a neutropenic febrile event is of paramount importance in preventing morbidity of bacterial infections. In a randomized study,144 the prophylactic use of fluconazole has been shown to reduce the incidence of systemic and superficial fungal infections. Fluconazole has not, however, been shown to affect the incidence of infections with resistant organisms such as Candida krusei or c. glabrata and invasive mold infections. Prophylaxis against other invasive fungal infections such as Aspergillus species is an area of debate with some studies supporting the use of newer azole agents145 or echinicandins.146 Herpes simplex virus reactivation is seen commonly in seropositive patients during the first 6 weeks after transplantation unless prophylaxis is administered. Acyclovir prophylaxis has been

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shown to prevent reactivation, reduce morbidity, and lower the incidence of antibiotic resistance and is routinely administered.147 Patients in whom HSV reactivation occurs should be treated with acyclovir or valacyclovir. For patients who develop resistance after multiple episodes of HSV reactivation, foscarnet may be required.148 Prophylaxis against Pneumocystis carinii (PCP) is recommended following engraftment and continued for a minimum of 6 months following transplantation or while taking immunosuppressive drugs. Trimethprim-sulfamethoxazole is the agent of choice for PCP prophylaxis given that it has the lowest breakthrough rate. Various other agents have been used including dapsone and aerosolized pentamidine.149 Cytomegalovirus (CMV) infections were once a common and fatal occurrence in the first 3 months following marrow transplantation. The routine prophylactic use of ganciclovir has largely eliminated CMV disease after transplantation. Randomized studies have clearly demonstrated efficacy in reducing the incidence of CMV disease by the prophylactic treatment of all CMV-seropositive patients.150 However, this strategy is problematic in that many patients will require discontinuation of therapy because of the development of neutropenia, which places the patients at increased risk for other infections. The creation of highly sensitive tests capable of detecting CMV reactivation before overt CMV disease has allowed for an alternative strategy to routine prophylaxis. These tests include direct detection of CMV pp65 antigen in peripheral blood leukocytes151 and detection of CMV DNA in leukocytes, plasma, or serum.152 Preemptive therapy when reactivation is first detected has been shown to result in similar rates of CMV-related death and survival when compared with universal ganciclovir prophylaxis starting at the time of engraftment. Patients receiving routine prophylaxis experienced less CMV disease in the first 100 days after transplantation; however, this benefit was offset by a higher rate of invasive fungal infections and late CMV disease.151 Thus, current recommendations support either approach for patients at risk for CMV disease (ie, all CMV-seropositive recipients and seronegative recipients with seropositive donors). For CMVseronegative patients with CMV-seronegative donors, the use of CMV-seronegative or leukocyte-reduced blood components are effective in preventing primary infections.153 Viral infections continue to pose significant challenges to clinicians managing posttransplant patients. In addition to community respiratory viruses, intestinal viruses are easily transmitted through person-to-person contact and have been associated with increased morbidity and mortality. Because of a lack of specific therapies for these agents, the focus must be on appropriate isolation of patients and the prevention of person-to-person transmission.

Long-Term Complications of High-Dose Therapies Several long-term complications occur following HSCT, requiring patients to maintain regular office visits and practitioners familiar with posttransplant follow-up guidelines to monitor patient progress. Regular eye screening for cataracts is required especially for those who received a TBI-containing regimen or steroids for

Chapter 35: HPCs: Allogeneic Transplantation

the treatment of GVHD.154 Several endocrine complications occur including hypothyroidism, stunted growth and development (in pediatric patients), and premature gonad failure/sterility.155 Patients who have undergone autologous HSCT are at increased risk for treatment-related MDS and AML, especially if they were subject to prior radiation (especially to the pelvis), were heavily pretreated, were slow PBPC mobilizers, or were subject to TBIcontaining conditioning regimens.156 Both autologous and allograft recipients are at higher risk for additional secondary cancers especially bone, head, neck, and connective tissue malignancies.157

Disease Indications Acute Myelogenous Leukemia and Myelodysplastic Syndromes AML and MDS currently constitute the most common indication for allogeneic transplantation. Treatment decisions pertaining to postinduction therapy are centered on disease risk for relapse as determined by cytogenetic abnormalities.158 Most would agree that suitable candidates with intermediate risk and all poor-risk candidates who have a suitable donor should undergo allogeneic transplantation while in first complete remission (CR).159 Patients who are in first relapse/second remission should be offered an allogeneic transplant regardless of their original cytogenetics provided that they have a suitable donor. Although the likelihood of success with allogeneic transplantation is substantially less for this group of patients when compared to those in first CR, no other therapy is known to offer long-term cures.160 Reduced-intensity conditioning regimens have allowed for allogeneic transplantation to be offered to older adults with AML and MDS with acceptable treatment-related mortality.161 This is extremely pertinent for disease that has an average age of onset in the seventh decade of life.

Acute Lymphocytic Leukemia Conventional chemotherapy offers prolonged remission and even a cure for most children and many adults. Accordingly, transplantation is not usually undertaken until after relapse.162 First remission transplantation has been reserved for adults with high-risk disease such as patients with t(4:15) or t(9:22) cytogenetics.163 Successful results of marrow transplants in first remission for these patients range from 30% to 60%, depending on patient age and donor type. The GVL effect may be less in ALL than is observed in CML or more indolent malignancies.164 The rarity of allogeneic donor lymphocyte infusion inducing remission for relapses after allogeneic transplantation supports this observation. Patients with active leptomeningeal leukemia should have this complication treated before transplantation, because few marrow transplant preparative regimens have agents that cross the blood-brain barrier.

allogeneic transplantation. A meta-analysis of four randomized trials of allogeneic transplantation in patients with CML in chronic phase found 10-year survival estimate of more than 60%.69 Patients with favorable prognostic factors (young age, favorable donor characteristics, and newly diagnosed disease) have a demonstrated overall survival of over 70%, albeit with a significant risk for acute and chronic GVHD.165 These results need to be viewed in the context of the promising Phase II trials with imatinib demonstrating a complete hematologic response in 95% and a major cytogenetic response in 65% of patients treated in chronic phase with a median follow-up of 4 years with minimal toxicity.166 For this reason, expert consensus opinion has recommended allogeneic transplantation be limited to patients who have not achieved a prompt complete hematologic response with initiation of imatinib or have evidence of persistent cytogenetic abnormalities. There is not enough evidence to determine whether newer, more potent tyrosine inhibitors will change these recommendations.167

Multiple Myeloma Multiple myeloma currently represents the most common indication for autologous HSCT. This is based upon multiple randomized studies demonstrating higher complete remission rates, event-free survival, and overall survival in patients undergoing high-dose therapy followed by autologous HSCT when compared to chemotherapy alone. A French study168 reported a CR and 5- year survival rates of 22% and 52% in the high-dose group vs 5% and 12% in the standard-dose study arm. Randomized studies have established melphalan 200 mg/m2 as the “standard” regimen for patients under the age of 65.169 The role of double autologous transplants was investigated in a protocol of the Intergroupe Francophone du Myelome. Attal et al170 randomly assigned 399 untreated patients younger than 60 years to receive melphalan 140 mg/m2 with TBI 8 Gy (single transplant) or melphalan 140 mg/m2 followed by a second transplant using the TBI/melphalan regimen.170 Response rate was similar, but overall and eventfree survival was significantly improved in the double autologous transplant study arm. The subset of patients who did not achieve at least a partial remission with the first procedure appeared to benefit the most with this strategy. It should be noted, however, that all studies that determined the key role of autologous transplantation in this disease were conducted before the development of new “target” therapies, which may change radically the way multiple myeloma is treated in the future. To date, no therapy is felt to be curative for this disease. Recently, autologous stem cell transplant followed by reduced-intensity conditioning allogeneic transplantation in patients with a sibling donor has been explored with some randomized studies suggesting improvement in survival over tandem autologous transplant.171

Lymphoma and Chronic Lymphocytic Leukemia Chronic Myelogenous Leukemia Before the advent of tyrosine kinase inhibitors such as imatinib, CML represented the most common disease group treated with

High-dose therapy followed by autologous HSCT is considered standard treatment for patients with diffuse large-cell lymphoma and Hodgkin disease who fail to achieve remission

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following initial therapy, or subsequently relapse after an initial remission with chemo-sensitive disease. The Parma study was a randomized trial comparing standard salvage chemotherapy in first relapse for large-cell lymphoma to standard chemotherapy plus autologous marow transplant.172 This trial demonstrated a definitive advantage for patients receiving an autologous transplant. Autologous HSCT is the accepted treatment for patients with chemo-sensitive relapsed or primary refractory Hodgkin disease.173 Patients who achieve a second CR following salvage treatment have the best outcomes with this approach with over 50% of patients achieving a long-term cure. There have been no randomized studies comparing TBI-containing to chemotherapyalone conditioning regimens, but generally radiationbased regimens are avoided in those patients who have previously received radiation therapy. Recently, the incorporation of the CD20 monoclonal antibody, rituximab, into chemomobilization and conditioning regimens have been shown to improve outcomes.23 The incorporation of radiolabeled antibodies into conditioning regimens is being explored.24 Allogeneic transplantation has been explored in patients with large-cell lymphoma and Hodgkin disease who have failed to achieve a suitable response to salvage chemotherapy or who have relapsed following an autologous HSCT with variable success. Treatment-related mortality has been high with full-intensity regimens; however, reduced-intensity regimens have been better tolerated and offer hope for cure in select patients.174,175 In addition, encouraging results have been reported with reducedintensity allogeneic transplants with indolent lymphomas and CLL.176,177

Breast Cancer Breast cancer once constituted the most common indication for high-dose chemotherapy followed by autologous HSCT. More recently, a series of randomized trials failed to demonstrate an improvement in long-term progression-free or overall survival in patients with high-risk178,179 or metastatic breast cancer.180 Apart from participation in a clinical trial, this approach is no longer justified.

Future Directions Fifty years have passed since Thomas and colleagues’ first attempts at allogeneic HSCT. The field has advanced so that this therapy can now be offered to more patients with less risk for acute and long-term toxicity. Despite these advances, the challenge remains the discovery of new strategies that will improve disease-free survival without associated treatment-related mortality. Autologous and allogeneic HSCT remain curative for a minority of patients with advanced, refractory disease. Broadly applied, dose-intensification has not been able to significantly affect outcomes for this group of patients because of the overlap between toxicity and efficacy. Undoubtedly tomorrow’s success will come through the incorporation of “targeted”

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therapies that offer the hope of decreasing relapse without adding additional toxicity. In addition, new strategies for harnessing the immunotherapeutic potential of a GVL effect while reducing the risk GVHD will make allogeneic transplantation a more widely applied modality.

Disclaimer The authors have disclosed no conflicts of interest.

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64. Kolb HJ, Bender-Gotze C. Late complications after allogeneic bone marrow transplantation for leukaemia. Bone Marrow Transplant 1990;6:61-72. 65. Santos GW, Tutschka PJ, Brookmeyer R, et al. Marrow transplantation for acute nonlymphocytic leukemia after treatment with busulfan and cyclophosphamide. N Engl J Med 1983;309:1347-53. 66. Tutschka PJ, Copelan EA, Klein JP. Bone marrow transplantation for leukemia following a new busulfan and cyclophosphamide regimen. Blood 1987;70:1382-8. 67. Devergie A, Blaise D, Attal M, et al. Allogeneic bone marrow transplantation for chronic myeloid leukemia in first chronic phase: A randomized trial of busulfan-cytoxan versus cytoxan-total body irradiation as preparative regimen. A report from the French Society of Bone Marrow Graft (SFGM). Blood 1995;85:2263-8. 68. Clift RA, Radich J, Appelbaum FR, et al. Long-term follow-up of a randomized study comparing cyclophosphamide and total body irradiation with busulfan and cyclophosphamide for patients receiving allogenic marrow transplants during chronic phase of chronic myeloid leukemia. Blood 1999;94:3960-2. 69. Socie G, Clift RA, Blaise D, et al. Busulfan plus cyclophosphamide compared with total-body irradiation plus cyclophosphamide before marrow transplantation for myeloid leukemia: Long-term follow-up of 4 randomized studies. Blood 2001;98:3569-74. 70. Hartman AR, Williams SF, Dillon JJ. Survival, disease-free survival and adverse effects of conditioning for allogeneic bone marrow transplantation with busulfan/cyclophosphamide vs total body irradiation: A meta-analysis. Bone Marrow Transplant 1998;22:439-43. 71. Ringden O, Remberger M, Ruutu T, et al. Increased risk of chronic graft-versus-host disease, obstructive bronchiolitis, and alopecia with busulfan versus total body irradiation: Long-term results of a randomized trial in allogeneic marrow recipients with leukemia. Nordic Bone Marrow Transplantation Group. Blood 1999;93:2196-201. 72. Lindley C, Shea T, McCune J, et al. Intraindividual variability in busulfan pharmacokinetics in patients undergoing a bone marrow transplant: Assessment of a test dose and first dose strategy. Anticancer Drugs 2004;15:453-9. 73. Vassal G, Deroussent A, Challine D, et al. Is 600 mg/m2 the appropriate dosage of busulfan in children undergoing bone marrow transplantation? Blood 1992;79:2475-9. 74. Gibbs JP, Gooley T, Corneau B, et al. The impact of obesity and disease on busulfan oral clearance in adults. Blood 1999;93:4436-40. 75. Grochow LB, Jones RJ, Brundrett RB, et al. Pharmacokinetics of busulfan: Correlation with veno-occlusive disease in patients undergoing bone marrow transplantation. Cancer Chemother Pharmacol 1989;25:55-61. 76. Hassan M, Oberg G, Bekassy AN, et al. Pharmacokinetics of highdose busulphan in relation to age and chronopharmacology. Cancer Chemother Pharmacol 1991;28:130-4. 77. Baker KS, Bostrom B, DeFor T, et al. Busulfan pharmacokinetics do not predict relapse in acute myeloid leukemia. Bone Marrow Transplant 2000;26:607-14. 78. Slattery JT, Clift RA, Buckner CD, et al. Marrow transplantation for chronic myeloid leukemia: The influence of plasma busulfan levels on the outcome of transplantation. Blood 1997;89:3055-60. 79. McCune JS, Gooley T, Gibbs JP, et al. Busulfan concentration and graft rejection in pediatric patients undergoing hematopoietic stem cell transplantation. Bone Marrow Transplant 2002;30:167-73. 80. Slattery JT, Sanders JE, Buckner CD, et al. Graft-rejection and toxicity following bone marrow transplantation in relation to busulfan pharmacokinetics. Bone Marrow Transplant 1995;16:31-42.

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81. Bolinger AM, Zangwill AB, Slattery JT, et al. An evaluation of engraftment, toxicity and busulfan concentration in children receiving bone marrow transplantation for leukemia or genetic disease. Bone Marrow Transplant 2000;25:925-30. 82. Chandy M, Balasubramanian P, Ramachandran SV, et al. Randomized trial of two different conditioning regimens for bone marrow transplantation in thalassemia—the role of busulfan pharmacokinetics in determining outcome. Bone Marrow Transplant 2005;36:839-45. 83. Gibbs JP, Liacouras CA, Baldassano RN, Slattery JT. Up-regulation of glutathione S-transferase activity in enterocytes of young children. Drug Metab Dispos 1999;27:1466-9. 84. Bolinger AM, Zangwill AB, Slattery JT, et al. Target dose adjustment of busulfan in pediatric patients undergoing bone marrow transplantation. Bone Marrow Transplant 2001;28:1013-18. 85. Bleyzac N, Souillet G, Magron P, et al. Improved clinical outcome of paediatric bone marrow recipients using a test dose and Bayesian pharmacokinetic individualization of busulfan dosage regimens. Bone Marrow Transplant 2001;28:743-51. 86. Deeg HJ, Storer B, Slattery JT, et al. Conditioning with targeted busulfan and cyclophosphamide for hemopoietic stem cell transplantation from related and unrelated donors in patients with myelodysplastic syndrome. Blood 2002;100:1201-7. 87. Radich JP, Gooley T, Bensinger W, et al. HLA-matched related hematopoietic cell transplantation for chronic-phase CML using a targeted busulfan and cyclophosphamide preparative regimen. Blood 2003;102:31-5. 88. Andersson BS, Madden T, Tran HT, et al. Acute safety and pharmacokinetics of intravenous busulfan when used with oral busulfan and cyclophosphamide as pretransplantation conditioning therapy: A phase I study. Biol Blood Marrow Transplant 2000;6:548-54. 89. Kashyap A, Wingard J, Cagnoni P, et al. Intravenous versus oral busulfan as part of a busulfan/cyclophosphamide preparative regimen for allogeneic hematopoietic stem cell transplantation: Decreased incidence of hepatic venoocclusive disease (HVOD), HVOD-related mortality, and overall 100-day mortality. Biol Blood Marrow Transplant 2002;8:493-500. 90. Kim SE, Lee JH, Choi SJ, et al. Morbidity and non-relapse mortality after allogeneic bone marrow transplantation in adult leukemia patients conditioned with busulfan plus cyclophosphamide: A retrospective comparison of oral versus intravenous busulfan. Haematologica 2005;90:285-6. 91. Mamlouk K, Saracino G, Berryman RB, et al. Modification of the Bu/ Cy myeloablative regimen using daily parenteral busulfan: Reduced toxicity without the need for pharmacokinetic monitoring. Bone Marrow Transplant 2005;35:747-54. 92. Andersson BS, Thall PF, Madden T, et al. Busulfan systemic exposure relative to regimen-related toxicity and acute graft-versus-host disease: Defining a therapeutic window for i.v. BuCy2 in chronic myelogenous leukemia. Biol Blood Marrow Transplant 2002;8: 477-85. 93. Thall PF, Champlin RE, Andersson BS. Comparison of 100-day mortality rates associated with i.v. busulfan and cyclophosphamide vs other preparative regimens in allogeneic bone marrow transplantation for chronic myelogenous leukemia: Bayesian sensitivity analyses of confounded treatment and center effects. Bone Marrow Transplant 2004;33:1191-9. 94. McDonald GB, Slattery JT, Bouvier ME, et al. Cyclophosphamide metabolism, liver toxicity, and mortality following hematopoietic stem cell transplantation. Blood 2003;101:2043-8.

95. de Lima M, Couriel D, Thall PF, et al. Once-daily intravenous busulfan and fludarabine: Clinical and pharmacokinetic results of a myeloablative, reduced-toxicity conditioning regimen for allogeneic stem cell transplantation in AML and MDS. Blood 2004;104:857-64. 96. Bornhauser M, Storer B, Slattery JT, et al. Conditioning with fludarabine and targeted busulfan for transplantation of allogeneic hematopoietic stem cells. Blood 2003;102:820-6. 97. Giralt S, Logan B, Rizzo D, et al. Reduced-intensity conditioning for unrelated donor progenitor cell transplantation: Long-term followup of the first 285 reported to the national marrow donor program. Biol Blood Marrow Transplant 2007;13:844-52. 98. Giralt S. Reduced-intensity conditioning regimens for hematologic malignancies: What have we learned over the last 10 years? Hematology Am Soc Hematol Educ Program 2005;384-9. 99. Okamoto S, Takahashi S, Tanosaki R, et al. Granisetron in the prevention of vomiting induced by conditioning for stem cell transplantation: A prospective randomized study. Bone Marrow Transplant 1996;17:679-83. 100. Spielberger R, Stiff P, Bensinger W, et al. Palifermin for oral mucositis after intensive therapy for hematologic cancers. N Engl J Med 2004;351:2590-8. 101. Andriole GL, Sandlund JT, Miser JS, et al. The efficacy of mesna (2- mercaptoethane sodium sulfonate) as a uroprotectant in patients with hemorrhagic cystitis receiving further oxazaphosphorine chemotherapy. J Clin Oncol 1987;5:799-803. 102. Anderson KC, Weinstein HJ. Transfusion-associated graft-versushost disease. N Engl J Med 1990;323:315-21. 103. Nemunaitis J, Rosenfeld CS, Ash R, et al. Phase III randomized, double-blind placebo-controlled trial of rhGM-CSF following allogeneic bone marrow transplantation. Bone Marrow Transplant 1995;15:949-54. 104. Dekker A, Bulley S, Beyene J, et al. Meta-analysis of randomized controlled trials of prophylactic granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor after autologous and allogeneic stem cell transplantation. J Clin Oncol 2006;24:5207-15. 105. Baron F, Baker JE, Storb R, et al. Kinetics of engraftment in patients with hematologic malignancies given allogeneic hematopoietic cell transplantation after nonmyeloablative conditioning. Blood 2004;104:2254-62. 106. Davies SM, Ramsay NK, Haake RJ, et al. Comparison of engraftment in recipients of matched sibling of unrelated donor marrow allografts. Bone Marrow Transplant 1994;13:51-7. 107. Davies SM, Kollman C, Anasetti C, et al. Engraftment and survival after unrelated-donor bone marrow transplantation: A report from the national marrow donor program. Blood 2000;96:4096-102. 108. Stucki A, Leisenring W, Sandmaier BM, et al. Decreased rejection and improved survival of first and second marrow transplants for severe aplastic anemia (a 26-year retrospective analysis). Blood 1998;92:2742-9. 109. Storb R, Etzioni R, Anasetti C, et al. Cyclophosphamide combined with antithymocyte globulin in preparation for allogeneic marrow transplants in patients with aplastic anemia. Blood 1994;84:941-9. 110. Bacigalupo A, Locatelli F, Lanino E, et al. Fludarabine, cyclophosphamide and anti-thymocyte globulin for alternative donor transplants in acquired severe aplastic anemia: A report from the EBMT-SAA Working Party. Bone Marrow Transplant 2005;36:947-50.

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111. Vassiliou GS, Webb DK, Pamphilon D, et al. Improved outcome of alternative donor bone marrow transplantation in children with severe aplastic anaemia using a conditioning regimen containing low-dose total body irradiation, cyclophosphamide and Campath. Br J Haematol 2001;114:701-5. 112. Champlin RE, Horowitz MM, van Bekkum DW, et al. Graft failure following bone marrow transplantation for severe aplastic anemia: Risk factors and treatment results. Blood 1989;73:606-13. 113. Molldrem JJ, Komanduri K, Wieder E. Overexpressed differentiation antigens as targets of graft-versus-leukemia reactions. Curr Opin Hematol 2002;9:503-8. 114. Filipovich AH, Weisdorf D, Pavletic S, et al. National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: I. Diagnosis and staging working group report. Biol Blood Marrow Transplant 2005;11:945-56. 115. Storb R, Deeg HJ, Whitehead J, et al. Methotrexate and cyclosporine compared with cyclosporine alone for prophylaxis of acute graft versus host disease after marrow transplantation for leukemia. N Engl J Med 1986;314:729-35. 116. Storb R, Deeg HJ, Fisher L, et al. Cyclosporine v methotrexate for graft-v-host disease prevention in patients given marrow grafts for leukemia: Long-term follow-up of three controlled trials. Blood 1988;71:293-8. 117. Storb R, Deeg HJ, Pepe M, et al. Graft-versus-host disease prevention by methotrexate combined with cyclosporin compared to methotrexate alone in patients given marrow grafts for severe aplastic anaemia: Long-term follow-up of a controlled trial. Br J Haematol 1989;72:567-72. 118. Storb R, Deeg HJ, Pepe M, et al. Methotrexate and cyclosporine versus cyclosporine alone for prophylaxis of graft-versus-host disease in patients given HLA-identical marrow grafts for leukemia: Longterm follow-up of a controlled trial. Blood 1989;73:1729-34. 119. Ratanatharathorn V, Nash RA, Przepiorka D, et al. Phase III study comparing methotrexate and tacrolimus (prograf, FK506) with methotrexate and cyclosporine for graft-versus-host disease prophylaxis after HLA-identical sibling bone marrow transplantation. Blood 1998;92:2303-14. 120. Nash RA, Antin JH, Karanes C, et al. Phase 3 study comparing methotrexate and tacrolimus with methotrexate and cyclosporine for prophylaxis of acute graft-versus-host disease after marrow transplantation from unrelated donors. Blood 2000;96:2062-8. 121. Przepiorka D, Weisdorf D, Martin P, et al. 1994 Consensus Conference on Acute GVHD Grading. Bone Marrow Transplant 1995;15:825-8. 122. Martin PJ, Schoch G, Fisher L, et al. A retrospective analysis of therapy for acute graft-versus-host disease: Secondary treatment. Blood 1991;77:1821-8. 123. MacMillan ML, Weisdorf DJ, Wagner JE, et al. Response of 443 patients to steroids as primary therapy for acute graft-versushost disease: Comparison of grading systems. Biol Blood Marrow Transplant 2002;8:387-94. 124. Cragg L, Blazar BR, Defor T, et al. A randomized trial comparing prednisone with antithymocyte globulin/prednisone as an initial systemic therapy for moderately severe acute graft-versus-host disease. Biol Blood Marrow Transplant 2000;6:441-7. 125. Van Lint MT, Uderzo C, Locasciulli A, et al. Early treatment of acute graft-versus-host disease with high- or low-dose 6-methylprednisolone: A multicenter randomized trial from the Italian Group for Bone Marrow Transplantation. Blood 1998;92:2288-93.

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126. Weisdorf D, Haake R, Blazar B, et al. Treatment of moderate/severe acute graft-versus-host disease after allogeneic bone marrow transplantation: An analysis of clinical risk features and outcome. Blood 1990;75:1024-30. 127. Storb R, Pepe M, Anasetti C, et al. What role for prednisone in prevention of acute graft-versus-host disease in patients undergoing marrow transplants? Blood 1990;76:1037-45. 128. Rossi SJ, Schroeder TJ, Hariharan S, First MR. Prevention and management of the adverse effects associated with immunosuppressive therapy. Drug Saf 1993;9:104-31. 129. Przepiorka D, Shapiro S, Schwinghammer TL, et al. Cyclosporine and methylprednisolone after allogeneic marrow transplantation: Association between low cyclosporine concentration and risk of acute graft-versus-host disease. Bone Marrow Transplant 1991;7:461-5. 130. Przepiorka D, Ippoliti C, Khouri I, et al. Tacrolimus and minidose methotrexate for prevention of acute graft-versus-host disease after matched unrelated donor marrow transplantation. Blood 1996;88:4383-9. 131. Storb R, Leisenring W, Anasetti C, et al. Long-term follow-up of allogeneic marrow transplants in patients with aplastic anemia conditioned by cyclophosphamide combined with antithymocyte globulin. Blood 1997;89:3890-1. 132. Champlin RE, Passweg JR, Zhang MJ, et al. T-cell depletion of bone marrow transplants for leukemia from donors other than HLAidentical siblings: Advantage of T-cell antibodies with narrow specificities. Blood 2000;95:3996-4003. 133. Lee SJ, Kim HT, Ho VT, et al. Quality of life associated with acute and chronic graft-versus-host disease. Bone Marrow Transplant 2006;38:305-10. 134. Lee SJ, Vogelsang G, Flowers ME. Chronic graft-versus-host disease. Biol Blood Marrow Transplant 2003;9:215-33. 135. Carreras E, Bertz H, Arcese W, et al. Incidence and outcome of hepatic veno-occlusive disease after blood or marrow transplantation: A prospective cohort study of the European Group for Blood and Marrow Transplantation. European Group for Blood and Marrow Transplantation Chronic Leukemia Working Party. Blood 1998;92:3599-604. 136. McDonald GB, Hinds MS, Fisher LD, et al. Veno-occlusive disease of the liver and multiorgan failure after bone marrow transplantation: A cohort study of 355 patients. Ann Intern Med 1993;118:255-67. 137. Richardson PG, Murakami C, Jin Z, et al. Multi-institutional use of defibrotide in 88 patients after stem cell transplantation with severe veno-occlusive disease and multisystem organ failure: Response without significant toxicity in a high-risk population and factors predictive of outcome. Blood 2002;100:4337-43. 138. Jules-Elysee K, Stover DE, Yahalom J, et al. Pulmonary complications in lymphoma patients treated with high-dose therapy autologous bone marrow transplantation. Am Rev Respir Dis 1992;146:485-91. 139. Hildebrandt GC, Olkiewicz KM, Corrion LA, et al. Donor-derived TNF-alpha regulates pulmonary chemokine expression and the development of idiopathic pneumonia syndrome after allogeneic bone marrow transplantation. Blood 2004;104:586-93. 140. Fukuda T, Hackman RC, Guthrie KA, et al. Risks and outcomes of idiopathic pneumonia syndrome after nonmyeloablative and conventional conditioning regimens for allogeneic hematopoietic stem cell transplantation. Blood 2003;102:2777-85.

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141. Lew MA, Kehoe K, Ritz J, et al. Ciprofloxacin versus trimethoprim/ sulfamethoxazole for prophylaxis of bacterial infections in bone marrow transplant recipients: A randomized, controlled trial. J Clin Oncol 1995;13:239-50. 142. Cruciani M, Rampazzo R, Malena M, et al. Prophylaxis with fluoroquinolones for bacterial infections in neutropenic patients: A metaanalysis. Clin Infect Dis 1996;23:795-805. 143. Guidelines for preventing opportunistic infections among hematopoietic stem cell transplant recipients. MMWR Recomm Rep 2000;49(RR-10):1-7. 144. Goodman JL, Winston DJ, Greenfield RA, et al. A controlled trial of fluconazole to prevent fungal infections in patients undergoing bone marrow transplantation. N Engl J Med 1992;326:845-51. 145. Ullmann AJ, Lipton JH, Vesole DH, et al. Posaconazole or fluconazole for prophylaxis in severe graft-versus-host disease. N Engl J Med 2007;356:335-47. 146. van Burik JA, Ratanatharathorn V, Stepan DE, et al. Micafungin versus fluconazole for prophylaxis against invasive fungal infections during neutropenia in patients undergoing hematopoietic stem cell transplantation. Clin Infect Dis 2004;39:1407-16. 147. Gluckman E, Lotsberg J, Devergie A, et al. Prophylaxis of herpes infections after bone-marrow transplantation by oral acyclovir. Lancet 1983;ii:706-8. 148. Iino T, Gondo H, Ohno Y, et al. Successful foscarnet therapy for mucocutaneous infection with herpes simplex virus in a recipient after unrelated bone marrow transplantation. Bone Marrow Transplant 1996;18:1185-8. 149. Vasconcelles MJ, Bernardo MV, King C, et al. Aerosolized pentamidine as pneumocystis prophylaxis after bone marrow transplantation is inferior to other regimens and is associated with decreased survival and an increased risk of other infections. Biol Blood Marrow Transplant 2000;6:35-43. 150. Winston DJ, Ho WG, Bartoni K, et al. Ganciclovir prophylaxis of cytomegalovirus infection and disease in allogeneic bone marrow transplant recipients. Results of a placebo-controlled, double-blind trial. Ann Intern Med 1993;118:179-84. 151. Boeckh M, Gooley TA, Myerson D, et al. Cytomegalovirus pp65 antigenemia-guided early treatment with ganciclovir versus ganciclovir at engraftment after allogeneic marrow transplantation: A randomized double-blind study. Blood 1996;88:4063-71. 152. Boeckh M, Gallez-Hawkins GM, Myerson D, et al. Plasma polymerase chain reaction for cytomegalovirus DNA after allogeneic marrow transplantation: Comparison with polymerase chain reaction using peripheral blood leukocytes, pp65 antigenemia, and viral culture. Transplantation 1997;64:108-13. 153. Bowden RA, Sayers M, Flournoy N, et al. Cytomegalovirus immune globulin and seronegative blood products to prevent primary cytomegalovirus infection after marrow transplantation. N Engl J Med 1986;314:1006-10. 154. Kempen-Harteveld ML, Struikmans H, Kal HB, et al. Cataract after total body irradiation and bone marrow transplantation: Degree of visual impairment. Int J Radiat Oncol Biol Phys 2002;52:1375-80. 155. Kauppila M, Koskinen P, Irjala K, et al. Long-term effects of allogeneic bone marrow transplantation (BMT) on pituitary, gonad, thyroid and adrenal function in adults. Bone Marrow Transplant 1998;22:331-7. 156. Stone RM, Neuberg D, Soiffer R, et al. Myelodysplastic syndrome as a late complication following autologous bone marrow transplantation for non-Hodgkin’s lymphoma. J Clin Oncol 1994;12:2535-42.

157. Curtis RE, Rowlings PA, Deeg HJ, et al. Solid cancers after bone marrow transplantation. N Engl J Med 1997;336:897-904. 158. Keating MJ, Smith TL, Kantarjian H, et al. Cytogenetic pattern in acute myelogenous leukemia: A major reproducible determinant of outcome. Leukemia 1988;2:403-12. 159. Estey E, de Lima M, Tibes R, et al. Prospective feasibility analysis of reduced-intensity conditioning (RIC) regimens for hematopoietic stem cell transplantation (HSCT) in elderly patients with acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (MDS). Blood 2007;109:1395-400. 160. Wong R, Shahjahan M, Wang X, et al. Prognostic factors for outcomes of patients with refractory or relapsed acute myelogenous leukemia or myelodysplastic syndromes undergoing allogeneic progenitor cell transplantation. Biol Blood Marrow Transplant 2005;11:108-14. 161. Martino R, Iacobelli S, Brand R, et al. Retrospective comparison of reduced-intensity conditioning and conventional high-dose conditioning for allogeneic hematopoietic stem cell transplantation using HLA-identical sibling donors in myelodysplastic syndromes. Blood 2006;108:836-46. 162. Tavernier E, Boiron JM, Huguet F, et al. Outcome of treatment after first relapse in adults with acute lymphoblastic leukemia initially treated by the LALA-94 trial. Leukemia 2007;21:1907-14. 163. Yanada M, Matsuo K, Suzuki T, Naoe T. Allogeneic hematopoietic stem cell transplantation as part of postremission therapy improves survival for adult patients with high-risk acute lymphoblastic leukemia: A metaanalysis. Cancer 2006;106:2657-63. 164. Collins RH Jr., Goldstein S, Giralt S, et al. Donor leukocyte infusions in acute lymphocytic leukemia. Bone Marrow Transplant 2000;26:511-16. 165. Crawley C, Szydlo R, Lalancette M, et al. Outcomes of reducedintensity transplantation for chronic myeloid leukemia: an analysis of prognostic factors from the Chronic Leukemia Working Party of the EBMT. Blood 2005;106:2969-76. 166. Kantarjian H, Sawyers C, Hochhaus A, et al. Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia. N Engl J Med 2002;346:645-52. 167. Baccarani M, Saglio G, Goldman J, et al. Evolving concepts in the management of chronic myeloid leukemia: recommendations from an expert panel on behalf of the European LeukemiaNet. Blood 2006;108:1809-20. 168. Attal M, Harousseau JL, Stoppa AM, et al. A prospective, randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma. Intergroupe Francais du Myelome. N Engl J Med 1996;335:91-7. 169. Moreau P, Facon T, Attal M, et al. Comparison of 200 mg/m2 melphalan and 8 Gy total body irradiation plus 140 mg/m2 melphalan as conditioning regimens for peripheral blood stem cell transplantation in patients with newly diagnosed multiple myeloma: Final analysis of the Intergroupe Francophone du Myelome 9502 randomized trial. Blood 2002;99:731-5. 170. Attal M, Harousseau JL, Facon T, et al. Single versus double autologous stem-cell transplantation for multiple myeloma. N Engl J Med 2003;349:2495-502. 171. Bruno B, Rotta M, Patriarca F, et al. A comparison of allografting with autografting for newly diagnosed myeloma. N Engl J Med 2007;356:1110-20. 172. Philip T, Guglielmi C, Hagenbeek A, et al. Autologous bone marrow transplantation as compared with salvage chemotherapy in relapses

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36

Umbilical Cord Blood: A Reliable Source of Stem and Progenitor Cells for Human Transplantation Suhag H. Parikh1 & Joanne Kurtzberg2 1

Assistant Professor, Pediatric Stem Cell Transplant Program, Duke University Medical Center, Durham, North Carolina, USA 2 Chief, Pediatric Blood and Marrow Transplant Program, Duke University Medical Center, Durham, North Carolina, USA

The first successful allogeneic hematopoietic stem cell transplantation was performed in 1968 by Dr. Robert Good. The patient, who had severe combined immunodeficiency (SCID), received marrow from a sibling as the source of stem cells. Since then, thousands of patients have been cured of life-threatening hematologic disorders—malignant and nonmalignant—establishing the field of marrow transplantation. Chapter 35 provides an overview of allogeneic transplantation with an emphasis on the traditional sources of hematopoietic stem cells—namely, marrow and peripheral blood. Over the years, the National Marrow Donor Program (NMDP) in the United States and registries in other parts of the world have recruited, typed, and maintained databases on millions of voluntary unrelated adult donors. Despite the large number of registered donors, approximately two-thirds of the patients in need of a transplant are still unable to find a suitable marrow donor in a timely manner. This problem is even more of a concern for patients belonging to ethnic minorities. The need to breach the HLA barrier—to be able to perform transplants with partially HLA-mismatched donors—was realized by early investigators. However, the outcomes of partially mismatched hematopoietic stem cell transplants from marrow or mobilized peripheral blood, with or without T-cell depletion, have been suboptimal. This is mainly because of a higher incidence of graft failure, severe graft-vs-host-disease (GVHD), and poor immune reconstitution, leading to increased infectionrelated mortality. There has been a desperate need for stem cell sources that could result in adequate engraftment with acceptable rates of GVHD in the partially mismatched transplant setting. The first part of this chapter focuses on the use of umbilical cord blood (UCB) as one such alternative source of hematopoietic stem and progenitor cells and describes the rapid emergence of its role in clinical hematopoietic transplantation. Recent research suggests that UCB applications may well extend beyond

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

the realm of hematopoietic reconstitution, with tremendous potential for the field of regenerative medicine. The latter part of the chapter provides an overview of the cord blood banking system.

Cord Blood: Biology and Transplantation The idea of using cord blood for hematopoietic transplantation arose from interactions among a group of physicians and scientists in the early 1980s. Using blood from near-term mouse donors, Dr. Ted Boyse at Memorial Sloan Kettering Cancer Center demonstrated reconstitution of hematopoiesis in lethally irradiated mice. In parallel, Dr. Hal Broxmeyer in New York and later in Indiana, performed early experiments characterizing the hematopoietic stem and progenitor cells in human UCB. He demonstrated that cord blood was a rich source of these cells, that they had higher proliferative capacity, and also that they could be readily cryopreserved.1 Hematopoietic stem and progenitor cells derived from cord blood are biologically different from those derived from marrow or mobilized peripheral blood, as evidenced by a large amount of data that has emerged from several groups studying stem cell biology. In-vitro studies of the sources show both quantitative differences, ie, a higher frequency of more primitive hematopoietic progenitor cells in UCB than in marrow, and functional differences, ie, a higher replating potential of UCB-derived colonies on semisolid cultures than of those derived from marrow. In-vivo studies show similar differences. Nonobese diabetic (NOD)/SCID repopulating cell assays indirectly measure the self-renewal and proliferative capacities of stem cells. Experiments have shown that the SCID-repopulating cell (SRC) frequency in UCB is threefold higher than in marrow and sixfold higher than in mobilized peripheral blood, suggesting that UCB contains a higher proportion of primitive hematopoietic stem and progenitor cells.2 It is important to distinguish the hematopoietic stem and progenitor cells derived from cord blood and/or marrow from “embryonic stem cells.” Embryonic stem cells are pluripotent or

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totipotent cells (cells capable of giving rise to all three basic germ layers—ectoderm, endoderm, and mesoderm) derived from the inner cell mass of the early embryo. With continued natural development of the embryo, the embryonic stem cells become more and more lineage committed, thus giving rise to cells of mostly one germ layer, but sometimes two. Such development results in a hierarchy of more restricted stem cells. Terms such as multipotent, tissue restricted, adult, or somatic stem cells are applied to these more committed stem cells. For example, liver stem cells give rise to hepatocytes; muscle stem cells give rise to muscle; and the hematopoietic stem or progenitor cells give rise to all three elements of blood—erythrocytes, leukocytes, and platelets. Marrow, as well as UCB, is rich in these more lineagerestricted multipotent hematopoietic cells, with some differences as alluded to above. Pluripotent embryonic stem cells, at the current level of development, are not yet in clinical use. One reason is the development of teratomas in animal models. Clearly, these cells are highly proliferative and further studies are needed before they are ready for clinical application. Some investigators have raised concerns regarding epigenetic developmental abnormalities. Human egg harvesting is an invasive procedure. Not only are human eggs difficult to obtain, but ethical issues and religious beliefs make this an extremely controversial topic, because it involves the loss of human embryos. Therefore, despite their enormous potential, clinical therapeutic use of embryonic stem cells is unlikely to become a reality in the near future. Conversely, marrow has been successfully used for hematopoietic transplantation for more than three decades. Furthermore, mobilized peripheral blood has been used, and now cord blood is increasingly being used as a cell source for transplantation. Taken together, these studies imply two important points: 1) that cord blood is a rich source of hematopoietic stem cells and 2) that there is a preponderance of more primitive types of stem cells in cord blood as compared to marrow or peripheral blood.

First Umbilical Cord Blood Transplantation: Proof of Principle In 1988, the use of cord blood as a source of hematopoietic stem cells was reported following an elaborate international, multiinstitutional collaboration, when a 6-year-old boy from the United States with Fanconi anemia underwent successful transplantation in Paris.3 The source of the donor cells was HLAidentical UCB from his unaffected sibling. Twenty years after the procedure, he is durably engrafted, immunologically competent, healthy, and cured of his hematologic condition. This outcome proved that cord blood harvested from a single donor contained a sufficient dose of stem cells for successful reconstitution of a pediatric patient’s aplastic marrow.

Matched Sibling Donor UCB Transplantation This success led to an increasing number of matched related cord blood transplant procedures in the following years (⬃60 transplants over the next 5 years). Apart from demonstrating

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acceptable rates of engraftment, it became clear from this early experience that among matched related donor transplants, cord blood caused less GVHD as compared to marrow, and that this property of cord blood might allow transplantation across genetic/HLA barriers. In 1992, the first public unrelated cord blood bank was established at the New York Blood Center by Dr. Pablo Rubinstein to explore the use of banked UCB from unrelated donors for hematopoietic stem cell transplantation.4

Unrelated-Donor UCB Transplantation In 1993, the first unrelated UCB transplantation was performed in a 3-year-old child with recurrent T-cell acute lymphoblastic leukemia. The outcomes of 25 successive transplants with unrelated UCB banked at the New York Blood Center, and transplanted at Duke University, were reported in 1996.5 Important observations in these patients and subsequent reports from other centers and registries including the New York Blood Center and the European Cord Blood Registry (Eurocord)6-8 demonstrated the following: 1. Unrelated cord blood could engraft in the marrow of children undergoing myeloablative therapy for leukemias and genetic diseases. 2. Reasonable outcomes could be achieved using partially HLA mismatched grafts. 3. The incidence and severity of acute and chronic GVHD was lower and milder than that seen with matched unrelated marrow transplants. 4. There was preservation of graft-vs-leukemia effects.9 5. Cell dose strongly correlated with clinical outcomes including time to engraftment and probability of overall engraftment and survival. 6. Engraftment times were observed to be slower than those of marrow or mobilized peripheral blood.

Prospective Study: COBLT Cord blood banking efforts in the United States were stimulated by the National Heart, Lung, and Blood Institute, which funded a prospective multicenter clinical trial of unrelated cord blood transplantation—the Cord Blood Transplantation Study (COBLT)—from 1997 to 2004. Three unrelated cord blood banks were established with funding provided through this study and all three banks established and followed common quality standards and standard operating procedures (SOPs). These SOPs addressed donor recruiting and screening. For screening, donors were evaluated for events in their medical history that would exclude them as donors (eg, multiple pregnancy, prematurity, placental deformity, self or a sibling diagnosed with cancer, prior receipt of a transplant, or demonstration of high risk behaviors likely to increase the chance of infection with blood-borne infectious diseases). SOPs were also established and validated for obtaining donor consent; obtaining medical histories; obtaining blood samples from maternal donors; cord blood collecting, processing, testing, cryopreservation, and storage; as well as searching for and releasing cord blood units for transplantation.10

Chapter 36: Umbilical Cord Blood

Twenty-six transplant centers participated in this prospective clinical trial designed to examine the safety and efficacy of unrelated cord blood transplantation in infants, children, and adults with malignancies; children with congenital immunodeficiency disorders; and children with inborn errors of metabolism. The study participants employed common preparative regimens, prophylaxis against GVHD, and supportive care measures. Results in children with malignant and nonmalignant conditions were favorable, with 55% survival in children with malignancies and 78% survival in children with nonmalignant conditions. Results in a very high-risk group of adults were inferior to those seen in children and in individuals receiving marrow from an unrelated donor. Subsequent studies in adults, reported by single centers or registries, revealed more encouraging results.11,12 The cumulative incidence of engraftment by Day 42 after transplantation was approximately 80% in all study strata including adults and children as well as children with malignant diseases, inborn errors of metabolism, and immunodeficiency syndromes. Factors adversely affecting engraftment or survival included lower cell doses, pretransplant cytomegalovirus seropositivity in the recipient, non-European ancestry, and greater HLA mismatching.

Retrospective Studies Over the nearly 15 years since the first unrelated-donor cord blood transplantation, more than 10,000 such procedures have been performed at more than 150 centers scattered throughout the world. Retrospective analyses of several of these patients have led to similar conclusions as the early studies. A minimum cell dose of 3 to 3.5 ⫻ 107 nucleated cells/kg has been defined as a prerequisite for optimal clinical outcomes.13 Efficacy of unrelated cord blood transplantation has been shown for children and adults with leukemias9,11 and children with a variety of nonmalignant disorders including hemoglobinopathies,14 immunodeficiencies,15,16 and inborn errors of metabolism.17 A majority of transplants have been mismatched at one or two HLA loci. Yet, a recent retrospective comparative analysis from the Center for International Blood and Marrow Transplant Research (CIBMTR) of unrelated donor graft sources—cord blood vs marrow—showed a comparable 5-year leukemia-free survival in children with acute leukemia who received allele-matched marrow vs one- or two-antigen mismatched cord blood transplants. The analysis suggests that 6/6 matched cord blood may yield superior survival, even though the comparison fell short of statistical significance.18 Unfortunately, there have not been any prospective randomized clinical trials comparing unrelated cord blood and marrow to date.

Inherited Metabolic Disorders and Unrelated UCB Transplantation Cord blood transplantation has been particularly effective in the treatment of young infants and children with certain inborn errors of metabolism, eg, mucopolysaccharidoses such as Hurler syndrome and leukodystrophies such as Krabbe disease.17,19,20 These diseases are examples of lysosomal storage

disorders, which result from single gene defects leading to specific enzyme deficiencies. These deficiencies cause defective lysosomal breakdown and accumulation of a toxic substrate, which in turn leads to progressive involvement of multiple organs, and eventually premature death. In these patients, durably engrafted cord blood cells of donor origin provide the missing or defective enzyme. Most patients with untreated Hurler syndrome usually die between 5 to 10 years of age from progressive cardiac and pulmonary involvement. They also suffer from severe bony abnormalities, corneal clouding, massive hepatosplenomegaly, and severe neuroregression. Engrafted cord blood cells have the ability to cross the blood-brain barrier and have been shown to effectively prevent neurodevelopmental progression, a beneficial outcome that is not expected with the alternative approach of recombinant enzyme replacement therapy.21 Unrelated cord blood transplantation, when performed before the age of 2 to 3 years, leads to correction of cardiac, pulmonary, liver, and neurologic damage and improves survival.19 In fact, a recent risk factor analysis of 146 patients from the European Group for Blood and Marrow Transplantation (EBMT) suggests that cord blood could be the preferred stem cell source in this disease.22 Similarly, in patients with leukodystrophies, cord blood transplantation in the presymptomatic phase of the disease can prevent demyelination in the central and, often, the peripheral nervous systems.20

Advantages and Disadvantages of Cord Blood Transplantation Cord blood transplantation has several advantages over marrow or peripheral blood stem cell transplantation, the most significant of which is that cord blood transplantation can be performed successfully from unrelated donor with one or two HLA mismatches (Table 36-1). In the setting of marrow or Table 36-1. Advantages and Disadvantages of Cord Blood Advantages ● Increased immune tolerance – Less restrictive HLA barriers increase access to transplantation therapy for patients lacking fully matched donors ● Decreased incidence and severity of GVHD ● Increased safety – Less risk of viral contamination – Easier access – Ready availability ● Minimal donor attrition ● Easier recruitment of minority donors ● Noncontroversial source of stem cells Barriers to Success ● Slower engraftment times – Possibly related to inadequate cell content in some single units, particularly for larger patients ● Cell dose limitations ● One-time usage ● Potential for transmission of genetic diseases ● Expensive infrastructure for banking

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peripheral blood transplantation, however, this same degree of HLA mismatching causes unacceptable rates of acute and chronic GVHD and is prohibitive. With the current level of cord blood inventories and allowing for up to two antigen mismatches, a cord blood donor can be found for ⬎90% of patients. There are other practical advantages to the use of allogeneic cord blood, such as increased certainty and promptness (⬍2 weeks vs 2 months) of obtaining the donor unit, less donor attrition, decreased risk of infections, absence of risk to mother or infant, and a higher likelihood of recruiting donors belonging to ethnic minorities. Last but not the least, cord blood may have more plasticity as suggested by the experience from transplantation in inherited metabolic disorders.19,22 There are also significant limitations dictated mainly by the kinetics of cord blood engraftment. The majority of single cord blood units may not have enough of a cell dose to be of benefit for adult patients. Delayed neutrophil and platelet engraftment often leads to more prolonged inpatient stay and higher acuity of supportive care, thus significantly increasing the cost of the transplant. Recent approaches combining two UCB units for a single transplant procedure have resulted in increased rates of engraftment in adults.23 There is also a limited ability to perform posttransplant donor-derived cellular therapy such as donor lymphocyte infusions.

Cord Blood Banking The advances in clinical cord blood transplantation would not be possible without parallel evolution of a formal system of cord blood banking throughout the world. This involves collection of

(A)

Cord Blood Collection Umbilical cord blood is the portion of the infant’s blood contained in the placenta, which typically would be discarded at birth. As discussed, cord blood is a rich source of multipotent hematopoietic stem and progenitor cells. Properly performed, cord blood collection can be accomplished without physical risk to the mother or infant donor, from the delivered placenta (ex utero) or during the third stage of labor (in utero). Many public cord blood banks employ dedicated staff to perform ex-utero collections away from the delivery room so that the privacy of the family is preserved and clinicians are not distracted from their usual practices [Fig 36-1 (A)]. Alternatively, obstetricians or midwives perform in-utero collections while waiting for the placenta to deliver. In either case, after sterile preparation, the umbilical vein is punctured with a 17-gauge needle attached to a sterile, closed-system collection bag containing citratephosphate-dextrose anticoagulant which is positioned lower than the placenta [Fig 36-1 (B)]. Blood flows from the placenta, through the cord into the bag over approximately 5 to 10 minutes [Fig 36-1 (C)]. Experienced collectors harvest an average of 110 mL from a single placenta. The cord blood unit is labeled and subsequently shipped to the cord blood bank for processing, testing, cryopreservation, and storage.24 There are two types of cord blood banks—public and private. Unrelated transplant programs employ public banks as their source of donor UCB units. These units are donated on a voluntary basis by women delivering healthy infants at term. Private banks, which are for-profit entities, store “directed donations”

(B)

(C)

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the cord blood and subsequent processing and cryopreservation until it can be used clinically.

Figure 36-1. Ex-utero cord blood collection. The placenta is delivered by usual obstetric techniques. After cutting and clamping the cord, operating room personnel place the placenta in a bucket and transport it to the collection room. There it is placed in a collection stand (A) on a clean chuck pad. The fetal side is placed down and the umbilical cord is pulled through an opening in the tray. The cord is tethered, cleansed with betadine and alcohol, and punctured with a 17-gauge needle attached to a collection bag (B). Blood drains by gravity over 5 to 10 minutes into the attached bag, which contains 25 mL of citratephosphate-dextrose anticoagulant (C). After the blood collection is complete, the needle is removed from the cord, the tubing is sealed, and the cord blood unit is transported to the processing laboratory for volume reduction, testing, cryopreservation, and long-term storage.

Chapter 36: Umbilical Cord Blood

collected by obstetricians from infants born into families who intend to use the cord blood for the infant from whom it came (autologous donation) or for another family member in need of future transplantation therapy. Cord blood collection in public cord blood banks today requires obtaining maternal consent. Guidelines for several aspects of cord blood banking are provided by the Institute of Medicine (IOM).25 By consenting, the infant’s mother agrees to the following: 1. The donation of her infant’s cord blood is voluntary. 2. She gives permission for her blood and the cord blood to be tested for blood-borne pathogens, eg, human immunodeficiency virus, hepatitis B and C viruses, syphilis, human T-cell lymphotropic virus, and West Nile virus, and agrees to give a detailed family medical history. 3. The cord blood is not being stored for personal use by the infant or other relatives and, instead, it will be listed on a public registry of unrelated donors and made available to patients in need of donors for unrelated transplantation. 4. She may be contacted in the future by the bank to obtain follow-up information on the infant’s health. 5. Measures will be used to protect her confidentiality and that of her infant. Unlike adult donor registries, the identities of cord blood donors are not revealed to the recipients and a recipient cannot contact the donor in the future. Most banks maintain a confidential link between the unit information (stored as a bar-code label) and the mother’s demographic information with the aim of being able to contact the family in the future, should the unit test positive for a new disease not anticipated by the family. Timing of maternal consent can vary. The vast majority of cord blood banks believe that consent should be obtained from the mother before collection of the cord blood, preferably in the late third trimester and before onset of labor. Despite efforts to recruit women during the third trimester of pregnancy and obtain their consent well before the onset of labor, many pregnant women in labor come to the hospital interested in cord blood donation but without having given prior consent. Some centers have addressed this situation by asking pregnant women in early labor to sign a short or “mini’ consent form allowing only the collection of the cord blood and maternal samples. Then after she recovers from the delivery someone meets with her to educate her and obtain full consent allowing for processing, testing, cryopreservation, and storage.

Cord Blood Storage As commercial, private cord blood banking has proliferated, its ethical justification has been widely debated.26,27 Generally, an initial storage fee of $1000-1500 is charged, followed by a yearly storage fee of approximately $100. While there are a few clear indications for this practice (eg, a sibling with cancer, a family history of hemoglobinopathy, marrow failure, congenital immunodeficiency syndrome, or inborn error of metabolism), the vast majority of families who store cord blood with private banks

pay to have access to stem cells in the future for use in treating degenerative diseases or problems related to injuries or aging. Currently, there is no evidence that this future use will be feasible or efficacious in such circumstances. Many private banks aggressively advertise their services. References in such advertising to rare and not-yet-tested applications for cord blood transplantation address the concerns of new parents wanting to provide every possible advantage for their newborn child.28 In addition to messages that may be exploitative, marketing may be inaccurate or misleading. One common reason offered by some private banks for storing autologous cord blood is to have a source of stem cells for transplantation if the child were to develop leukemia. However, most children with childhood leukemias can be cured with conventional chemotherapy alone and in those who do not respond to this approach, allogeneic transplantation is the treatment of choice. Furthermore, leukemic cells have been found in autologous cord blood of infants who later present with leukemia from 1 to 11 years of age. With increasing use of cord blood transplantation, public banks face other challenges. Procuring and providing units for public use in unrelated allogeneic transplantation has involved funding from third party sources for the creation of the inventory. Currently, there are approximately 14 public cord blood banks in the United States and approximately 30 more worldwide. All of these banks struggle financially because the revenues gained from sale of UCB units for transplantation are not sufficient to support the basic operations of a bank that is in the process of building inventory. There is also no requirement for public cord blood banks to list their inventories on a single registry available to all transplant centers. Thus, transplant centers must be knowledgeable about and search multiple banks and registries to find the best donor for each patient. In 2004, after appropriation of $20,000,000 by the US Congress to increase the inventory of cord blood units in US public banks, the Health Resources Services Administration (HRSA) asked the IOM to perform a study to determine the best way to organize public cord blood banking and distribution to patients undergoing unrelated transplantation, the results of which were published in April 2005.25 In brief, the IOM recommended that HRSA contract with eligible banks to procure approximately 150,000 new, ethnically diverse, unrelated donor cord blood units over the next 5 years. The units will need to meet quality standards as defined by an advisory board, the Food and Drug Administration, and other accrediting agencies. They will also have to be listed on a computerized, Web-based system created to allow searching of all unrelated cord blood and adult donors from a single point of access (SPA). Since the publication of the IOM study, the US Congress passed legislation that appropriated funds to establish a National Cord Blood Program. The WC “Bill” Young Stem Cell Transplantation Act, is federally funded and administered through HRSA. Selected cord blood banks have been chosen to participate in the National Cord Blood Inventory, which is

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tasked with building a high-quality, ethnically diverse inventory of 150,000 new UCB units to be listed on a combined registry for adult and cord blood donors. Under this new program, patient advocacy must be provided and transplant outcomes collected and evaluated. There is also provision for a SPA for patients and doctors searching for a suitable stem cell HLA match—be it from marrow, peripheral blood, or cord blood. The legislation also instituted mandatory reporting of transplant outcomes by all transplant centers in the United States to the SCTOD (Stem Cell Transplant Outcomes Database), a contract awarded to the CIBMTR. HRSA administers contracts for the banks directly, whereas the other programs (adult stem cell and cord blood coordinating centers, SPA, and SCTOD) were awarded to, and are all administered through, the NMDP.

Future Directions Umbilical cord blood from related and unrelated donors, matched or mismatched at one or two antigens, is now widely regarded as an alternate donor source to matched marrow or peripheral blood for allogeneic transplantation in children as well as adults for a variety of malignant and nonmalignant disorders. This use of cord blood dramatically increases the accessibility of hematopoietic stem cell transplantation. Retrospective analysis of large patient datasets does not show a survival difference and prospective randomized clinical trials comparing these graft sources are needed. Despite its favorable impact on the field of hematopoietic transplantation, time to engraftment remains a weakness and strategies to improve engraftment rates and speed of engraftment are needed. Several new approaches are being investigated, including combining two cord blood units23 and combining a cord blood unit with cells from a haploidentical donor.29 Investigations are also ongoing to study reducedintensity conditioning regimens in order to decrease the toxicity associated with transplantation. A federal mandate has provided an impetus for the cord blood banking community to increase the cord blood inventories throughout the country and to streamline efforts into creation of a national cord blood banking program. Increasing inventories, especially for minorities, will result in availability of better matched cord blood units and improved outcomes in this segment of the population. These developments in cord blood banking are thus expected to complement the growth of clinical allogeneic cord blood transplantation. There has been increasing new evidence in recent years suggesting presence of pluripotent cells in cord blood that hold vast potential in treatment of degenerative disorders involving several organs other than hematopoietic system, such as liver, brain, heart, eyes, endocrine organs, and bones.30

Disclaimer The authors have disclosed no conflicts of interest.

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References 1. Broxmeyer HE, Douglas GW, Hangoc G, et al. Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci U S A 1989;86:3828-32. 2. Mayani H, Lansdorp PM. Biology of human umbilical cord blood-derived hematopoietic stem/progenitor cells. Stem Cells 1998;16:153-65. 3. Gluckman E, Broxmeyer H, Auerbach A, et al. Hematopoietic reconstitution in a patient with Fanconi’s anemia by means of umbilical-cord blood from an HLA-identical sibling. N Engl J Med 1989;321:1174-8. 4. Rubinstein P, Dobrila L, Rosenfield RE, et al. Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution. Proc Natl Acad Sci U S A 1995;92:10119-22. 5. Kurtzberg J, Laughlin M, Graham ML, et al. Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. N Engl J Med 1996;335:157-66. 6. Wagner J, Rosenthal J, Sweetman R, et al. Successful transplantation of HLA-matched and HLA-mismatched umbilical cord blood from unrelated donors: Analysis of engraftment and acute graft-versushost disease. Blood 1996;88:795-802. 7. Rubinstein P, Carrier C, Scaradavou A, et al. Outcomes among 562 recipients of placental-blood transplants from unrelated donors. N Engl J Med 1998;339:1565-77. 8. Gluckman E, Rocha V, Boyer-Chammard A, et al. Outcome of cordblood transplantation from related and unrelated donors. N Engl J Med 1997;337:373-81. 9. Rocha V, Cornish J, Sievers EL, et al. Comparison of outcomes of unrelated bone marrow and umbilical cord blood transplants in children with acute leukemia. Blood 2001;97:2962-71. 10. Fraser J, Cairo M, Wagner E, et al. Cord Blood Transplantation Study (COBLT): Cord blood bank standard operating procedures. J Hematother 1998;7:521-61. 11. Laughlin MJ, Barker J, Bambach B, et al. Hematopoietic engraftment and survival in adult recipients of umbilical-cord blood from unrelated donors. N Engl J Med 2001;344:1815-22. 12. Rocha V, Labopin M, Sanz G, et al. Transplants of umbilical-cord blood or bone marrow from unrelated donors in adults with acute leukemia. N Engl J Med 2004;351:2276-85. 13. Gluckman E, Rocha V, Arcese W, et al. Factors associated with outcomes of unrelated cord blood transplant: Guidelines for donor choice. Exp Hematol 2004;32:397-407. 14. Hall J, Martin P, Wood S, et al. Unrelated umbilical cord blood transplantation for an infant with beta-thalassemia major. J Pediatr Hematol Oncol 2004;26:382-5. 15. Parikh SH, Szabolcs P, Prasad VK, et al. Correction of chronic granulomatous disease after second unrelated-donor umbilical cord blood transplantation. Pediatr Blood Cancer 2007;49:982-4. 16. Bhattacharya A, Slatter MA, Chapman CE, et al. Single centre experience of umbilical cord stem cell transplantation for primary immunodeficiency. Bone Marrow Transplant 2005;36:295-9. 17. Prasad VK, Kurtzberg J. Emerging trends in transplantation of inherited metabolic diseases. Bone Marrow Transplant 2008;41:99-108. 18. Eapen M, Rubinstein P, Zhang MJ, et al. Outcomes of transplantation of unrelated donor umbilical cord blood and bone marrow in children with acute leukaemia: A comparison study. Lancet 2007;369:1947-54.

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19. Staba SL, Escolar ML, Poe M, et al. Cord-blood transplants from unrelated donors in patients with Hurler’s syndrome. N Engl J Med 2004;350:1960-9. 20. Escolar ML, Poe MD, Provenzale JM, et al. Transplantation of umbilical-cord blood in babies with infantile Krabbe’s disease. N Engl J Med 2005;352:2069-81. 21. Muenzer J, Fisher A. Advances in the treatment of mucopolysaccharidosis type I. N Engl J Med 2004;350:1932-34. 22. Boelens JJ, Wynn RF, O’Meara A, et al. Outcomes of hematopoietic stem cell transplantation for Hurler’s syndrome in Europe: A risk factor analysis for graft failure. Bone Marrow Transplant 2007;40:225-33. 23. Barker JN, Weisdorf DJ, DeFor TE, et al. Transplantation of 2 partially HLA-matched umbilical cord blood units to enhance engraftment in adults with hematologic malignancy. Blood 2005;105:1343-7. 24. Kurtzberg J, Lyerly AD, Sugarman J. Untying the Gordian knot: Policies, practices, and ethical issues related to banking of umbilical cord blood. J Clin Invest 2005;115:2592-7.

25. Meyer E, Hanna K, Gebbie K, et al, editors. Cord blood: Establishing a national hematopoietic stem cell bank program. Washington, DC: National Academic Press, 2005: 320. 26. Sugarman J, Kaalund V, Kodish E, et al. Ethical issues in umbilical cord blood banking. Working Group on Ethical Issues in Umbilical Cord Blood Banking. JAMA 1997;278:938-43. 27. Burgio GR, Gluckman E, Locatelli F. Ethical reappraisal of 15 years of cord-blood transplantation. Lancet 2003;361:250-2. 28. Annas GJ. Waste and longing—The legal status of placental-blood banking. N Engl J Med 1999;340:1521-4. 29. Magro E, Regidor C, Cabrera R, et al. Early hematopoietic recovery after single unit unrelated cord blood transplantation in adults supported by co-infusion of mobilized stem cells from a third party donor. Haematologica 2006;91:640-8. 30. Harris D, Rogers I. Umbilical cord blood: A unique source of pluripotent stem cells for regenerative medicine. Curr Stem Cell Res Ther 2007;2:301-9.

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PART IV

37

Surgery Patients

Alternatives to Transfusion: Perioperative Blood Management Lynne Uhl Assistant Professor of Pathology, Harvard Medical School, and Director, Division of Laboratory and Transfusion Service, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA

Recognition of blood supply limitations and concern for potential deleterious effects of transfusion have promoted the concept of integrated programs for blood management in the surgical setting.1 Perioperative transfusions account for more than half of the estimated 20 million blood components transfused annually.2 Logically, if measures are taken to reduce this utilization, both the pressure on resource allocation as well as the risks associated with allogeneic transfusion could be reduced. The scope of blood management is broad and includes several different options, some of which can be initiated before patient hospitalization, others of which occur during the intraoperative period or the postoperative period (Table 37-1). This chapter covers the various aspects of blood management, which present alternatives to allogeneic transfusion. Table 37-1. General Principles of Blood Management 1. Formulate a plan of care for avoiding and controlling blood loss tailored to the clinical management of individual patients, including anticipated and potential procedures. 2. Employ a multidisciplinary treatment approach to blood conservation using a combination of interventions. 3. Manage blood utilization proactively and be prepared to address potential complications. 4. Promptly investigate and treat anemia, preferably preoperatively. 5. Exercise clinical judgment and be prepared to modify routine practice when appropriate. 6. Consult promptly with senior specialists experienced in blood conservation at an early stage if there is physiologic deterioration or if complications arise. 7. Restrict blood drawing for laboratory tests. 8. Decrease or avoid the perioperative use of anticoagulants and antiplatelet agents. Reprinted with permission from Goodnough and Shander.1 Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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Blood Management in the Preoperative Period Blood management strategies should begin during the preoperative phase of patient management. During the preoperative evaluation of a patient, it is important to carefully review a patient’s personal and family history to assess for potential bleeding risks. For example, family history of von Willebrand disease or specific factor deficiency (eg, Factor XI deficiency) identifies a patient who is potentially at risk for significant blood loss. Also important is the review of medication history for presence of drugs that interfere with either primary or secondary hemostasis (eg, aspirin, aspirin-containing compounds, antiplatelet agents, anticoagulants). Additionally, evidence of anemia, either through history or laboratory assessment, should be determined. If present, measures to increase red cell mass before surgery should be recommended (eg, nutritional supplementation with iron).1

Autologous Blood Donation The practice of preoperative autologous blood donation (PAD) gained widespread use in the early 1980s when there was a growing concern about transfusion-transmitted hepatitis and human immunodeficiency virus.3-5 However, several reviews of PAD programs suggest that their role in transfusion management has declined, an observation confirmed by national data on blood procurement (Table 37-2).6-9 The decline is attributed to reduced risk for transfusion-transmitted disease and improved surgical techniques leading to reduced blood loss, as well as the acceptance of lower transfusion thresholds.6 Further questions about the efficacy of PAD were raised in the systematic review published by the Cochrane Collaboration.10 Results of this review showed that although PAD is associated with an overall reduction of allogeneic blood exposure, patients participating in PAD have a higher likelihood of transfusion (allogeneic and autologous blood) compared with patients who do not participate in PAD. Reasons for this include lower preoperative hemoglobin concentrations, resulting in increased probability

Chapter 37: Alternatives to Transfusion: Perioperative Blood Management

Table 37-2. Collection and Transfusion of Autologous Blood (in Thousands of Units) in the United States between 1999 and 20069 1999

2001

Percent Change 1999-2001

2004

Percent Change 2001-2004

2006

Percent Change 2004-2006

Collected Autologous % of total Total

659 4.8 13,649

619 4.1 15,076


4.8

458 3.0 15,019


26.0

335 2.1 16,023

–26.9

Transfused Autologous % of total Total

367 3.0 12,389

359 2.6 13,898


2.3

270 1.9 14,182


24.6

189 1.3 14,650

–30.0

of requiring intraoperative and/or postoperative blood transfusion and, possibly, more liberal use of transfusion based on the fact that there is “blood in the bank.”11 The findings of Billote et al,12 in particular, suggest that PAD has limited utility for reducing the need for allogeneic transfusion, even for procedures associated with significant blood loss. In this well-designed, randomized, controlled study the investigators examined the usefulness of PAD for reducing the need for allogeneic transfusion in patients undergoing unilateral primary total hip replacement. The patient population consisted of surgical candidates who were not anemic (predonation hemoglobin greater than or equal to 12 g/dL) and the collection protocol required that the last unit be collected no later than 2 weeks before surgery. Additionally, patients with significant comorbidities (including severe or unstable cardiac disease, uncontrolled hypertension, symptomatic carotid or vertebral artery stenosis, a history of bleeding diathesis, or bacteremia) were excluded from the study. Standardized transfusion guidelines for allogeneic and autologous transfusion used intraoperatively, included: 1) acute blood loss (25% blood volume), 2) tachycardia attributed to hemoglobin-responsive hypoxia and unresponsive to intravenous fluid administration, 3) a hemoglobin concentration 7 g/dL in healthy patients, and 4) a hemoglobin concentration of 8 g/dL in patients with significant comorbid disease (eg, heart disease). In the postoperative period, indications for autologous transfusion included a hemoglobin concentration of 11 g/dL in the immediate postoperative recovery period and 10 g/dL in the subsequent postoperative period. As summarized in Table 37-3, important findings of this study included the observation that no patient required allogeneic transfusion; furthermore, patients randomly assigned to the nondonation cohort did not require transfusion, whereas those in the PAD cohort received a total of 42 autologous Red Blood Cell (RBC) units. Also significant was that 34 (41%) of the 82 autologous units collected were not transfused.13 This report has led many in the field of transfusion medicine to question the role of PAD in blood conservation, particularly because it is associated with a relatively high wastage of units.

Table 37-3. Randomized Controlled Trails Examining Autologous vs Allogeneic RBC Use in Patients Undergoing Hip Replacement Surgery Variable

Number of patients Number of autologous units transfused 0 1 2 Allogeneic units transfused

Cohort Autologous

Allogeneic

42

54

13 10* 19* 0

0 0 0 0

*34 (41%) of the 82 available autologous units were not transfused. Reprinted with permission from Blajchman. 13

Considerations for a Successful PAD Program Traditionally, PAD has been advocated for patients who are undergoing procedures associated with significant blood loss (procedures associated with 90% likelihood of requiring transfusion), such as orthopedic joint replacement, cardiothoracic procedures, vascular surgery, and radical prostatectomies.14 In contrast, PAD is unnecessary for patients scheduled for procedures with little anticipated blood loss, for example, vaginal hysterectomies.14 To optimize the success of PAD, several factors should be considered. Studies have shown the endogenous erythropoietic response to PAD is modest, and can be negatively affected by poor nutritional status and/or anemia of chronic disease. Thus, an assessment for history of chronic disease or chronic blood loss, as well as general nutritional status should be performed to determine if PAD is appropriate. Based on several studies (Table 374), the standard phlebotomy schedule of 1 unit/week in patients who are otherwise healthy (nonanemic and iron replete) can lead to an 11% to 19% expansion of red cell mass (equivalent to 1.01.75 RBC units).16,17 More aggressive phlebotomy programs (eg, 2 units/week) promote a more robust erythropoietic response (equating to 2-3 RBC units) that is enhanced with the addition of exogenous erythropoietin administration.18-21

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Table 37-4. Erythropoiesis during Autologous Blood Donation Patients (n)

Baseline Red Cell Mass (mL)

Blood Removed (Donated) Units Requested

Donated

Blood Produced

RBC (mL)

Red Cell Mass mL

Increase %

Iron Therapy

Ref

Standard phlebotomy 108

1,884

3

2.7

522

351

19%

PO

Kasper et al16

22

1,936

3

2.8

590

220

11%

None

Kasper et al17

45

1,881

3

2.9

621

331

17%

PO

Kasper et al17

41

1,918

3

2.9

603

315

16%

PO, IV

Kasper et al17

Aggressive phlebotomy 30

2,075

3

3.0

540

397

19%

None

Weisbach et al18

30

2,024

3

3.1

558

473

23%

PO

Weisbach et al18

30

2,057



2.9

522

436

21%

IV

Weisbach et al18

24

2,157

6

4.1

683

568

26%

PO

Goodnough et al19,20

23

2,257

6

4.6

757

440

19%

PO

Goodnough et al21

Data expressed as means. PO  oral; IV  intravenous * Reprinted with permission from Goodnough et al.15

Complications and Adverse Effects of PAD The incidence of donor reactions in the autologous blood donor population appears to be similar to that of allogeneic donors, ranging from 2% to 5%.22,23 As with allogeneic donations, risk factors include first-time donation, female gender, and decreasing age.23 The majority of reactions are mild and generally vasovagal in origin11; however, recent assessments question whether the side effects merit the time and effort put into autologous donation, particularly in the pediatric population.24 A recent study demonstrated a dramatic reduction (21%) in donor reactions by the simple ingestion of 0.5 L of water within 10 minutes of the donation episode.25 The transfusion of autologous blood has many of the same complications as transfusion of allogeneic units, including bacterial contamination, hemolysis resulting from errors in the administration of units, and volume overload. Advantages and disadvantages of PAD are summarized in Table 37-5. Because mortality from allogeneic blood transfusion is now more likely the result of administrative error26 rather than blood-transmitted infection,27 the risks of banked autologous blood units are similar to banked allogeneic blood units.

Erythropoietin Erythropoietin, as discussed in Chapter 2, is a key regulator of erythropoiesis. Briefly, endogenous erythropoietin is released by renal cortical interstitial cells in response to tissue hypoxia.28 The released hormone then exerts an effect on colony-forming unit erythroid precursors in the marrow, promoting cellular differentiation and maturation.28 Additional actions include enhanced hemoglobin synthesis and release of reticulocytes into the systemic circulation.28

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Table 37-5. Autologous Donation Advantages

Disadvantages

1. Prevents transfusion-transmitted disease

1. Risk of bacterial contamination or volume overload remains

2. Prevents red cell alloimmunization

2. Does not eliminate risk of administrative error with ABO incompatibility

3. Supplements the blood supply

3. More costly than allogeneic blood

4. Provides compatible blood for patients with alloantibodies

4. Wastage of blood not transfused

5. Prevents some adverse transfusion reactions

5. Causes perioperative anemia and increased likelihood of transfusion

As noted above, the erythropoietic response to blood loss anemia is modest. The discovery that compensatory hematopoiesis is better enhanced through the administration of exogenous erythropoietin (recombinant erythropoietin) spawned several clinical trials investigating the role of erythropoietin therapy in the preoperative setting. Patients scheduled to undergo surgical procedures associated with significant blood loss (eg, major orthopedic, cardiovascular, and urologic surgical procedures) were specifically evaluated.29-35 As summarized in Table 37-6, the clinical trials in the orthopedic patient population, using a variety of dosing regimens, demonstrated benefit; preoperative hemoglobin concentrations increased and allogeneic transfusions were reduced. Patients with preoperative hemoglobin concentrations ranging between 10 and 13 g/dL appeared to benefit the most from preoperative

Chapter 37: Alternatives to Transfusion: Perioperative Blood Management

Table 37-6. Efficacy of Perioperative Recombinant Erythropoietin in Major Elective Orthopedic Surgery Study (Number of Patients)

Orthopedic Procedure

EPO Dose

Oral Iron

Key Findings

Canadian Study29 (198)

Hip arthroplasty

300 IU/kg  14 or 9 days vs placebo

300 mg beginning 21 days before surgery

Dose-dependent increase in hemoglobin concentration and reticulocyte count Decrease in risk of exposure to allogeneic blood in patients treated with erythropoietin

Faris et al30 (185)

Hip and knee arthroplasty

300 IU/kg or 100 IU/kg  15 days vs placebo

325 mg TID while on study

Dose-dependent increase in hemoglobin concentration and reticulocyte count Decrease in risk of exposure to allogeneic blood in patients treated with erythropoietin

de Andrade et al31 (290)

Hip and knee arthroplasty

300 IU/kg or 100 IU  15 days vs placebo

Yes

Dose-dependent increase in hemoglobin concentration and reticulocyte count Decrease in risk of exposure to allogeneic blood in patients treated with erythropoietin

Goldberg et al32 (140)

Hip and knee arthroplasty

600 IU/kg, weekly for 3 weeks before surgery and once on day of surgery 300 IU/kg  15 days

Yes

Increase in hemoglobin concentration Equivalent response with weekly vs daily dosing regimen in reducing allogeneic transfusion

* Modified with permission from Faris and Ritter.28

administration of erythropoietin.31 On the basis of the results of these clinical trials, erythropoietin therapy gained US regulatory approval in 1996 for perioperative use in patients scheduled for elective, noncardiovascular surgical procedures. Several trials in orthopedic patient populations have confirmed the positive impact of erythropoietin administration on reducing allogeneic red cell transfusion, particularly when coupled with iron repletion.36,37 However, despite the evidence of benefit, the use of erythropoietin has not been widely adopted. Deterrents to the general use of erythropoietin include the inconvenience of self-administration, restrictive reimbursement by insurance companies, concern for thrombosis (particularly in the cardiovascular surgical patient population38), and lack of demonstrable cost-effectiveness with respect to patient morbidity and mortality.39 However, as the enthusiasm for PAD declines, it is possible that more general use of erythropoietin in the preoperative setting will increase.

acellular fluid (in general, a combination of crystalloid and colloid) in order to maintain a normovolemic state. The practice stemmed from the early days of open-heart surgery when “blood pooling,” the intentional removal of whole blood, was employed as a means to sequester platelets, thus avoiding contact activation via exposure to the extracorporeal circuit and consequent development of postoperative coagulopathies.40,41 Soon thereafter, it was recognized that additional potential benefits of ANH included a reduction of red cell loss, because the hematocrit of the blood shed during intraoperative procedures is less. For example, in theory, a 2-L blood loss in a patient with a 45% hematocrit, or the equivalent of 900 mL of red cells, could be reduced to 400 mL of red cells following hemodilution to a hematocrit of 20%. Several reports illustrating the mathematical modeling of hemodilution suggest that the benefit of ANH, in terms of reduction in significant red cell loss, is realized only in cases where extreme hemodilution (eg, a reduction in hematocrit to 21% or lower) is undertaken and the surgical blood loss volume is large (in excess of 1.5 L) (Fig 37-1).43-45

Intraoperative/Postoperative Strategies Acute Normovolemic Hemodilution Acute normovolemic hemodilution (ANH) is the immediate preoperative removal of whole blood and its replacement with an

Safety and Efficacy of ANH: Clinical Studies ANH is quite a straightforward procedure. Following induction of anesthesia, blood is withdrawn into standard blood collection bags containing anticoagulant-preservative solution. Upon

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Table 37-7. Compensatory Mechanisms Following Hemodilution and the Creation of an Acute Anemia

3.0

Units saved

2.5 2.0 1.5

Increased cardiac output

●

Increased stroke volume

●

Slight increase in heart rate (not clinically significant)

●

Reduced blood viscosity and systemic vascular resistance leading to decreased afterload

●

Increase blood flow to oxygen-dependent tissues—beyond the cardiac output increase

●

Increase in oxygen extraction in end organs

●

Increased coronary blood flow

1.0 0.5 0 0

2,000 4,000 6,000 8,000 Estimated blood loss (mL)

10,000

Figure 37-1. The theoretic relationship between the number of Red Blood Cell units saved as a function of estimated blood loss following acute normovolemic hemodilution to a minimum hematocrit of 28% (open symbols) or a hematocrit of 18% (shaded symbols) for a patient with a blood volume of 5 liters and an initial hematocrit of 45%. Replacement strategies were either to infuse mL-for-mL of blood lost (circle symbols) or to transfuse so as to maintain the hematocrit at the minimal hematocrit (square symbols). (Reprinted with permission from Goodnough et al.42)

completion of the blood collection process, the units are labeled with the patient’s name and stored at room temperature within the operative suite. The collected units of blood are then reinfused to the patient at the close of the procedure, in reverse order. The first unit collected, and therefore the last unit transfused, has the highest hematocrit and concentration of clotting factors and platelets. Although ANH decreases the arterial oxygen content of the circulating blood, compensatory mechanisms including increased cardiac output and decreased systemic vascular resistance might be expected, in theory at least, to maintain adequate tissue oxygen delivery (Table 37-7).46 Mathematical models suggest that ANH is not very effective in reducing the amount of red cells lost, and may put some patients at risk for untoward complications related to iatrogenically induced ischemia.43 Clinical studies, as well, have shown little sparing of allogeneic RBC transfusion with the use of ANH. In a recent meta-analysis, Siegel et al47 examined 42 studies of ANH that met the inclusion criteria of being randomized, controlled trials of either large-volume or low-volume ANH in a surgical population. The studies were conducted from 1972 through 2002, and a majority involved patients undergoing cardiothoracic surgery, followed by trials in hip arthroplasty. It was evident from this analysis that there was an insignificant reduction in the relative risk (RR) for allogeneic transfusion for patients managed with ANH [RR, 0.96; 95% confidence interval (CI), 0.90-1.01]. A study of selected patients undergoing hip arthroplasty demonstrated no significant reduction in the number of allogeneic transfusions in patients randomly assigned to ANH vs standard transfusion therapy.48 The authors appropriately point out that their data may have been affected by transfusion practices that were more conservative than expected (ie, acceptance of a lower hemoglobin concentration), inability to accomplish ANH in all patients randomly assigned to the ANH arm because of technical

570

●

Used with permission from Shander and Rijhwani.46

difficulties (primarily poor venous access precluding blood collection), and protocol violations (particularly in the ANH arm). The study of Sanders et al49 in which patients scheduled to undergo major gastrointestinal surgery for which total blood losses were anticipated to be 40%, also failed to demonstrate benefit of ANH vs standard transfusion practice with respect to reduced allogeneic blood transfusion in the operative or postoperative period. Still other studies have supported the use of ANH as a means to avoid allogeneic transfusion. Casati et al50 reported on a study of patients undergoing off-pump coronary artery bypass surgery who were randomly assigned to undergo either ANH or standard transfusion management. The protocol included ANH of 17% total blood volume (moderate ANH), use of red cell recovery and infusion, and a restrictive transfusion strategy (hemoglobin of 8 g/dL). Patients undergoing ANH received statistically significantly fewer RBC units and were more likely to have been transfused as a consequence of surgical bleeding rather than postoperative anemia. The authors concluded that ANH offers the potential to reduce allogeneic red cell exposure in this particular surgical population. The mixed reports regarding the clinical effectiveness of ANH as a conservation strategy result, in large part, from the heterogeneity in the methods of ANH (large volume vs moderate volume), the surgical procedures, and the transfusion management protocols. This inconsistency signals the need for larger and more rigorous clinical trials examining the benefit of ANH.51 Although efficacy of ANH is not clearly established, it poses little risk of harm to the patient. Furthermore, it appears to be a low-cost procedure compared to other alternatives directed at procurement of autologous blood.51

Intraoperative Autologous Blood Recovery and Reinfusion Intraoperative red cell recovery and reinfusion of autologous blood represent another approach to reduction in allogeneic transfusion. In general, their use has been restricted to surgical procedures in which 1) the estimated blood loss is anticipated to be more than 20% of a patient’s blood volume or 2) greater than 10% of patients undergoing the procedure require transfusion

Chapter 37: Alternatives to Transfusion: Perioperative Blood Management

Table 37-8. General Indications for Use of Intraoperative Blood Recovery Procedures* Specialty

Surgical Procedure

Cardiac

Valve replacement Redo bypass grafting

Orthopedics

Major spine Bilateral knee replacement Revision hip replacement

Urology

Radical retropubic prostatectomy Cystectomy Nephrectomy

Neurosurgery

Giant aneurysm

Vascular

Thoracoabdominal aortic aneurysm repair Abdominal aortic aneurysm repair

Other

Jehovah’s Witness Unexpected massive blood loss Red cell antibodies

Comments

Individualized by surgeon Limited to patients with prior radiation therapy When tumor involved major vessels

Table 37-9. Possible Contraindications to Intraoperative Blood Recovery* Pharmacologic agents Clotting agents (Avitene, Surgicel, Gelfoam, etc) Irrigating solutions (Betadine, antibiotics meant for topical use) Methylmethacrylate Anticoagulants Contaminants Urine Bone chips Fat Bowel contents Infection Amniotic fluid Cellular stroma Activated leukocytes, platelets, complement, plasmin Malignancy

Should be individualized by surgeon and patient characteristics When accepted by patient

Hematologic disorders Sickle cell disease Thalassemia Miscellaneous Carbon monoxide (electrocautery smoke) Catecholamines (pheochromocytoma) Oxymetazoline (Afrin) *Reprinted with permission from Waters.52

*Reprinted with permission from Waters.52

(and the mean transfusion requirement is 1 unit). Table 37-8 lists commonly accepted indications. Recovery techniques include collection of blood by direct suctioning from the operative field as well as recovery of blood from surgical sponges. Following recovery, the blood may or may not be further manipulated. Devices available for postcollection processing of shed blood fall into two main categories: 1) hemofiltration, whereby shed blood is drawn into the autotransfusion device, which is equipped with an integral filtration device; the collected product is then simply filtered (ie, there is no washing of the product) and reinfused to the patient52 and 2) red cell wash systems, whereby shed blood is collected and subsequently processed by automated devices that wash the shed blood free of cellular debris and other contaminants as well as concentrate the recovered red cells.52,53

Efficacy The evidence that use of cell recovery and reinfusion promotes avoidance of allogeneic blood transfusion is not solid. A recent review of 51 published trials on cell recovery and reinfusion involving primarily cardiac and orthopedic patients revealed several methodologic problems in this area of investigation, including small numbers of study subjects, heterogeneity in transfusion practice (protocol-driven vs transfusion at the investigators’ discretion), and either no reference to whether the study was blinded, or clear indications that the study was not blinded.54 Despite these concerns, the authors, after applying rigorous review criteria,

determined that use of red cell recovery and reinfusion reduced the relative risk for perioperative allogeneic RBC transfusion exposure by 39% (RR  0.61; 95% CI, 0.52 to 0.71). Furthermore, their analysis showed a more pronounced reduction for allogeneic red cell exposure in orthopedic patients vs cardiac patients (58% compared to 23%, respectively). A critical question is whether a reduction in the relative risk for allogeneic red cell exposure equates to a meaningful reduction in volume of allogeneic red cell transfusion. Unfortunately, of the 51 studies analyzed, only 27 reported data on the volume of allogeneic red cells transfused. Based on these available data, use of cell recovery and reinfusion reduced the average number of allogeneic RBC transfusions by only 0.67 unit/patient (95% CI, 0.89 to 0.45 unit). However, when analyzed in terms of a carefully constructed economic model, the cost/benefit ratio of intraoperative recovery supported its use as an effective measure to reduce allogeneic red cell exposure.55

Complications Although seemingly straightforward, transfusion of recovered blood is not without potential risks, and for some clinical situations, the practice is contraindicated (Table 37-9). However, it is important to recognize that many contraindications are relative, not absolute, because there are limited data to support the danger of cell recovery in these situations.52 For example, studies of tumor surgeries in which cell recovery has been employed as a blood conservation measure have not demonstrated an increase

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in either incidence of metastatic disease or death, but have resulted in reduced allogeneic red cell transfusion.57-59 The literature is peppered by reports of disseminated intravascular coagulation and acute renal failure secondary to reinfusion of recovered blood that was either filtered or inadequately washed, improper use of the devices, inadvertent use of hypo- or hypertonic wash solutions resulting in compromised products, and air embolism caused by inadequate venting of collection/ infusion bags.52 Patient factors appear to play a major role in risk for complications; for example, concern for bacterial contamination of recovered blood is probably valid in a case of major abdominal trauma with significant bowel injury, but not as much in elective cases of abdominal surgery, where the surgical setting is much more controlled.60 Similarly, the concern for renal damage secondary to hemolyzed red cells following recovery may be warranted in patients with underlying renal pathology or in patients who are hypotensive, thus at risk for suboptimal renal perfusion.60 Such concerns may be more valid for postoperatively recovered blood, where hemofiltration devices are more commonly used.61

Postoperative Autologous Blood Recovery and Reinfusion Postoperative recovery of blood involves the collection of blood from surgical drains followed by reinfusion, with or without processing. Blood recovered by hemofiltration (in contrast to systems where recovered products are washed as part of the recovery process) can be dilute, partially hemolyzed, and contain high concentrations of cytokines and cellular debris.62-64 The safety and the benefit of the use of unwashed blood obtained from surgical drains following orthopedic surgery remain in question.65 Although the results of a recent randomized clinical trial66 evaluating the effectiveness and safety of postoperative recovered blood demonstrated a reduction in allogeneic transfusion in the study arm (vs control), this study was plagued by several confounding variables, including mixed surgical types with unequal randomization, lack of characterization of the recovered product, and variable transfusion thresholds.65 Similar concerns about heterogeneity of study population and variable transfusion practices were also highlighted by Carless et al54 in their systematic review of perioperative cell recovery and suggest the need for further study of the role of such devices in perioperative blood management.

Summary The blood needs of surgical patients can be managed by many alternatives to allogeneic transfusion. However, several factors (eg, patient baseline hematocrit, cardiac performance, anticipated surgical blood loss) affect their practicality of use and cost-effectiveness. Hence, effective use of these alternatives, either alone or in combination, requires the prospective identification of appropriate surgical candidates who will need transfusion and

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who may truly benefit from blood conservation. The primary impetus to avoid allogeneic transfusion is no longer based on the safety of the blood supply, but rather on evidence that blood conservation is safe and of value for individual patients.

Disclaimer The author has disclosed a financial relationship with Genzyme Corporation.

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36. Karkouti K, McCluskey SA, Evans L, et al. Erythropoietin is an effective clinical modality for reducing RBC transfusion in joint surgery. Can J Anaesth 2005;52:362-8. 37. Wong CJ, Vandervoort MK, Vandervoort SL, et al. A cluster-randomized controlled trial of a blood conservation algorithm in patients undergoing total hip joint arthroplasty. Transfusion 2007;47:832-41. 38. Goodnough L. Preoperative management and preparation for transfusion-free surgery. In: Jabbour N, ed. Transfusion-free medicine and surgery. 1st ed. Oxford: Blackwell; 2005:60-74. 39. Fergusson DA, Hebert P. The health(y) cost of erythropoietin in orthopedic surgery. Can J Anaesth 2005;52:347-51. 40. Cooley DA, Beall AC Jr, Grondin P. Open-heart operations with disposable oxygenators, 5 per cent dextrose prime, and normothermia. Surgery 1962;52:713-9. 41. Petry AF, Jost T, Sievers H. Reduction of homologous blood requirements by blood-pooling at the onset of cardiopulmonary bypass. J Thorac Cardiovasc Surg 1994;107:1210-4. 42. Goodnough LT, Monk TG, Brecher ME. Autologous blood procurement in the surgical setting: Lessons learned in the last 10 years. Vox Sang 1996;71:133-41. 43. Brecher ME, Rosenfeld M. Mathematical and computer modeling of acute normovolemic hemodilution. Transfusion 1994;34:176-9. 44. Goodnough LT, Monk TG, Brecher ME. Acute normovolemic hemodilution should replace the preoperative donation of autologous blood as a method of autologous-blood procurement. Transfusion 1998;38:473-6. 45. Weiskopf RB. Efficacy of acute normovolemic hemodilution assessed as a function of fraction of blood volume lost. Anesthesiology 2001;94:439-46. 46. Shander A, Rijhwani TS. Acute normovolemic hemodilution. Transfusion 2004;44(Suppl):26S-34S. 47. Segal JB, Blasco-Colmenares E, Norris EJ, Guallar E. Preoperative acute normovolemic hemodilution: A meta-analysis. Transfusion 2004;44:632-44. 48. Bennett J, Haynes S, Torella F, et al. Acute normovolemic hemodilution in moderate blood loss surgery: A randomized controlled trial. Transfusion 2006;46:1097-103. 49. Sanders G, Mellor N, Rickards K, et al. Prospective randomized controlled trial of acute normovolaemic haemodilution in major gastrointestinal surgery. Br J Anaesth 2004;93:775-81. 50. Casati V, Benussi S, Sandrelli L, et al. Intraoperative moderate acute normovolemic hemodilution associated with a comprehensive blood-sparing protocol in off-pump coronary surgery. Anesth Analg 2004;98:1217-23. 51. Shander A, Perelman S. The long and winding road of acute normovolemic hemodilution. Transfusion 2006;46:1075-9. 52. Waters JH. Indications and contraindications of cell salvage. Transfusion 2004;44(Suppl):40S-4S. 53. Freischlag JA. Intraoperative blood salvage in vascular surgery— worth the effort? Crit Care 2004;8(Suppl):S53-S56. 54. Carless PA, Henry DA, Moxey AJ, et al. Cell-salvage for minimising perioperative allogeneic blood transfusion. Cochrane Database Syst Rev 2006;(4):1-109. 55. Davies L, Brown TJ, Haynes S, et al. Cost-effectiveness of cell salvage and alternative methods of minimising perioperative allogeneic blood transfusion. Health Technol Assess 2006;10:1-228. 56. Waters J, Cheng D, Shande A, et al, eds. Perioperative blood management. 1st edition. Bethesda, MD: AABB, 2006:64.

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57. Zulim RA, Rocco M, Goodnight JE, et al. Intraoperative autotransfusion in hepatic resection for malignancy: Is it safe? Arch Surg 1993;128:206-11. 58. Klimberg I, Sirois R, Wajsman Z, Baker J. Intraoperative autotransfusion in urologic oncology. Arch Surg 1986;121:1326-9. 59. Hart OJ, III, Klimberg IW, Wajsman Z, Baker J. Intraoperative autotransfusion in radical cystectomy for carcinoma of the bladder. Surg Gynecol Obstet 1989;168:302-6. 60. Dzik WH, Sherburne B. Intraoperative blood salvage: Medical controversies. Transfus Med Rev 1990;4:208-35. 61. Hansen E, Pawlik M. Reasons against the retransfusion of unwashed wound blood. Transfusion 2004;44(Suppl):45S-53S. 62. Sinardi D, Marino A, Chillemi S, et al. Composition of the blood sampled from surgical drainage after joint arthroplasty: Quality of return. Transfusion 2005;45:202-7.

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63. Dalen T, Bengtsson A, Brorsson B, Engstrom KG. Inflammatory mediators in autotransfusion drain blood after knee arthroplasty, with and without leucocyte reduction. Vox Sang 2003;85:31-9. 64. Duchow J, Ames M, Hess T, Seyfert U. Activation of plasma coagulation by retransfusion of unwashed drainage blood after hip joint arthroplasty: A prospective study. J Arthroplasty 2001;16:844-9. 65. Waters JH, Dyga RM. Postoperative blood salvage: Outside the controlled world of the blood bank. Transfusion 2007;47:362-5. 66. Moonen AF, Knoors NT, van Os JJ, et al. Retransfusion of filtered shed blood in primary total hip and knee arthroplasty: A prospective randomized clinical trial. Transfusion 2007;47:379-84.

38

Blood Components to Achieve Hemostasis for Surgery and Invasive Procedures Walter H. Dzik Co-Director, Blood Transfusion Service, Massachusetts General Hospital, and Associate Professor of Pathology, Harvard Medical School, Boston, Massachusetts, USA

Blood components are frequently transfused to patients undergoing invasive procedures and surgery. As illustrated by the cases below, decisions regarding transfusion before invasive procedures can be complex. Case 1 A 49-year-old man is transferred to your hospital because of fever, altered mental status, and thrombocytopenia (platelet count 35,000/µL). Because the diagnosis of thrombotic thrombocytopenic purpura (TTP) was considered by the referring institution, neither the referring physicians nor your clinical team want to administer platelet transfusions. Although a diagnostic lumbar puncture (LP) was considered important by the referring hospital, the platelet count was considered too low to safely perform an LP and so it was not done. You are called to organize plasma exchange, not only to treat the patient, but also to produce a higher platelet count so that an LP can be safely performed. Comment: There is absolutely no evidence that a diagnostically important LP should be delayed because of a platelet count of 35,000/µL. In fact, there is evidence demonstrating that an LP is safe at this level of thrombocytopenia (discussed below). You recommend that the LP be done without administration of platelets. The procedure results in no hemorrhagic complication. The results of the LP demonstrate acute bacterial meningitis, the diagnosis of which was unfortunately delayed by the failure to do an important diagnostic test in a patient with fever and altered mental status. The patient does not have TTP. Case 2 Following an extended period of endotracheal intubation, a 60year-old man, currently in the intensive care unit (ICU), needs a tracheotomy tube and feeding gastrotomy tube placed at the bedside. The platelet count is 40,000/µL and the international normalized ratio (INR) is 1.3. The patient has either medicationrelated or immune-mediated consumptive thrombocytopenia and does not respond to platelet transfusions. He has recently Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

received intravenous immunoglobulin. You are told that the surgeon “absolutely refuses” to do the procedure without the prior infusion of four doses of platelets (equivalent to 24 units) with an additional two doses available to “run in” during the procedure. You are also supporting the transfusion needs of a trauma patient (gunshot wound) currently in the operating room (OR); another OR patient who is receiving a heart transplant; and a patient who is scheduled for an aortic aneurysm repair later this evening. You have 12 doses of platelets available and inform the ICU team that you cannot commit six doses of platelets to a bedside tracheotomy-gastrotomy case. The medical ICU team replies that your decision will mean that the patient will not receive his urgently needed tracheotomy and they request that you come up to the floor to explain to a concerned family why you are denying the patient his essential care. Comment: There is no evidence that the request for preprocedure platelets is medically justified. The use of infusions “run in” during the procedure is largely theatrical. You are correct to manage a limited inventory of platelets by assigning them to active surgical patients who are more likely to benefit from platelet transfusions. The response of the medical ICU team, who attempt to twist the dynamic implying that you are denying the patient essential care, is completely inappropriate. Nevertheless, this sort of attempted coercion occurs commonly. You discuss the issue with the individuals assigned to do the tracheotomy and review with them the published risks on bleeding (described below). Weighing the risks and benefits, they decide to proceed with the bedside tracheotomy and gastrotomy without any preprocedure blood infusions. The procedure goes smoothly with an estimated blood loss of
10 mL. Case 3 A previously healthy 64-year-old man with pneumonia and sepsis requires placement of a central line for hemodynamic monitoring and vascular access. The patient has an INR of 1.6. Because of hypoxia, decreased urine output, and presumed decreased left ventricular performance, circulatory overload is a concern. Despite this, the presiding house-staff physician requests 4 units of fresh frozen plasma (FFP) to “correct the coagulopathy” before central line placement. When you question this decision, the house-staff member acknowledges that he

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is “nervous” about placing the line and that “he would feel more comfortable if the patient got FFP first.” Comment: There is no evidence that preprocedure FFP will benefit this patient undergoing central line placement. Transfusion of this patient, for whom volume overload is a legitimate concern, in order to increase the comfort level of the physician is not appropriate.

Current Challenges As illustrated by the cases presented above, three basic challenges confront our understanding of the proper transfusion support of patients undergoing surgery or invasive procedures. First, there is no consensus on what extent of bleeding should be prevented or should demand treatment. For example, no clinician would transfuse a patient to prevent or to “treat” a 2-cm superficial skin hematoma at the site of insertion of a central venous catheter. While such procedure-related bleeding is not desirable, it is acceptable and represents largely a temporary cosmetic adverse event of little consequence. In contrast, all clinicians would wish to avoid a hemothorax resulting in a collapsed lung, transfusion of 4 units of Red Blood Cells (RBCs), and placement of a chest tube for drainage. The challenge is to understand where the degree of reasonable and acceptable bleeding ends and where the extent of excessive bleeding begins. How much bleeding is acceptable differs for patients depending upon their entire medical status, the nature and urgency of the procedure, and the expected benefit to be gained. The amount of acceptable bleeding differs for each anatomic site. Opinions about excessive bleeding are also influenced by each clinician’s experience and expectations. A second challenge in transfusion support is the contrast between the use of blood components for prevention of bleeding and the use of blood therapies for the treatment of bleeding. Although transfusions given as therapy to bleeding patients have an unquestionable rationale, the same cannot be said for transfusions given to prevent bleeding before surgery or invasive procedures. Blood components given before an invasive procedure are likely to benefit patients who have severe derangements of hemostasis or who offer a clear history of bleeding complications following similar procedures. However, blood component therapy given to patients before procedures solely on the basis of abnormal laboratory tests is of uncertain value. Indeed, there is scant medical evidence, if any, that preprocedure therapies are of any benefit when given to patients with mild to moderate abnormalities of laboratory tests of hemostasis. There is also no evidence that a prophylactic strategy is superior to a treatment-of-bleeding strategy. From a risk-to-benefit perspective, prophylactic transfusion strategies can verge on the absurd. Most clinicians have witnessed patients receiving 10 units of platelets and plasma in an attempt to avoid the potential loss of one-tenth of one unit of blood, a hemorrhage that would not have required treatment had it occurred. Nevertheless, prophylactic infusion of blood components is an extremely common medical practice. If indeed prophylactic treatments are of no value for most patients, and if such

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treatments cause their own complications in a fraction of recipients, then large numbers of individuals are suffering adverse consequences of such treatments with no attendant benefit. A third challenge in the area of procedure-related bleeding is to understand the relative value of blood component therapies compared with other agents that promote hemostasis. Although transfusions of platelets or FFP are often used, some patients would fare better were they to receive topical hemostatic agents, medications such as fibrinolytic inhibitors, or ligation of a bleeding vessel by a well-placed suture. Thus, the decision-making for each patient should consider the following three questions: 1) What extent of bleeding is acceptable? 2) What is the evidence to suggest that any treatment should be given before the procedure? and 3) What is most appropriate therapy to achieve hemostasis? An overview of these considerations is shown in Fig 38-1.

Preprocedure Blood Components as Prophylaxis Against Bleeding The last decade has seen explosive growth in the number and variety of invasive procedures performed outside of the OR. In fact, far more invasive procedures are performed outside the operating room than within the OR. Many patients who undergo invasive procedures will have abnormalities on routine laboratory tests of hemostasis. It is very common, although medically unproven, for clinicians to administer blood components to such patients in the expectation of reducing the risk or severity of procedure-related bleeding. The decision to transfuse blood components as prophylaxis before a bedside procedure is subject to three major criticisms.

Mild-to-Moderate Abnormalities of Common Laboratory Tests Do Not Identify Patients at Increased Risk of Procedure-Related Bleeding In an excellent review of hemostasis during surgery,1 Kitchens summarizes the predictive value of coagulation screening tests in three large survey studies representing nearly 4500 patients.2-4 These studies found that, in the absence of any history of bleeding, the positive predictive value of an abnormal coagulation screening test as a means to forecast bleeding was only 3%. Thus, the great majority of patients (97%) with abnormal coagulation screening tests will have no increased bleeding at surgery. In a systematic review of the published evidence that a prolonged INR would predict bleeding at the time of an invasive procedure, Segal and Dzik5 found no evidence to conclude that abnormal preprocedure test results should be used as an indication to transfuse blood components. A similar conclusion was drawn by Holland and Sarode.6 A common clinical error is to declare that a patient has a coagulopathy because the patient has an abnormal INR or activated partial thromboplastin time (aPTT). This error is epidemic and patients are routinely referred to as “coagulopathic” based

Chapter 38: Blood Components to Achieve Hemostasis

Approach to blood components and bleeding

Prophylaxis before an invasive procedure

Treatment of bleeding

Localized

* Mild-to-moderate abnormalities of laboratory tests of hemostasis do NOT predict bleeding.

Generalized

* Topical agents collagen, cellulose, gelatin thrombin sealants

* Usual doses of FFP fail to “correct” laboratory tests of hemostasis.

* There is no evidence that the extent of bleeding is less for patients given blood components before an invasive procedure compared with those given blood components as treatment to stop bleeding.

* Skin and mucous membrane oozing

* Purpura and soft tissue bleeding

* Treating wound fibrinolysis

* Small vessel bleeding at surgery

Figure 38-1. Overview of blood support for surgery and invasive procedures.

% coagulation factors 100%

INR and coagulation reserve

Zone of normal hemostasis Figure 38-2. The nonlinear relationship between the concentration of coagulation factors and coagulation test results. The concentration of factors (y-axis) is shown as a function of the international normalized ratio (INR) or prothrombin time (PT, in seconds). In general, a concentration of 30% is adequate for biologic hemostasis. Note that when starting at an elevated INR, a small increase in the concentration of factors will have a large impact on the measured INR. In contrast, an equivalent rise in the concentration of factors will have a negligible effect on mildly elevated INR values. The curve is a generalized view and will vary for individual patients.

50% 30%

Zone of therapeutic anticoagulation

20% 10%

PT (sec) 12 INR

solely on an elevated laboratory test. Clinicians will claim that the indication to transfuse blood was “to correct the patient’s coagulopathy.” Each of these commonly expressed viewpoints fails to consider evidence that an abnormal laboratory test does not, by itself, imply a breakdown in physiologic hemostasis. Indeed, mild to moderate prolongations of the INR or aPTT are completely consistent with normal hemostasis. This is because of the physiologic reserve of coagulation factors and the fact that the laboratory tests are designed to give results outside the range of normal before factor deficiencies reach levels of significant physiologic depletion.7 See Fig 38-2.

1.0

13

15.5

19 21.8

1.3

1.7 2.0

24 2.2

30

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3.0

Six aspects of the INR and aPTT make these tests poor predictors of hemorrhage. 1. The relationship between coagulation factors and the prothrombin time (PT) and aPTT is nonlinear. The exponential relationship between the concentration of clotting factors and the test result is shown in Fig 38-2. This relationship may not be fully appreciated by those who expect a simple dose-response effect from infusion of clotting factors. 2. Mildly abnormal test results occur among patients with biologically normal coagulation. In the same way that physiologically adequate tissue oxygenation does not require that the

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hemoglobin concentration be in the normal range, physiologic hemostasis does not require that the INR or aPTT value be in the normal range. 3. INR and aPTT tests overestimate deficiencies in the upper limb of the cascade and underestimate deficiencies in the lower limb. The INR assay is dominated by the level of Factor VII in the sample. Patients who have mildly reduced levels of Factor VII that are well within the range for adequate hemostasis and who have normal levels of other factors, will have a prolonged INR result. Reduced, but adequate, levels of Factor VII are very common in hospitalized patients because of decreased hepatic synthesis, vitamin K depletion, antibiotics, subclinical impairment of the enterohepatic circulation, and catabolic states with decreased protein synthesis. A very common flaw in the literature is to equate correction of the INR with correction of a hemostatic defect. This misunderstanding has given undo merit to “treatments” that elevate the Factor VII level and produce a dramatic effect on the INR regardless of whether they improve physiologic hemostasis. The same principle is true for the aPTT. 4. The tests overestimate the extent of coagulation factor depletion if more than one factor is reduced. Burns et al8 observed the following in a series of clever in-vitro mixing studies: When plasma containing 50% activity of a single clotting factor was mixed with an equal volume of normal plasma (yielding a mixture with 75% activity of the single deficient factor), the INR and aPTT of the mixture was normal. However, when two plasma samples, each deficient in different factors, were combined with normal plasma samples such that the resulting mixture contained 75% activity of two coagulation factors and 100% of the rest, the resulting INR and aPTT values were prolonged. Thus, for patients with multiple mild factor deficiencies—such as those with liver disease or vitamin K depletion—coagulation tests are prolonged out of proportion to the degree of hemostatic defect that would be assumed based on inherited single-factor deficiencies. With multiple mild deficiencies, patients with factor levels well within the range of normal will show abnormal test results. 5. Whereas the INR and aPTT are useful tests to analyze the defect in patients with disorders of fibrin formation, neither test was designed to predict bleeding. Given that clinical hemostasis depends on the complex interrelationships between the blood vessel wall, cellular elements of the blood, platelet number and function, fibrin formation, and fibrinolysis, it is not surprising that the INR and aPTT prove unable to predict who will bleed during surgery. The uncertainty that attends any invasive procedure is stressful for clinicians. This stress coupled with the understandable strong desire to prevent bleeding complications may have seduced clinicians into conferring upon these laboratory tests the power to foresee the future—a power they never have had, and never will. 6. Most important, these tests are insufficient to assess global hemostasis. The INR and aPTT focus on fibrin formation in vitro using a highly artificial assay, rabbit brain extracts, synthetic phospholipids, and without the contribution from biologically important molecules such as protein C and thrombomodulin.

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INRpost  INRpre 1.5 121 Patients 1.0

324 Units FFP

0.5 0 0.5 1

2

3

4

4

Units of FFP transfused Figure 38-3. “Box-and-whiskers” plot of the median change in international normalized ratio (INR) resulting from transfusion of Fresh Frozen Plasma (FFP). The change in INR measured within 8 hours of transfusion of FFP is shown. All patients had INR values
1.7 before transfusion. Used with permission from Abdel-Wahab.10

This can lead to incorrect conclusions regarding hemostasis. For example, Mannucci et al9 have shown that thrombin generation in patients with cirrhosis is incorrectly measured by standard INR and aPTT tests.

FFP Given Before a Procedure Fails to Correct the Abnormal Coagulation Test Result For patients with mild to moderate prolongation of the INR or aPTT, there is ample evidence to demonstrate that routine doses of FFP fail to “normalize” the coagulation test. This fact does not prove, by itself, that such transfusions have no value. Rather, the observed findings challenge the commonly expressed opinion that the indication for preprocedure FFP is “to correct” the “coagulopathy” as demonstrated by an elevated INR. AbdelWahab and colleagues10 reported the effect of FFP on the INR in 121 patients transfused with 324 units of FFP. All patients had INR values ranging from 1.1 to 1.85 before transfusion and repeat INR values measured within 8 hours of receiving 1 to 6 units of FFP. Transfusion of FFP resulted in normalization of INR values in
1% of patients and decreased the INR value halfway to normal in 15% of patients. The median decrease in INR as a result of transfusion was a negligible 0.07. See Fig 38-3. Holland and Brooks11 extended these findings to a larger cohort of adult and pediatric patients treated at the University of Oklahoma. They observed that transfusion of FFP had no effect on the INR values for patients with INRs in the range of 1.7. At higher levels of INR before transfusion, FFP exerted an effect. The effect of a unit of FFP on the INR was linear (r2  0.82) for INR values above 1.7 and was represented by an INR change of 0.37 to 0.47. Thus, for a patient with an INR equal to 3, each unit of FFP would be expected to reduce the INR by 0.64. Other studies have also found that FFP fails to normalize laboratory tests.12-17 The effect of FFP transfusion on the INR is seen in Fig 38-2. Because the volume of distribution of different

Chapter 38: Blood Components to Achieve Hemostasis

coagulation factors is larger than the intravascular space, 2 units of FFP would be expected to increase the concentration of coagulation factors by 5% to 15%. When the pretransfusion INR is below 1.7, a 10% increase in coagulation factors will have a trivial effect on the measured PT. This fact leads to the following common sequence of events. Because a patient’s INR exceeds the local threshold value, FFP is infused and a bedside procedure is performed without checking for any effect of transfusion on the INR. The procedure goes smoothly and the successful outcome is attributed to the transfusion. However, had the INR been measured after the FFP infusion, the clinician would have observed that the posttreatment INR was still above the clinician’s own personal threshold. This simple finding combined with the successful procedure outcome makes two points: first, that the treatment failed to “correct” the abnormality; and second, that the procedure was safely performed despite an INR value that was above the clinician’s own personal threshold. There is no evidence that prophylactic transfusions given before an invasive procedure will limit the extent of bleeding more effectively than therapeutic transfusions given after a procedure. Kitchen’s review noted that only 3% of patients with abnormal coagulation screening tests actually experience surgery-related bleeding. If one were to accept the undocumented opinion that 4 units of FFP and 6 units of platelets given before a procedure would actually decrease the extent of bleeding at the time of the procedure, then 100 candidate patients with abnormal tests would collectively require 1000 units of components as preprocedure prophylaxis. Of these 100 patients, 97 would receive transfusion unnecessarily, encountering a fixed transfusion risk for no benefit. Alternatively, if no preprocedure products were infused, then three patients would experience bleeding that could then be treated with blood components. For the same resource, each one of these three patients would have available as many as 133 units of FFP and 200 units of platelets per patient, representing a quantity of blood support that would certainly be more than adequate to treat bleeding. Thus, from the perspective of both the patient who bleeds and the patient who does not bleed, it would seem that treating well the patient who is actually bleeding is more valuable than an approach that transfuses the vast majority of blood components to individuals who do not need them.

Toxicity Resulting from Preprocedure Transfusions If the benefit from prophylactic transfusions is negligible, then even small transfusion risks drive risk-to-benefit considerations away from prophylactic transfusion. Recent studies highlight the susceptibility of ICU patients to toxicities of transfusion. These patients frequently undergo bedside procedures and receive preprocedure transfusions. Khan et al18 published an important paper documenting that FFP and platelet transfusions are associated with the development of acute lung injury in ill medical patients. They studied 841 consecutive critically ill patients admitted to intensive care at the Mayo Clinic. Of these, 298 (35%) were transfused. Acute lung injury developed more

commonly (25% vs 18%, p  0.025) in transfused patients. Transfused patients remained two-fold more likely to develop acute lung injury than nontransfused patients even when other risk factors for lung injury were accounted for. Of interest, the risk of lung injury was associated with transfusion of FFP or platelets rather than RBCs. Because other studies19 have found that the most common indication for FFP outside of surgery was prophylaxis before an invasive procedure, the findings of Khan et al underscore the potential adverse effects on critically ill patients from unnecessary exposure to FFP and platelets. A follow-up study from the same medical center confirmed concerns that FFP and platelet transfusions were associated with acute lung injury in intensive care patients. Gajic et al20 reported on 901 transfused patients. Of these, an astounding 74 (8%) developed objective signs of acute lung injury within 6 hours of transfusion. When adjusted for patient characteristics, the risk of transfusion-related acute lung injury (TRALI) was significantly higher for each of the following blood donor characteristics: female donor, history of multiple pregnancies, positive test for granulocyte or HLA antibodies, and concentration of phosphatidylcholine in the product. This paper suggests that TRALI is far more common among intensive care patients than has been previously recognized. The high frequency of TRALI in susceptible patients should, by itself, call for a reappraisal of the risk-to-benefit ratio surrounding the transfusion of blood components to nonbleeding patients.

Preprocedure Bleeding Risk for Specific Bedside Procedures Central Venous Catheter Insertion Serious complications of central venous catheter placement are rare and include pneumothorax, air embolus, and hemothorax.21 Significant bleeding following subclavian vein catheterization occurs in patients with perfectly normal hemostasis and results from inadvertent laceration of the subclavian artery.22 Among patients with advanced liver disease, the insertion of a central venous catheter for transvenous hepatic biopsy provides an excellent model to demonstrate the failure of abnormal coagulation tests to predict bleeding complications at the time of central line placement. Goldfarb and Lebrec23 reported results on central line insertions in 1000 consecutive patients with liver disease, all of whom had abnormalities of laboratory coagulation tests. Only one patient experienced a significant hematoma. Foster et al24 performed central line insertions on 259 patients with advanced liver disease awaiting transplantation. All patients had serious laboratory derangements including a mean platelet count of 49,000/µL, aPTT equal to 97 seconds, and INR mean of 2.0. No preprocedure products were given and no important bleeding complications occurred. In an interesting report by Petersen et al,25 516 consecutive patients received internal jugular cannulations before cardiac surgery. Of these, 252 (49%) were deliberately fully

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abnormal screening tests are not an indication for preprocedure FFP or platelet transfusions were reported by others.27-29

anticoagulated with heparin before surgery with a target aPTT of 1.5 times control. Central lines were placed while the patients were anticoagulated. For another 264 patients, anticoagulation was reversed before line insertion. An observer, unaware of the anticoagulation status of each patient, assessed the insertion site. Of the 22 hematomas observed, 13 were found in anticoagulated patients and nine were in nonanticoagulated persons. None of the patients with hematomas required treatment beyond topical care. Of note, among 22 episodes of inadvertent puncture of the carotid artery, the incidence of subcutaneous hematoma formation was the same in both the anticoagulated patients (four of 12 patients with carotid puncture) and the nonanticoagulated patients (three of 10 patients with carotid puncture). Fisher et al26 reported results among 658 cannulations that were evenly balanced among internal jugular and subclavian vein insertions. All patients had advanced liver disease and abnormal coagulation tests. The median INR was 2.4 (range, 1-16) and the median platelet count was 81,000/µL (range, 9000-1,088,000/µL). In 580 cases, the INR was 1.5; in 531 cases the platelet count was
150,000/µL; and in 453 cases both abnormalities were present. Preprocedure FFP or platelets were not given routinely. One patient, with near normal coagulation tests, had a bleeding complication attributed to technical mishap. Superficial skin hematomas and mild oozing at the puncture site were observed. Logistic regression analysis identified INR values 5.0 (n  137) or platelet counts
50,000/µL (n  146) as statistically associated with a higher incidence of superficial hematomas or site oozing, respectively. The greatest predictor (odds ratio, 8.0) of a procedure-related hematoma was a failed guidewire attempt (n  54). This paper underscored the fact that technical mishaps remain the dominant cause of hematoma formation, and that bleeding complications other than skin hematoma or oozing are rare regardless of the coagulation test results. The paper suggests that patients with an INR  5 or a platelet count
50,000/µL are at a higher risk for simple skin hematoma or oozing following central line placement. Similar studies documenting that

Liver Biopsy Closed liver biopsy has generally been considered one of the higher risk bedside invasive procedures. Several features may account for this reputation: patients with liver disease often have multiple derangements of coagulation tests and are known to have truly impaired global hemostasis; biopsies are performed without the aid of direct pressure on the wound site; patients undergoing biopsy may have vascular malignancies. Indeed, sensitive studies using ultrasound analysis have detected small intrahepatic hematomas in as many as one-quarter of the patients who undergo a liver biopsy.30 For these reasons, there is a strong desire to identify which patients are at increased risk for bleeding. Some programs will not perform closed biopsies on patients with certain locally determined abnormalities of hemostatic screening tests. These patients may receive an open surgical biopsy, a transjugular biopsy, a laparoscopic biopsy,31 or a biopsy followed by gelfoam plugging.32 More than 25 years ago, Ewe et al33 performed an informative and unique study in which patients underwent liver biopsy using a laparoscope. Following the procedure, the investigators left the laparoscope in place and measured the duration of bleeding from the site of the biopsy. Among 200 consecutive patients with a variety of nonmalignant liver disorders, most patients had at least one abnormal coagulation test but none were treated with blood components before the procedure. The study showed that there was no correlation between the length of time the liver bled after puncture and the preprocedure laboratory values. See Fig 38-4. Terjung et al34 reviewed bleeding outcomes among 629 percutaneous liver biopsies performed in 574 patients. No patient had a severe derangement of clotting tests, but some patients had INR values as high as 1.4 and platelet counts below 60,000/µL. Preprocedure transfusions were only rarely given (4%). Bleeding Coagulation time (% activity)

Liver bleeding time (min) 30

Liver bleeding time (min)

Platelet count

18 15

12

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4.5 min average 6

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8 4.5 min average

x 4’37” 4

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% 10

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Prothrombin time (quick)

Figure 38-4. Lack of correlation between bleeding from the site of liver biopsy and preprocedure coagulation test or platelet count. The duration of bleeding from the site of puncture of the liver (y-axis) is plotted as a function of the preprocedure platelet count (left-hand panel) or the preprocedure coagulation test (expressed as % activity, right-hand panel). There is no correlation between preprocedure laboratory test results and the duration of bleeding. Used with permission from Ewe.33

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was defined as: “clinically overt” (n  10, 1.6%); a postprocedure decline in hemoglobin 2 g/dL (n  45, 7.1%); or a hematoma detected after the procedure among 292 patients receiving ultrasound (n  31, 11%). Using multiple logistic regression analysis, no correlation was found between bleeding risk and the mild to moderate abnormalities in the preprocedure INR, aPTT, platelet count, or bleeding time. In a prospective study, Makris et al35 measured INR, aPTT, thrombin time, fibrinogen, bleeding time, and platelet count in 104 patients before liver biopsy. All patients underwent computed tomography scanning 24 hours after the procedure to assess bleeding. More than 50% of patients had one or more abnormalities of laboratory screening test results. INR values ranged as high as 2.0 and platelet counts were as low as 50,000/µL. Only two patients demonstrated procedure-related bleeding and both of these had normal preprocedure laboratory test results. Other studies examined the role of preprocedure laboratory testing or preprocedure infusion of blood components to prevent bleeding. All studies (except one) found no evidence for the predictive value of screening tests or the clinical value of preprocedure blood component prophylaxis.36-40 One study suggested that a prolonged preprocedure skin bleeding time assay was associated with bleeding after biopsy, but this small study involved only nine patients with bleeding, three of whom had received marrow transplants.41 Given the strong (albeit unfounded) reliance on the INR as a key measure of hemostasis in liver disease and given the dependence of the INR test on Factor VII, it is not surprising that investigators might explore the role of recombinant Factor VIIa given before liver biopsy. Jeffers et al42 studied 63 patients with a mean INR of 2.0 who received recombinant Factor VIIa in doses ranging from 5 to 120 µg/kg 10 minutes before biopsy. Hemostasis at 10 minutes failed in 30% of patients, most of whom were given a postprocedure dose of 80 µg/mL. No toxicity was observed. This study unfortunately lacked a control group and no follow-up testing for hematoma formation was performed. Although the ability of recombinant Factor VIIa to improve the INR in patients with liver disease is undeniable, the fundamental question is whether preprocedure treatment with it is superior to treatment with FFP or to no treatment at all. This would be best answered in a prospective, randomized, controlled clinical trial. The evidence for and against the use of recombinant Factor VIIa for patients without hemophilia was reviewed in a recent Cochrane report.43

Thoracentesis and Paracentesis Given the lack of evidence in favor of preprocedure transfusions for central venous catheter placement or for closed liver biopsy, it is not surprising that published evidence also fails to support the use of preprocedure prophylaxis for less invasive procedures such as thoracentesis or paracentesis. McVay and Toy44 reported a retrospective review of outcomes in 608 consecutive procedures (391 paracenteses, 207 thoracenteses, and 10 both procedures). Patients were not transfused before the procedure.

No patient had serious bleeding. The proportion of procedures that were accompanied by a decline in hemoglobin was the same in the group with normal INR/aPTT values as in the group with abnormal INR/aPTT values. A decline in hemoglobin following the procedure also did not correlate with the preprocedure platelet count. The change in hemoglobin was not different among 87 patients with both thrombocytopenia and prolonged coagulation times compared to those with normal values. Bleeding following paracentesis was examined by Webster et al,45 who reported a retrospective analysis of 179 outpatients. Four patients developed intra-abdominal or abdominal wall bleeding that required a transfusion of RBCs. The INR values of these four were indistinguishable from those of the 175 patients who did not bleed.

Gastrointestinal Endoscopy and Biopsy The use of aspirin or nonsteroidal anti-inflammatory drugs (NSAIDs) and procedure-related bleeding was addressed by Shiffman et al.46 Of 694 patients who underwent either upper endoscopy with biopsy or colonoscopy with biopsy or polypectomy, half had recently taken either aspirin or NSAIDs. Four patients had serious bleeding requiring hospitalization or treatment—two in the group taking NSAIDs/aspirin and two in the group not taking these medications. After colonoscopy, minor bleeding requiring no therapy was significantly more common in the group taking NSAIDs (6%) than among those taking no medication (2%) (odds ratio, 3).

Procedures on the Upper Airway, Bronchoscopy, and Transbronchial Lung Biopsy Special care to provide good hemostasis is prudent when procedures are being performed on the oropharynx, trachea, or bronchi, given that excessive bleeding can be rapidly fatal. Tonsillectomy was an early subject for studies assessing preoperative screening. As with other surgical procedures, the predictive value of an isolated abnormal coagulation screening test for bleeding at tonsillectomy or adenoidectomy is very poor. The majority of patients who bleed during these procedures have normal presurgical tests.47,48 Tracheotomy is commonly performed in very ill patients in intensive care, many of whom may have abnormal laboratory tests for coagulation. Auzinger et al49 partially addressed the approach to such patients in a prospective study of procedurerelated bleeding in a cohort of 60 patients with severe liver disease undergoing percutaneous tracheotomy. Patients ranged in age from 18 to 60 years of age and had high severity of illness scores. All but two patients had derangements of laboratory coagulation tests or platelet count. Coagulation abnormalities could not be corrected in 25 patients and 13 patients had platelet counts
30,000/µL. Only one patient had excessive bleeding (defined as 150 mL), which was external (extratracheal). No patient required open surgical revision. Kluge et al50 reviewed a 6-year experience with tracheotomy performed in 42 patients with thrombocytopenia. The mean platelet count was 26,000/µL.

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Two patients (
5%) demonstrated excess bleeding. Beiderlinden and colleagues51 found a low incidence of procedure-related bleeding in 136 patients of whom 18 were known to have a significant coagulopathy. Similar results were found by Ben Nun et al52 who reported the absence of significant procedure-related bleeding in 157 patients of whom 12 had coagulation abnormalities that could not be normalized despite factor replacement or cessation of anticoagulation. Although these studies all include small numbers of patients with abnormal coagulation tests and do not exclude the possibility that bleeding complications might be slightly higher among patients with measured defects in coagulation, the publications taken together certainly refute the notion that mild to moderate prolongation of the INR or aPTT or mild to moderate thrombocytopenia represent absolute contraindications to safe tracheotomy. Bronchoscopy and biopsy was directly addressed in an early study by Kozak and Brath.53 They reviewed 274 patients undergoing 305 fiberoptic bronchoscopy and biopsy procedures at a tertiary care institution. Abnormal coagulation screening tests were found in 10% (n  28) of patients. Overall, 35 patients bled, and 32 of these had normal preprocedure laboratory values. Three patients had severe bleeding and each of them had normal preprocedure test results. Although the authors noted no utility for prebronchoscopy tests of hemostasis, the number of patients with defined hemostatic abnormalities was too few to develop risk ratios with small confidence intervals (CIs). Further evidence that preprocedure coagulation tests are not a suitable guideline for preprocedure transfusion comes from the work of Diette et al.54 They reported a retrospective review of 720 fiberoptic bronchoscopies performed over a 1-year period at Johns Hopkins. Using multivariate analysis, they found that bleeding did not correlate with coagulation parameters or platelet count, and that performing a transbronchial biopsy did not independently increase the risk of bleeding. Postprocedure hemoptysis and bleeding (25 mL) were associated with prior lung transplant (odds ratio, 3.4; 95% CI, 1.3-8.6) and long procedure time (odds ratio, 2.5; 95% CI, 1.2-5.0). The higher incidence of bleeding among lung transplant recipients may relate to anatomic causes such as fragile tissue from poor nutrition or chronic exposure to steroids, mucositis from immune suppression and superinfection, or elevated pulmonary vascular pressures. Bjortuft et al55 monitored blood loss following 105 consecutive transbronchial lung biopsies and found no correlation with the INR, aPTT, bleeding time, or platelet count. Herth et al56 studied whether aspirin ingestion increased the extent of bleeding at the time of bronchoscopy and transbronchial lung biopsy. Of 1217 patients, 285 were taking aspirin at the time of the procedure. Bleeding complications occurred in 57 patients (4.7%). The incidence of minor (1.8 vs 2.9%), moderate (1.1 vs 1.4%), and severe (0.8 vs 0.9%) bleeding was not statistically different in those who took aspirin and those who did not. This report provides clinical evidence that there is no requirement to discontinue aspirin ingestion before transbronchial biopsy, nor is the history of recent or current aspirin use an appropriate indication for preprocedure

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platelet transfusion. Laboratory tests failed to correlate with bleeding in a separate report by Weiss et al.57 In an interesting animal study, Brickey et al58 did transbronchial biopsies in 18 swine who were treated with escalating doses of warfarin. The object of the study was to determine the INR level at which excess bleeding would occur following the invasive procedure. Excess bleeding was defined as 100 mL blood loss among 50% of animals. To the surprise of the investigators, the warfarin-treated animals did not develop excessive postprocedure bleeding despite the fact that 11 of the 18 animals had INR levels 7 at the time of the biopsy. Three highly anticoagulated animals died from spontaneous, nonpulmonary hemorrhage from ruptured ovarian cysts. The authors concluded that INR elevation did not correlate with bleeding following transbronchial lung biopsy in this animal model.

Renal Biopsy Davis and Chandler59 examined the predictive value of hemostasis tests obtained before biopsy of renal allografts. It should be noted that a renal allograft is placed in the pelvic area close to the skin surface and is accessible to biopsy. The template bleeding time, INR, aPTT, platelet count, and thromboelastograph results were examined in 120 patients. Bleeding was defined either as 4% decline in hematocrit or as ultrasound evidence of a perirenal hematoma. Overall, 21% of patients showed evidence of mild bleeding, two showed evidence of hematoma on ultrasound, and none required transfusion for bleeding. Most patients with bleeding had normal test results before biopsy. A weakness of this study is that only a few patients had moderate to severe abnormalities of coagulation. Procedure-related bleeding among 70 patients with combined liver and kidney disease who underwent transjugular renal biopsy was reported by Jouet et al.60 The published literature is not sufficient to draw any strong conclusions regarding hemostatic defects and bleeding at the time of renal biopsy. Because patients undergoing a renal biopsy may have platelet dysfunction from renal insufficiency, it would seem that desmopressin might be a very useful agent to administer before biopsy.

Epidural Anesthesia, Diagnostic Lumbar Puncture, Neurosurgical Procedures Because bleeding in the closed space of the subarachnoid or epidural region can produce paraparesis or paraplegia following lumbar puncture, avoiding preventable bleeding is more important for lumbar punctures and epidural anesthesia than for most invasive procedures. Nevertheless, several studies provide evidence that thrombocytopenia is not a major contraindication to diagnostic LP. Howard et al61,62 reported an extensive experience with 5000 LP procedures performed in 958 children with leukemia. These children received repeat LPs for administration of intrathecal chemotherapy. Despite the fact that platelets in these children may have been dysfunctional because of leukemia or antibiotics, there were few hemorrhagic complications. Some 170 LPs were performed in patients with platelet counts between 10,000 and 20,000/µL;

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742 LPs were performed in patients with platelet counts between 20,000 and 50,000/µL; and 858 LPs were performed in patients with platelet counts between 50,000 and 100,000/µL. Platelet transfusions were not given before the procedure. No patient developed spinal hematoma or a clinical bleeding complication. The authors defined a “bloody” LP as one with 500 red cells/µL. In contrast, normal blood has ⬃ 5  106 red cells/µL. Using this very conservative definition of procedure-related bleeding, they noted that the likelihood of finding 500 red cells/µL in the spinal fluid was slightly higher in patients with platelet counts
100,000/µL compared to those with counts 100,000/µL (odds ratio, 1.5; 95% CI, 1.2-1.8). However, among patients with counts
100,000/µL, the likelihood of an LP with 500 red cells/µL did not increase as the platelet count declined to levels as low as 10,000/µL. The authors concluded that a platelet count of 10,000/µL was adequate to perform a routine LP for administration of intrathecal chemotherapy. A smaller but similar retrospective study extended these same conclusions to adults with leukemia. Vavricka et al63 reviewed records of 195 LPs performed on 66 adult patients (age 18-68) over a 6-year period. No patient developed spinal hematoma or clinical hemorrhagic complications. As found by Howard et al,61,62 patients with platelet counts
100,000/µL were more likely to have microscopic bleeding (500 red cells/µL). The authors recommended that prophylactic transfusions be given only if the platelet count were
20,000/µL. These studies provide evidence that an LP, if of diagnostic importance, should not be delayed or withheld because of thrombocytopenia. Other studies, with smaller numbers of patients, also found no relationship between hemorrhagic complications of LP and the platelet count. For example, Waldman et al64 used a small (25-gauge) needle to administer morphine via a caudal block to 19 patients with platelet counts
50,000/µL without bleeding complications. Rasmus et al65 reported that none of 14 women with platelet counts ranging from 15,000/µL to 100,000/µL developed complications from epidural anesthesia given at the time of childbirth. Hew-Wing et al66 reviewed the literature on the issue of epidurals and thrombocytopenia and could find no case report of a thrombocytopenic patient who developed a hematoma after epidural anesthesia. However, they did identify one report of eight cases of spinal subdural hematoma complicating LP among leukemia patients.67 In contrast to thrombocytopenia, the chance of peridural hematoma appears to be increased among patients with fulldose anticoagulation who undergo placement or removal of an epidural catheter for spinal anesthesia. In patients with low-dose anticoagulation undergoing vascular surgery, Rao and El-Etr68 did not find evidence of epidural hematomas following placement of either an epidural catheter (n  3164) or a subarachnoid catheter (n  847) using a 17-gauge needle. The mean heparin dose was 10,500 units per 24 hours. Catheters were left in the spinal space for 24 hours and were then removed while patients were still being treated with heparin. No patients developed peridural hematomas. In a similar manner, low-dose heparin was not associated with spinal hematoma in over 5000 patients

undergoing spinal or epidural anesthesia.69 However, with fulldose anticoagulation, the risk may increase. For example, Ruff and Dougherty70 reported five cases of paraplegia among 342 patients who received a diagnostic LP and then were administered full-dose heparin (exact dose not stated). In addition, the introduction of low-molecular-weight heparin treatment in the United States was accompanied by an observed increase in reported spinal hematomas among patients with epidural catheters, estimated to occur at a rate of 1 in 10,000 patients.71 In this author’s experience, neurosurgeons are often strong advocates for preprocedure transfusions before invasive procedures. Some neurosurgeons will flatly refuse to perform procedures they believe to be otherwise clinically indicated solely based on mild to moderate abnormalities of the preprocedure laboratory results. Against this position is the report by Schramm et al72 who reviewed outcomes among 1211 patients having neurosurgical procedures at the Royal Melbourne Hospital in Australia. Consecutive neurosurgical procedures were examined for a period of 1 year to identify preoperative risks for bleeding. The authors tabulated not only clinical features such as the presence of malignancy or renal failure, liver disease, trauma, history of alcohol use, malabsorption, history of prior surgical bleeding, and medications, but also results of laboratory tests including the INR, aPTT, and platelet count. Three groups of surgical procedures were examined: craniotomies (n  675), spinal surgeries (n  400), and other (n  136). The authors found that 17% of preoperative laboratory tests were abnormal. Using logistic regression analysis to identify factors associated with bleeding, they found that neither prolongation of the INR nor depression of the platelet count predicted excessive surgical bleeding. However, two independent predictors of bleeding were found. First, procedure-related bleeding was associated with a small percentage of patients (n  14; 1%) who gave a history of bleeding and who demonstrated a prolonged aPTT (odds ratio  24; 95% CI, 4-150). These patients may have had von Willebrand disease. Second, procedure-related bleeding was found among a larger number of patients (n  675; 56%) in association with craniotomy regardless of all other factors examined (odds ratio, 10; 95% CI, 2-54).

Angiography The increasing use of interventional radiology and cardiology has created a new cohort of patients at risk for arterial bleeding following angiography. Not surprisingly, there is little evidence that preprocedure blood components are indicated for angiographic procedures. Darcy et al73 prospectively studied 1000 consecutive patients undergoing femoral arterial puncture for diagnostic or therapeutic vascular procedures. Analysis was restricted to patients with INR values
1.5. The incidence of hematoma formation was the same in patients without any abnormality of the preprocedure INR (9.7%) as those with abnormal results (8.1%). Neither the INR nor the aPTT was predictive of hematoma formation and each test had a
10% positive predictive value. However, hematoma formation was more common among patients with platelet counts
100,000/µL, with

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the use of catheters larger than 5 Fr, and among patients with a documented history of bleeding disorders.

Recommendations Prospective, controlled trials in which patients with similar degrees of abnormal hemostasis are randomly assigned to receive or not receive preprocedure blood components are needed to be able to make firm recommendations on the value of prophylactic transfusions before bedside invasive procedures. Given the low incidence of any form of hemorrhagic complications even among patients with known defects, such studies should be designed as multicenter trials in order to accrue a sufficient number of patients to have adequate statistical power. In the absence of such studies, the current literature can serve to suggest some guidelines for the clinical approach to bedside procedures. There is a substantial lack of evidence that mild to moderate abnormalities of the INR or aPTT can be or should be used as an indication to administer preprocedure FFP. Profound thrombocytopenia (
20,000/µL), although a poor predictor of bleeding, arises more commonly than prolongation of the INR or aPTT as an independent risk factor for procedure-related bleeding. Indeed, severe thrombocytopenia (
20,000/µL) represents not only a more correctable defect but also a greater absolute defect in hemostasis than mild to moderate prolongation of coagulation times. On the basis of the published literature, preprocedure transfusions are not routinely required for patients with an INR
3 accompanied by a platelet count 50,000/µL; nor are they required for patients with 20,000/µL platelets accompanied by an INR
1.7. For patients with mixed coagulation disorders (for example, patients with platelet counts in the range of 20,000 to 50,000/µL accompanied by an INR in the range of 1.7 to 3.0) individual decisions regarding the risk of bleeding are needed. These decisions may be influenced by experience of the operator, technical factors, anatomy, and disease-related comorbidities. When deciding on the clinical approach to a particular patient, clinicians and laboratorians should recognize that any strategy of prophylactic blood transfusions triggered solely by laboratory screening test results is not supported by published evidence. Clinicians must recognize that mild to moderate abnormalities of laboratory screening tests do not necessarily define a clinical defect in hemostasis. In addition, the decision to transfuse blood components for hemostasis is completely defensible when transfusions are given to arrest bleeding, but are difficult to justify as prophylaxis. As is true throughout clinical medicine, the best decisions are made in the context of each patient’s full clinical picture rather than on the basis of laboratory values alone.

Transfusion of Blood Components to Treat Bleeding The principal question in addressing transfusion therapy for the bleeding patient is to decide whether the bleeding is primarily localized or generalized.

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Local Treatments for Local Bleeding Upon removing a large kitchen knife from a stab wound to the chest and seeing copious blood emerge from the wound, no physician would elect to treat the problem by ignoring the wound and infusing FFP in the antecubital vein. Yet, countless patients with local bleeding are treated this way every day. Patients with bleeding varices, bleeding ulcers, bleeding from central lines, or bleeding postoperatively from surgical sites are frequently given blood component infusions to stop local bleeding, when a direct approach to the site of bleeding would be more effective. Systemic infusion of blood components for local bleeding is most common in patients who have mild abnormalities of coagulation tests, presumably under the unsupported assumption that the local bleeding results from hemostatic defects reflected by these laboratory tests. While systemic infusion of FFP or platelets may contribute to improved hemostasis at the site of tissue injury and bleeding, transfusions are secondary to a direct approach to hemostasis. Suture ligation of a damaged vessel remains the single most effective hemostatic treatment for localized bleeding and, if technically possible, should always be considered the treatment of choice. For bleeding from vessels that cannot be ligated, a variety of other choices exist including: electrocautery, injection sclerosis of vessels, argon-laser beam coagulation, and direct packing and compression.

Topical Agents: Collagen, Cellulose, and Gelatin A variety of topical agents are used routinely during surgery to improve hemostasis, especially at sites of bleeding that cannot be approached by ligature. None of these agents are perfect and all rely upon creating a surface for platelet adhesion and fibrin clot formation. Although useful for oozing, they are washed away by vigorous bleeding and are no substitute for suture. The available compounds are sterile and can be left inside the body, where they dissolve. There is very little known toxicity to these agents. Commercially available agents include products based on collagen (Avitene, C.R. Bard, Murray Hill, NJ; FloSeal, Baxter, Deerfield, IL), cellulose (Surgicel, Johnson & Johnson, New Brunswick, NJ), and gelatin (Gelfoam, Pfizer, New York, NY). For more information, the reader is referred to a fine review by Jackson.74 Topical Sealants In some situations, topical application of blood components to the site of bleeding can be therapeutic. A common example is the use of “fibrin sealant.” Fibrin sealant consists of the simultaneous application of topical fibrinogen (usually supplied as 1-2 units of cryoprecipitate) plus topical thrombin. Fibrin sealant will not stanch massive bleeding, but it can prove very useful for sites where a suture cannot be placed. Fibrin sealants are also provided commercially. Currently, Tisseel VH (Baxter) is licensed for use in the United States. It consists of separate vials containing either purified human fibrinogen plus aprotinin or purified human thrombin. Fibrin sealants may also be packaged with a

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pump used to spray the sealant on a bleeding surface. Fibrinbased sealants have also been incorporated into dressings that may be applied to the site of bleeding to provide not only direct pressure but also local high concentrations of fibrin for clot formation. Fibrin sealant dressings are under active investigation for use by the military and for trauma applications. Fibrin sealant sprays have been used in cardiac or vascular surgery, especially to address bleeding along lines of vascular anastamosis. Liquid fibrin sealants are used in burn care, liver surgery, maxillofacial surgery, neurosurgery, and some orthopedic surgery.74

Topical Thrombin Topical thrombin of bovine origin is available as a powder, packaged as 5000 units or 20,000 units. It is usually reconstituted in a diluent and applied at concentrations of 1000-2000 units/mL. It may also be applied directly in powdered form to the site of bleeding. Thrombin is also marketed as a liquid in a spray pump bottle. Recently, the Food and Drug Administration approved both a human thrombin (Evithrom, Johnson & Johnson) and a recombinant thrombin (Recothrom, ZymoGenetics, Seattle, WA). The use of bovine-derived thrombin has been associated in a minority of patients with the development of antibodies directed against bovine coagulation proteins in the preparation. These antibodies include Factor V antibodies and thrombin antibodies. In some patients, these antibodies can cross-react with human coagulation proteins, resulting in the development of a coagulation inhibitor. In some of these patients, the inhibitor can be clinically significant and represents a serious adverse effect of bovine thrombin. However, clinically significant inhibitors are very infrequent and this risk of inhibitor formation should be further reduced with the advent of human and recombinant thrombin. Thus, direct thrombin application remains a useful adjunct to topical hemostasis. Topical Antifibrinolytics In surgical cases, such as trauma surgery, some patients may have substantial diffuse oozing with no defined vessel to ligate. For such bleeding, direct packing and closure with subsequent return to the OR for definitive hemostasis can be lifesaving. In addition to simple packing with sterile gauzes, some clinicians believe there is benefit to packing bleeding sites with gauzes made wet with applied thrombin, antifibrinolytics (eg, Amicar, Xanodyne Pharmaceuticals, Cincinnati, OH), or platelets. The author’s institution uses gauzes applied with antifibrinolytics in selected patients. Treating Wound Fibrinolysis The serosal cavities of the body are lined by tissues that are rich in fibrinolytic proteins such as tissue plasminogen activator. When blood enters such cavities it undergoes rapid clot lysis. This accounts for the observed finding that blood in the pericardial space, the pleural space, the peritoneal space, or in joint spaces exists as a liquid that can be extracted with a needle. Blood extracted from such cavities will not clot ex vivo because it has

already undergone complete clot lysis. An example of this is seen with the unclottable blood collected into canisters from chest tube drains. A common problem after chest surgery is bleeding into the pleural and mediastinal space. In some patients, if blood drainage is incomplete, the local environment surrounding the bleeding wound site can become bathed in fibrinolytic blood. Thus, wound bleeding into intrapleural, intramediastinal, or intra-abdominal sites can result in an unfavorable cycle in which bleeding into the cavity undergoes clotting and lysis and the fibrinolytic process breaks down clots and results in more bleeding. The principal treatment for this condition is removal or drainage of blood from the body cavity, a therapy underscored by the time-honored surgical practice of washing out retained blood. However, when this is not possible, wound fibrinolysis may occur and simple infusion of blood components is not likely to be effective. For these patients, a short course of a systemic antifibrinolytic agents (eg, Amicar) may be beneficial.

Treatment of Generalized Bleeding Skin and Mucous Membrane Oozing Visible, annoying, distasteful, frightening, and unsightly skin and mucous-membrane bleeding often demands attention even if not life-threatening. Often a sign of thrombocytopenia or impaired platelet function, skin and mucous-membrane bleeding may also result from drug eruptions, mucositis, burns, or other damage to skin. Platelet transfusions may be given to patients with petechiae under the assumption that skin bleeding is a harbinger of more serious life-threatening bleeding to come. It is difficult to prove or refute this contention, but petechiae or mucosal bleeding in conjunction with thrombocytopenia would seem a far more logical indication for prophylactic platelet transfusion than a platelet count alone. For patients who are refractory to platelet transfusions as a result of alloimmunization, oral or intravenous antifibrinolytics have been used. Purpura and Soft-Tissue Bleeding Purpura and soft-tissue bleeding may imply a serious hemostatic defect and are nearly always worthy of investigation. An exception is “senile purpura”—a largely cosmetic problem limited to the extensor surfaces of the forearms in elderly patients with visibly fragile skin. Purpura generally suggests an abnormality in fibrin formation and treatment is directed at the underlying defect. When no specific hemostatic abnormalities are found, the cause may prove to be vasculitis, drug purpura, amyloidosis, or even factitious. Vasculitic bleeding can range from dermal bleeding, as seen in palpable purpura, to life-threatening hemorrhage as seen in pulmonary vasculitis. Rare coagulation defects, such as Factor XIII deficiency, dysfibrinogenemias, or conditions with excessive fibrinolysis may result in purpura without striking abnormalities found on routine laboratory tests. Aneurysms and vascular anomalies can sometimes present as confusing cases of unexpected bleeding. Patients with persistent recurrent gastrointestinal bleeding may have Osler-Weber-Rendu disease (hereditary hemorrhagic telangiectasias). In some patients, the

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external telangiectasias that provide the clue to the diagnosis are subtle and limited to the oral mucosa. Vitamin C deficiency (scurvy) typically results in gum bleeding and perifollicular petichiae, but more substantial bleeding with large plate-like areas of skin and soft-tissue purpura may also occur. Unrecognized von Willebrand syndrome or platelet storage pool disorders can result in unexpected bleeding, although most patients with serious examples of these conditions will give a history of easy bruising, heavy menstrual bleeding, iron deficiency, or prior surgical hemorrhage.

Small Vessel Bleeding During Surgery Diffuse small vessel bleeding during surgery is not uncommon. In many cases, such generalized bleeding is a sign of a recognizable and correctable hemostatic disorder. Anesthesiologists and surgeons caring for patients undergoing major surgery need access to basic coagulation tests with rapid turnaround time. Usually the patient with diffuse oozing at the end of surgery has developed dilutional thrombocytopenia or dilutional depletion of coagulation factors and these conditions can be corrected by transfusion. In other cases, the results of intraoperative coagulation testing point more toward disseminated intravascular coagulation. This condition should be anticipated if the patient has experienced prolonged hypotension with tissue hypoperfusion and ischemia. In this author’s experience, a commonly overlooked cause of diffuse small vessel bleeding is fibrinolysis. With hypotension, pressors, and acidosis, patients will release tissue plasminogen activator from vascular sites. The plasmin that is formed localizes preferentially to fibrin. Therefore, even before a patient will demonstrate fibrinogenolysis (with low systemic fibrinogen levels), the patient will first develop fibrinolysis with dissolution of previously formed clot. In this early fibrinolytic response, there is no measurable abnormality of the circulating blood except for the presence of increased concentrations of D-dimers as a sign of fibrin clot dissolution. Indeed, some degree of fibrinolysis is nearly universal in surgery. Thus, the observation in the OR that the patient is oozing from surgical sites that were previously well clotted should be taken as a sign of the possibility of increased clot lysis. Fibrinolysis is not corrected by infusions of FFP, which may only serve to add plasminogen and create more plasmin. If the bleeding is sustained and if there are no contraindications to the use of an antifibrinolytic agent, then a single bolus dose or a loading dose followed by a brief infusion of an antifibrinolytic may prove very useful.

Conclusion James Blundell administered the first human blood transfusions in the early years of the 19th century to treat patients who were actively bleeding. When viewed from an evidence-based perspective, little has changed in 200 years. Blood component therapy remains a remarkable lifesaving treatment of unquestionable value in patients with significant bleeding. Transfusion

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of blood to bleeding patients rests on solid principles of resuscitation, tissue oxygenation, and the repletion of vital elements of hemostasis. In stark contrast, transfusion given as prophylaxis based on abnormal screening tests offers unfavorable risk-tobenefit and cost-to-benefit ratios.75 Clinical research using the outcome measure of bleeding should be conducted to refine the indications for blood use at surgery and before invasive procedures. Better tests of hemostasis are needed. In the meantime, many lives will be saved by transfusion given to bleeding patients. In some cases, these transfusions will directly arrest bleeding by replacing depleted elements of hemostasis. In many more cases, transfusion will serve as an exquisite life-support measure while other interventions are used to stop hemorrhage.

Disclaimer The author has disclosed no conflicts of interest.

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Chapter 38: Blood Components to Achieve Hemostasis

13. Gazzard BG, Henderson JM, Williams R. The use of fresh frozen plasma or a concentrate of factor IX as replacement therapy before liver biopsy. Gut 1975;16:621-5. 14. Youssef WI, Salazar F, Dasarathy S, et al. Role of fresh frozen plasma infusion in correction of coagulopathy of chronic liver disease: A dual phase study. Am J Gastroenterol 2003;98:1391-4. 15. Williamson LM, Llewelyn CA, Fisher NC, et al. A randomized trial of solvent/detergent-treated and standard fresh-frozen plasma in the coagulopathy of liver disease and liver transplantation. Transfusion 1999;39:1227-34. 16. Lerner RG, Nelson J, Sorcia E, et al. Evaluation of solvent/detergenttreated plasma in patients with a prolonged prothrombin time. Vox Sang 2000;79:161-7. 17. Johnson CA, Snyder MS, Weaver RL. Effects of fresh frozen plasma infusions on coagulation screening tests in neonates. Arch Dis Child 1982;57:950-2. 18. Khan H, Belsher J, Yilmaz M, et al. Fresh-frozen plasma and platelet transfusions are associated with development of acute lung injury in critically ill medical patients. Chest 2007;131:1308-14. 19. Dzik W, Rao A. Why do physicians request fresh frozen plasma? Transfusion 2004;44:1393-4. 20. Gajic O, Rana R, Winters JL, et al. Transfusion-related acute lung injury in the critically ill: Prospective nested case-control study. Am J Respir Crit Care Med 2007;176:886-91. 21. Adhya S, Laha SK. Central venous catheterization. N Engl J Med 2007;357:944-5. 22. Vanherweghem JL, Cabolet P, Dhaene M, et al. Complications related to subclavian catheters for hemodialysis. Am J Nephrol 1986;6:339-43. 23. Goldfarb G, Lebrec D. Percutaneous cannulation of the internal jugular vein in patients with coagulopathies: An experience based on 1,000 attempts. Anesthesiology 1982;56:321-3. 24. Foster PF, Moore LR, Sankary HN, et al. Central venous catheterization in patients with coagulopathy. Arch Surg 1992;127:273-5. 25. Petersen GA. Does systemic anticoagulation increase the risk of internal jugular vein cannulation? (letter) Anesthesiology 1991;75:1124. 26. Fisher NC, Mutimer DJ. Central venous cannulation in patients with liver disease and coagulopathy—a prospective audit. Intensive Care Med 1999;25:481-5. 27. Mumtaz H, Williams V, Hauer-Jensen M, et al. Central venous catheter placement in patients with disorders of hemostasis. Am J Surg 2000;180:503-5. 28. Doerfler ME, Kaufman B, Goldenberg AS. Central venous catheter placement in patients with disorders of hemostasis. Chest 1996;110:185-8. 29. DeLoughery TG, Liebler JM, Simonds V, Goodnight SH. Invasive line placement in critically ill patients: Do hemostatic defects matter? Transfusion 1996;36:827-31. 30. Minuk GY, Sutherland LR, Wiseman DA, et al. Prospective study of the incidence of ultrasound-detected intrahepatic and subcapsular hematomas in patients randomized to 6 or 24 hours of bed rest after percutaneous liver biopsy. Gastroenterology 1987;92:290-3. 31. Iqbal M, Creger RJ, Fox RM, et al. Laparoscopic liver biopsy to evaluate hepatic dysfunction in patients with hematologic malignancies: A useful tool to effect changes in management. Bone Marrow Transplant 1996;17:655-62. 32. Fandrich CA, Davies RP, Hall PM. Small-gauge gelfoam plug liver biopsy in high-risk patients: Safety and diagnostic value. Australas Radiol 1996;40:230-4.

33. Ewe K. Bleeding after liver biopsy does not correlate with indices of peripheral coagulation. Dig Dis Sci 1981;26:388-93. 34. Terjung B, Lemnitzer I, Dumoulin FL, et al. Bleeding complications after percutaneous liver biopsy: An analysis of risk factors. Digestion 2003;67:138-45. 35. Makris M, Nakielny R, Toh CH, et al. A prospective investigation of the relationship between haemorrhagic complications of percutaneous needle biopsy of the liver and coagulation screening tests (abstract). Br J Haematol 1992;81:51. 36. Sharma S, McDonald GB, Banaji M. The risk of bleeding after percutaneous liver biopsy: Relation to the platelet count. J Clin Gastroenterol 1982;4:451-3. 37. McGill DB, Rakela J, Zinsmeister AR, Ott BJ. A 21 year experience with major hemorrhage after percutaneous liver biopsy. Gastroenterology 1990;99:1396-400. 38. McVay PA, Toy PTCY. Lack of increased bleeding after liver biopsy in patients with mild hemostatic abnormalities. Am J Clin Pathol 1990;94:747-53. 39. Caturelli E, Squillante MM, Andriulli A, et al. Fine-needle liver biopsy in patients with severely impaired coagulation. Liver 1993;13:270-3. 40. Dillon JF, Simpson KJ, Hayes PC. Liver biopsy bleeding time: an unpredictable event. J Gastroenterol Hepatol 1994;9:269-71. 41. Boberg KM, Brosstad F, Egeland T, et al. Is a prolonged bleeding time associated with an increased risk of hemorrhage after liver biopsy? Thromb Haemost 1999;81:378-81. 42. Jeffers LJ, Chalasani N, Balart L, et al. Safety and efficacy of recombinant factor VIIa in patients with liver disease undergoing laparoscopic liver biopsy. Gastroenterology 2002;123:118-26. 43. Stanworth SJ, Birchall J, Doree CJ, Hyde C. Recombinant factor VIIa for the prevention and treatment of bleeding in patients without haemophilia. Cochrane Database Syst Rev 2007;(2):CD005011. 44. McVay PA, Toy PTCY. Lack of increased bleeding after paracentesis and thoracentesis in patients with mild coagulation abnormalities. Transfusion 1991;31:164-71. 45. Webster ST, Brown KL, Luchey MR, Nostrant TT. Hemorrhagic complications of large-volume abdominal paracentesis. Am J Gastroenterol 1996;91:366-8. 46. Shiffman ML, Farrel MT, Yee YS. Risk of bleeding after endoscopic biopsy or polypectomy in patients taking aspirin or other NSAIDs. Gastrointest Endos 1994;40:458-62. 47. Kang J, Brodsky L, Danziger I, et al. Coagulation profile as a predictor for post-tonsillectomy and adenoidectomy hemorrhage. Int J Pediatr Otorhinolaryngol 1994;28:157-65. 48. Zwack G, Derkay CS. The utility of pre-operative hemostatic assessment in adenotonsillectomy. Int J Pediatr Otorhinolaryngol 1997;39:67-76. 49. Auzinger G, O’Callaghan GP, Bernal W, et al. Percutaneous tracheostomy in patients with severe liver disease and a high incidence of refractory coagulopathy: A prospective trial. Critical Care 2007;11: R110. 50. Kluge S, Meyer A, Kuehnelt P, et al. Percutaneous tracheostomy is safe in patients with severe thrombocytopenia. Chest 2004;126:547-51. 51. Beiderlinden M, Groeben H, Peters J. Safety of percutaneous dilational tracheostomy in patients ventilated with high positive endexpiratory pressure. Intensive Care Med 2003;29:944-8. 52. Ben Nun A, Altman E, Best LA. Extended indications for percutaneous tracheostomy. Ann Thorac Surg 2005;80:1276-9.

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53. Kozak EA, Brath LK. Do “screening” coagulation tests predict bleeding in patients undergoing fiberoptic bronchoscopy with biopsy? Chest 1994;106:703-5. 54. Diette GB, Wiener CM, White P. The higher risk of bleeding in lung transplant recipients from bronchoscopy is independent of traditional bleeding risks: Results of a prospective cohort study. Chest 1999;115:397-402. 55. Bjortuft O, Brosstad F, Boe J. Bronchoscopy with transbronchial biopsies: Measurement of bleeding volume and evaluation of the predictive value of coagulation tests. Eur Respir J 1998;12:1025-7. 56. Herth FJ, Becker HD, Ernst A. Aspirin does not increase bleeding complications after transbronchial biopsy. Chest 2002;122:1461-4. 57. Weiss SM, Hert RC, Gianola FJ, et al. Complications of fiberoptic bronchoscopy in thrombocytopenic patients. Chest 1993;104:1025-8. 58. Brickey DA, Lawlor DP. Transbronchial biopsy in the presence of profound elevation of the international normalized ratio. Chest 1999;115:1667-71. 59. Davis CL, Chandler WL. Thromboelastography for the prediction of bleeding after transplant renal biopsy. J Am Soc Nephrol 1995;6:1250-5. 60. Jouet P, Meyrier A, Mal F, et al. Transjugular renal biopsy in the treatment of patients with cirrhosis and renal abnormalities. Hepatology 1996;24:1143-7. 61. Howard S, Amar G, Ribeiro RC, et al. Safety of lumbar puncture for children with acute lymphoblastic leukemia and thrombocytopenia. JAMA 2000;284:2222-4. 62. Howard S, Gajjar AJ, Cheng C, et al. Risk factors for traumatic and bloody lumbar puncture in children with acute lymphoblastic leukemia. JAMA 2002;288:2001-07. 63. Vavricka SR, Walter RB, Irani S, et al. Safety of lumbar puncture for adults with acute leukemia and restrictive prophylactic platelet transfusion. Ann Hematol 2003;82:570-3.

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64. Waldman SD, Feldstein GS, Waldman JH, et al. Caudal administration of morphine sulfate in anticoagulated and thrombocytopenic patients. Anesth Analg 1987;66:267-8. 65. Rasmus KT, Rottman RL, Kotelko DM, et al. Unrecognized thrombocytopenia in parturients: A retrospective review. Obstet Gynecol 1989;73:943-6. 66. Hew-Wing P, Rolbin SH, Hew E, Amato D. Epidural anaesthesia and thrombocytopenia. Anaesthesia 1989;44:775-7. 67. Edelson RN, Chernik NL, Posner JB. Spinal subdural hematomas complicating lumbar puncture. Occurrence in thrombocytopenic patients. Arch Neurol 1974;31:134-7. 68. Rao TK, El-Etr AA. Anticoagulation following placement of epidural and subarachnoid catheters: An evaluation of neurologic sequelae. Anesthesiology 1981;55:618-20. 69. Schwander D, Bachmann F. Heparine et anesthesies medullaires: Analyse de decision. Ann Fr Anesth Reanim 1991;10:284-96. 70. Ruff RI, Dougherty JH. Complications of lumbar puncture followed by anticoagulation. Stroke 1981;12:879-81. 71. Horlocker TT. Thromboprophylaxis and neuraxial anesthesia. Orthopedics 2003;26(Suppl 2):243-9. 72. Schramm B, Leslie K, Myles PS. Coagulation studies in pre-operative neurosurgical patients. Anaesth Intensive Care 2001;29:388-92. 73. Darcy MD, Kanterman RY, Kleinhoffer MA, et al. Evaluation of coagulation tests as predictors of angiographic bleeding complications. Radiology 1996;198:741-4. 74. Jackson MR. Topical hemostatic agents for localized bleeding. In: Kitchens CS, Alving BM, Kessler CM, eds. Consultative hemostasis and thrombosis, 2nd ed. Philadelphia, PA: WB Saunders, 2007:503-8. 75. Stanworth SJ. The evidence-based use of FFP and cryoprecipitate for abnormalities of coagulation tests and clinical coagulopathy. Hematology Am Soc Hematol Educ Program 2007;179-86.

39

Transfusion Therapy in the Care of Trauma and Burn Patients Ellen C. Omi1 & Richard L. Gamelli2 1

Assistant Professor of Surgery, Division of Critical Care, Trauma and Burn, Loyola University Medical Center, Stritch School of Medicine, Maywood, Illinois, USA 2 The Robert J. Freeark Professor and Chairman, Department of Surgery, Director, Burn and Shock Trauma Institute, Chief, Burn Center, Loyola University Medical Center, Stritch School of Medicine, Maywood, Illinois, USA

In 1915, Lewisohn published the first reports of collecting blood in sodium citrate at Mount Sinai Hospital in New York, and by 1917, the first military blood bank was set up by Major Oswald H. Robertson of the United States Army. What drove the technology in blood transfusion and storage was the demand for transfusion during World War I.1 Although the first described blood transfusion was performed in the early 19th century, widespread acceptance of transfusion therapy did not occur until Landsteiner described the ABO blood groups. Experience gained during World War I and the Spanish Civil War led to technologies to collect, store, and transfuse blood in emergencies. Bernard Fantus in 1937 opened the first blood bank in the United States at Cook County Hospital in Chicago. The use of the preservative acid-citrate-dextrose was implemented by the end of World War II, further expanding the ability to store blood.2 During this same period, the importance of resuscitation in the treatment of shock and trauma was recognized and the use of crystalloids began. The ability to store blood in large quantities was a boon for trauma surgery. The liberal use of group O Rh-negative blood during the Vietnam War significantly reduced mortality.3 Coupled with rapid transport time, aggressive fluid resuscitation, and early operative intervention for patients not responding to resuscitation, this advance marked the beginning of modern trauma surgery. The lessons learned from wartime experience with the resuscitation of injured patients were applied to the civilian injured, and by the late 1960s, organized prehospital emergency services were rapidly transporting patients to trauma centers. The use of blood for the resuscitation and treatment of thermally injured patients was advocated as early as the 1920s and 1930s.4 Blood and blood components have since been part of various treatment regimens and burn formulas for the initial resuscitation of burn patients. The ready availability of blood and blood components has improved survival among elderly patients and patients with associated medical problems who otherwise would not have tolerated a low hemoglobin level. Intraoperative transfusion has Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

allowed wider, more aggressive débridement procedures, thereby decreasing the number of surgical procedures needed by a patient. The current indications for transfusion in trauma and burn care recognize the merits of this form of treatment but acknowledge the practical considerations regarding transfusion. The limitations of the blood supply are apparent. In the United States, more than 14 million units of Whole Blood and Red Blood Cells (RBCs) are transfused yearly to approximately 5 million recipients, with most transfusions being administered in the perioperative period. RBCs have a limited shelf life and are discarded after 42 days. In addition, the incidence of transfusion reactions and of the transmission of hepatitis was well documented by the 1970s. The risk of transmission of human immunodeficiency virus or hepatitis C virus are each estimated to be 1 in approximately 2,000,000 units of blood.5 On a yearly basis, approximately 35 patients die per year of hemolytic transfusion reactions (mostly from ABO-incompatible transfusion), transfusion-related acute lung injury, or bacterial contamination.6 Concerns have also been raised about the possible immunosuppressive effects of transfusion, citing associations between transfusion and infections or recurrence of malignancies.7,8 In addition, it is known that 50% of severely injured patients who survive the immediate posttrauma phase are at increased risk for the development of the systemic inflammatory response syndrome and may be vulnerable to immunomodulatory effects of transfusions. Although a transfusion is still a lifesaving treatment in the appropriate circumstances, the foregoing observations have led to a more judicious use of blood and blood components as the risk-to-benefit considerations are more fully understood. Current blood use in treating trauma and burns, therefore, must combine judgment and careful assessment of the risk-to-benefit ratio.

Shock Definition of Shock Shock is defined as inadequate organ perfusion and tissue oxygenation. After trauma, the most common type of shock is hemorrhagic. Neurogenic shock after high cervical spinal injuries and septic shock after penetrating trauma also are possible. After burns,

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hypovolemic shock is caused by fluid sequestration at the site of injury and in unburned injured tissue as a systemic response to injury and by evaporative loss caused by loss of the dermal barrier. A critical goal in the care of trauma and burn patients is optimization of oxygen delivery and prevention of tissue hypoxia. Although it would be ideal if there were a simple laboratory value that could identify the need for transfusion to optimize oxygen delivery, such a magic bullet does not exist. The decision to administer transfusion must be based on an understanding of the role of hemoglobin in the definition of oxygen delivery. Oxygen delivery (DO2) is the product of arterial oxygen content (CaO2) and cardiac output (CO): DO2 ⫽ CaO2 ⫻ CO. Arterial oxygen content is a measure of both hemoglobin-bound and free oxygen. It is calculated with the following equation:

Oxygen consumption

Section II: Part IV

Flow dependent Trauma Normal

330 450 Oxygen delivery mL/min/m2

600

CaO2 ⫽ (1.34 ⫻ SaO2 ⫻ Hb ⫻ 10) ⫹ (0.003 ⫻ PaO2)

Figure 39-1. Unrecognized flow-dependent oxygen consumption (VO2) under normal conditions and in a trauma state. Critical oxygen delivery (DO2) in anesthetized adult is 330 mL/minute/m2. The normal oxygen delivery index is 450 mL/minute/m2. The survivor response after normal resuscitation is 600 mL/minute/m2. Used with permission from Moore et al.13

The left term of the equation (1.34 ⫻ SaO2 ⫻ Hb ⫻ 10) calculates the amount of oxygen bound to hemoglobin. The constant 1.34 is an estimate of the mean volume of oxygen in milliliters that can be bound to 1 g of fully saturated normal hemoglobin, SaO2 is oxygen saturation, and Hb is the hemoglobin in g/dL. Ten is a correction factor converting g/dL to g/L. The right term of the equation (0.003 ⫻ PaO2) is the estimation of the dissolved component of arterial oxygen content. PaO2 is the partial pressure of arterial oxygen and the constant 0.003 is the solubility coefficient of oxygen in blood at normal body temperature and atmospheric pressure. From this equation, it is apparent that dissolved oxygen contributes little to total CaO2 at atmospheric pressure. This contribution would be increased with the use of hyperbaric oxygen, which increases the amount of oxygen dissolved in the blood. Oxygen consumption (VO2) is a measure of total oxidative metabolism and is a product of the arteriovenous oxygen difference (CaO2 ⫺ CvO2) and the cardiac output (Q). In most cases, oxygen consumption is independent of hemoglobin concentration over a wide range of oxygen delivery values; compensation occurs through increases in cardiac output and oxygen uptake by the tissues. The oxygen extraction ratio, the ratio between oxygen consumption and oxygen delivery, is approximately 25% to 30% under normal conditions. However, different tissues have a variable ability to increase oxygen extraction, further increasing tissue oxygen availability in those tissues. The heart is at maximal oxygen extraction under baseline conditions and depends on increased delivery to meet increasing oxygen demands. It has been shown in experiments in dogs that, in the normovolemic state, cardiac output progressively increases with decreases in hematocrit to a level of 8.6% ⫾ 0.4%, after which time the dogs went into heart failure.9 This occurs through reductions in blood viscosity and an increase sympathetic nervous activity.10 The change in blood viscosity decreases total peripheral vascular resistance, increases postcapillary venular flow, and increases venous return.10 The increased sympathetic activity results in increased cardiac contractility,11,12 and increased venomotor tone.10 As the hematocrit decreases from

the normal value to 30%, oxygen delivery increases to approximately 110% of baseline level.10 It has been demonstrated in dog models that these compensatory mechanisms allow maintenance of oxygen delivery and uptake until the hematocrit decreases to approximately 10%, after which there is a marked decline.11 The relationship between oxygen delivery and oxygen consumption is biphasic (Fig 39-1). At less than a critical level of oxygen delivery, consumption is related to delivery in a linear manner. At greater than critical oxygen delivery, consumption is independent of delivery, the flow-independent portion of the curve.10 Analysis of patients under anesthesia showed that the critical value of oxygen delivery differentiating flow-dependent and flow-independent oxygen consumption is 333 mL/minute/m2.14 Shoemaker et al15 compared survivors with nonsurvivors of elective surgery. They found that nonsurvivors had lower myocardial performance indicators, higher pulmonary artery pressures and pulmonary vascular resistance, and lower oxygen delivery than did the survivors. A study by Boyd et al16 showed a reduced mortality rate and reduced incidence of postoperative complications if inotropic agents were used to increase cardiac output with a target oxygen delivery of 600 mL/minute/m2. After major trauma, several factors alter the DO2/VO2 relation. First, VO2 increases to 150 mL/minute/m2 to repay ongoing oxygen debts and to support the systemic inflammatory response syndrome. At the same time, peripheral oxygen extraction is impaired, which decreases the slope of the flow-dependent portion of this relation (Fig 39-1). It is possible that a trauma patient with a normal DO2 of 450 mL/minute/m2 may be on the flow-dependent portion of the curve.17 Bishop et al18 found that resuscitating to supranormal values of vital signs, urine output, and pulmonary artery catheter parameters decreased the risk of multiple organ failure and death, but the results of this study have not been duplicated despite numerous attempts.19-21 In fact, some studies have shown an increase in the mortality of critically ill surgical and medical patients with the use of dobutamine to augment oxygen delivery and consumption parameters.22 What seems to be a common observation in these studies is

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Chapter 39: Transfusion Therapy in the Care of Trauma and Burn Patients

Table 39-1. Blood Losses and Classes of Shock Based on Patient’s Initial Presentation Class I

Class II

Class III

Class IV

Blood loss (mL)

Up to 750

750-1500

1500-2000

⬎2000

Blood loss (% blood volume)

Up to 15

15-30

30-40

⬎40

Pulse rate (beats/min)

⬍100

⬎100

⬎120

⬎140

Blood pressure

Normal

Normal

Decreased

Decreased

Pulse pressure (mm Hg)

Normal or Increased

Normal

Normal

Normal

Respiratory rate (breath/min)

14-20

20-30

30-40

⬎35

Urine output (mL/hour)

⬎30

20-30

5-15

Negligible

Mental status

Slightly anxious

Mildly anxious

Anxious, confused

Confused, obtunded

Fluid replacement (3:1 rule)

Crystalloid

Crystalloid

Crystalloid and blood

Crystalloid and blood

Patient is a 70-kg man. These guidelines are based on the 3-for-1 rule. This rule derives from the empiric observation that most patients in hemorrhagic shock need as much as 300 mL of electrolyte solution for each 100 mL of blood loss. Applied blindly, these guidelines can result in excessive or inadequate fluid administration. Used with permission from American College of Surgeons Committee on Trauma.27

that achieving adequate or supranormal oxygen delivery serves more as a prognosticator of survival than an endpoint of resuscitation. Trauma patients who do not respond to resuscitation, or to augmentation with inotropes to supranormal hemodynamic parameters, are at higher risk of mortality when compared to the patients who are resuscitated successfully or respond to inotropic support. In the care of critically ill patients in whom maximal increases in cardiac output have occurred without the necessary improvements in oxygen delivery needed to reach the flow-independent portion of the curve, transfusion is considered to optimize oxygen delivery. However, this strategy may not be effective for patients in whom oxygen delivery is adequate.23

Hemorrhagic Shock and Classification Hemorrhage results in decreased cardiac output and total body ischemia, which cannot be corrected until circulating volume is restored. Acute loss of intravascular volume results in increased vascular tone and redistribution of blood flow to the heart and brain at the expense of cutaneous, splanchnic, and renal vascular beds. Acidosis develops as tissues switch from aerobic to anaerobic metabolism. This acidosis initially facilitates the unloading of oxygen at the tissues with a shift of the oxygen dissociation curve to the right. Urine output decreases as the kidneys work to retain water and sodium in response to decreased renal perfusion. Without transfusion therapy, this correction requires the movement of fluid and protein from the interstitium to the plasma, or transcapillary plasma refill.24 Initially triggered by a decrease in capillary hydrostatic pressure, this results in movement of protein-free fluid from the interstitium to the plasma. A second phase involves movement of protein into the plasma space in support of plasma oncotic pressure. This results in restoration of plasma volume and protein concentration with reduced oxygen-carrying capacity resulting from loss of red cell mass

by hemorrhage—that is, normovolemic anemia. Transcapillary refill is capable of sustaining a relatively fixed level of plasma volume, equal to approximately two-thirds of the initial plasma volume, irrespective of the rate of bleeding. Laboratory studies have shown that plasma refill reaches 33% by 0.5 hour after hemorrhage, and 50% by 3 hours, allowing fairly rapid restoration of circulating blood volume.25,26 The amount of transcapillary refill is a function of the severity of pressure-driven hemorrhage but does not correlate with arterial pressure or cardiac output. In evaluating injured patients, it rapidly becomes apparent that trauma patients are an extremely heterogeneous population. Each patient’s resuscitative needs are different but can be broadly anticipated on the basis of initial hemodynamic status and the response to resuscitative efforts. The American College of Surgeons Committee on Trauma,27 in an effort to guide and standardize the initial resuscitation of trauma patients, has defined four classes of shock (Table 39-1). Whereas crystalloid resuscitation alone is adequate for Class I and II shock, early consideration should be given to administering blood to patients in Class III and IV shock.

The Trauma Patient There are two distinct clinical settings in the care of the trauma patient. The first is the prehospital setting and the second is the in-hospital setting. Treatment of hemorrhagic shock starts in the prehospital setting with control of external bleeding. Once the patient is in the trauma bay, in-hospital management begins and it is perhaps best for the purposes of this chapter to approach this setting in multiple phases. The first phase is the initial resuscitation followed by either an intraoperative phase to stop bleeding or to definitively treat injuries, or a phase of observation, as in the management of solid organ injury. The

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“intraoperative” phase can also take place in the angiography suite where bleeding can be controlled, most often in the presence of retroperitoneal and pelvic bleeding from pelvic fractures. The final phase of the care of the trauma patient is the recovery phase. Each of these phases differs with respect to the triggers and indications for transfusion therapy depending on the types of injuries that are inflicted and the type of patient that has sustained the injuries.

Initial Resuscitation Phase of the Trauma Patient Initial evaluation of a trauma patient, or the primary survey (ABCDEs), requires an assessment of the airway (A), breathing and ventilation (B), circulation (including the control of external hemorrhage) (C), neurologic status or disability (D), and exposure (E) of the patient. The initial resuscitation fluid of choice after trauma is an isotonic crystalloid solution, usually lactated Ringer’s solution, and is often initiated in the prehospital clinical setting. The American College of Surgeons Committee on Trauma Advanced Trauma27 recommends insertion of two largebore intravenous catheters and immediate infusion of 2000 mL warmed crystalloid solution (or 20 mL/kg for a child). Because shock after trauma most often is hypovolemic, the response to initial fluid resuscitation determines subsequent resuscitative measures. The risk of hypothermia must be recognized and prevented during the resuscitation phase. The response to fluid resuscitation determines which, if any, subsequent resuscitative measures are needed. A patient with minimal injury and normal vital signs with initial crystalloid resuscitation is called a responder to fluid resuscitation. The condition of these patients, by definition Class I or II hemorrhagic shock, remains hemodynamically normal without ongoing resuscitation. Such patients need evaluation and management of the injuries, but rarely need transfusion during the initial evaluation. A patient in compensated shock, defined as hypovolemia coexistent with normotension but with serious metabolic derangement, often is difficult to identify. The conventional endpoints of resuscitation—heart rate, blood pressure, and urine output—are crude assessments of the adequacy of resuscitation. Early identification of compensated shock is facilitated by recognition of a transient response to fluid resuscitation. Patients in Class III shock often have hypotension and tachycardia, but vital signs improve or normalize in response to initial fluid resuscitation. During observation, such patients have physiologic signs of shock and need aggressive ongoing resuscitation to maintain relatively normal vital signs. Early identification of the site of hemorrhage and prompt surgical intervention minimize the period of hypoperfusion and the subsequent risk of multiple organ dysfunction. Transfusion with crossmatched or type-specific blood is indicated. A patient in Class IV, or uncompensated, shock has pallor, diaphoresis, and apathy—clinical signs and symptoms that must be correctly interpreted. These patients also have tachycardia, hypotension, and tachypnea not responsive to aggressive fluid resuscitation with crystalloid and blood transfusion. Any injured

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or burned patient who is cool and tachycardic is in shock until proven otherwise. These patients are described as nonresponders, in that their vital signs do not improve with aggressive resuscitative techniques. In this population, early identification of the site of hemorrhage and surgical intervention are paramount to survival. Early transfusion with group O RBCs is indicated. The decision regarding early administration of blood, that is, in the trauma department, should be reserved for patients who have a transient response to initial resuscitation or those who have no response to resuscitation. These two groups have lost more than 30% of their blood volume or are still actively bleeding. Laboratory values of hemoglobin or hematocrit do not typically play a role in the determination of the need for blood transfusion in this patient population, but rather the clinical signs and symptoms of hemorrhage and response to resuscitation are the main determinants of the patient’s estimated blood loss. Transfusion of blood in the trauma department must not delay definitive surgical intervention, and these patients should be transported to the operating room urgently. In a retrospective study with 1000 trauma patients, Knottenbelt28 found an interesting correlation between initial low hemoglobin level and mortality. He found that patients who came to medical attention in shock and died had significantly lower hemoglobin levels than did patients who did not arrive for treatment in shock. The mean hemoglobin levels of patients who died of hemorrhagic causes were significantly lower regardless of initial blood pressure. In this study, 48% of patients received no prehospital resuscitation, indicating that hemodilution was not the cause of a low initial hemoglobin level. Knottenbelt hypothesized that patients with a low hemoglobin level may have suffered blood loss exceeding their compensatory mechanisms. He concluded that a low initial hemoglobin level indicated severe, ongoing blood loss. Once the decision to administer RBC transfusion has been reached, there are several options for the type of blood used. Blood ideally should by typed and crossmatched before transfusion, although this can take as long as 1 hour in many blood banks. This is an unacceptably long delay for a patient in Class III or IV shock. An alternative is transfusion of type-specific blood. Transfusion of type-specific blood is associated with a processing delay of 20 to 30 minutes, which in some circumstances still may be an unacceptable delay. In most trauma units, group O RBC units are immediately available and the most rapidly available choice in the care of trauma patients in hemodynamically unstable condition not responding to crystalloid resuscitation. Numerous studies have shown that administration of group O RBC units is a safe and rapid means by which to restore circulating red cell volume in a patient in severe shock. It is of paramount importance to assess the adequacy of resuscitation; however, the search continues for valid markers. Many believe that base deficit accurately reflects the hemodynamic and tissue perfusion changes associated with shock and resuscitation.29 Serum lactate is a reliable marker of hypoperfusion in hemorrhagic shock, because failure to clear lactate within 24 hours of injury has been associated with increased mortality.30 Although serum markers may be helpful for guiding

Chapter 39: Transfusion Therapy in the Care of Trauma and Burn Patients

the treatment of trauma patients, they are at best only a global measure of perfusion. Organ-specific monitoring, therefore, may be useful in ensuring adequate end-organ perfusion. Ivatury et al31 have proposed gastric tonometry for such monitoring. They documented improved survival among patients whose gastric pH normalized within 24 hours. Other methods of measuring endpoints of resuscitation in the trauma patient include sublingual pCO2 monitoring, tissue oxygen and carbon dioxide electrodes, and near infared spectroscopy (NIRS) for the measurement of skeletal muscle oxyhemoglobin levels. Baron et al demonstrated in trauma patients with torso injuries that sublingual capnometry was able to differentiate between patients with varying amounts of blood loss32 and was able to predict survival in hypotensive trauma patients equivalent to lactic acid levels and base deficit.33 Measurement of skeletal muscle tissue oxygenation by two probes place within the deltoid muscle include the polarographic tissue oxygen monitor (Licox, Integra, Plainsboro, NJ) and the NIRS (InSpectra, Hutchinson Techology, Hutchinson, MN). Each of these have been studied in the trauma patient.34 They have not yet found a role in guiding resuscitation, but are predictive of postinjury complications. NIRS measures skeletal muscle oxyhemoglobin levels. It has been shown to be predictive of multiple organ failure in the trauma patient and has the added advantage that it is noninvasive.35 Although these devices have the potential to help guide resuscitation, currently they are only adjuncts to other endpoints of resuscitation in the trauma patient and no particular one has been adopted widely.

Intraoperative Phase of the Trauma Patient General Issues The initial surgical treatment of a trauma patient involves rapid control of bleeding. In the case of severe abdominal trauma, this may necessitate use of “damage control” techniques or rapid packing of the abdomen to achieve hemostasis and rapid control of injuries to hollow viscera.36 “Damage control” allows the quick, sometimes temporary control of bleeding so the anesthesia team can administer intravenous fluids and rapidly restore intravascular volume. The use of heated ventilator circuits prevents heat and moisture loss, but does not provide heat gain. The amount of heat lost from a normal person at room temperature is negligible, but it does become important in environments where inspired air is low temperature, such as the prehospital setting in cold climates.37 Warmed intravenous fluids reduce the risk of hypothermia by preventing ongoing heat loss and provide some heat gain.38 Ten liters of warmed intravenous fluids infused to a 70-kg male will increase the body temperature about 1.4ºC. This contributes to the prevention of subsequent coagulopathy and acidosis associated with reduced body temperature and the infusion of large volumes of crystalloid and blood, which may be much cooler than body temperature. The use of “damage control” operative techniques in the care of the trauma patient is necessary when these physiologic factors are present. The decision to perform intraoperative transfusion can be somewhat complex and involves both the surgeon and the

anesthesiologist. Despite a traditional hemoglobin-based transfusion trigger of 10 g/dL, numerous studies have documented the safety of a lower hemoglobin level.39,40 In a study of Jehovah’s Witness patients with anemia undergoing elective surgery, Spence et al39 found that active bleeding was an independent predictor of survival only when hemoglobin level decreased to less than 4 g/dL. Low hemoglobin levels became an independent predictor of mortality only when they decreased to less than 3 g/dL. The application of these data to the care of a trauma patient must be tempered with the recognition that hemoglobin levels obtained during active hemorrhage may be falsely elevated because the intravascular and interstitial spaces have not equilibrated. In addition, the compensatory mechanisms of chronic anemia require more time to develop than that afforded by acute hemorrhage. Although results of these studies support the concept that the use of an arbitrary hemoglobin transfusion trigger is probably inappropriate, the intraoperative decision to administer transfusion must be based on an assessment of the patient’s overall physiologic condition and the severity of ongoing blood loss. Autotransfusion of the patient’s blood has become a widely accepted practice in elective surgery and can be used as an adjunct to transfusion in the care of trauma patients. The benefits of this practice include the conservation of the blood supply and avoidance of complications related to allogeneic transfusion. Although intraoperative blood recovery and reinfusion has been successfully used in cases of thoracic trauma, its use in patients with abdominal injuries has not been universally applicable because of the risk of contamination from injuries to hollow viscera. The blood is typically anticoagulated with citrate or heparin, washed with normal saline, and then reinfused. Systems relying on filtration, rather than washing, were associated with disseminated intravascular coagulation and other complications and are generally no longer in use, with the possible exception of the reinfusion of mediastinal drainage fluid, which is largely defibrinated, following cardiac surgery.

Solid Organ Injury At the beginning of the 1900s, the mortality rate for nonoperative management of splenic injuries was 100%; thus, splenectomy was readily accepted as the standard of care for splenic rupture. However, King and Schumaker reported on overwhelming postsplenectomy sepsis among infants who had undergone splenectomy.41 Despite this report, the practice of splenectomy remained unchallenged through the first half of the 20th century, until pediatric surgeons began to realize that children in hemodynamically stable condition with splenic injuries could be safely observed. With the advent of improved imaging techniques, including computed tomography (CT) of the abdomen and pelvis, splenic injuries can be identified in patients with normal hemodynamic values. The pediatric experience has been extended to the treatment of adults who have sustained trauma but who are in hemodynamically stable condition. Benefits of splenic preservation include avoidance of overwhelming postsplenectomy sepsis and avoidance of unnecessary laparotomy.

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In the 1990s, these principles were extended to the nonoperative treatment of selected patients with liver trauma.42 In determining whether a patient needs operative therapy for a splenic injury, the most important factor is hemodynamic stability. Patients in hemodynamically unstable condition (approximately 35% of adult patients with splenic injury and 3% in children42) need exploration and splenectomy. Splenorrhaphy is an option, but with the nonoperative management of many splenic injuries, it is not utilized in the adult trauma patient with failed nonoperative management. Generally, surgery is indicated in the worst of the splenic injuries that have failed nonoperative management and require removal. Splenorrhaphy is generally reserved for preservation of the spleen when it is an incidental finding upon laparotomy and not a source of major hemorrhage, especially in the immunologically immature pediatric patient.43,44 However, 65% of patients with splenic injury arrive in hemodynamically stable condition and are candidates for nonoperative treatment. Nonoperative management fails when hemodynamic instability necessitates exploration, or hemoglobin level decreases, necessitating transfusion, but there is some variation between practitioners regarding the criteria for operative intervention. Most patients with splenic injury do not need transfusion. However, children under the age of 5 have a higher risk of postsplenectomy sepsis. The relative risk of a 1- or 2-unit transfusion (if the patient is in otherwise stable condition) seems a reasonable alternative to splenectomy.45 Adjuncts to improve the success of nonoperative management of splenic injuries include the use of angiography. Schurr et al46 described a contrast blush on helical CT scans of the spleen. This finding corresponded to a traumatic pseudoaneurysm on an angiogram and can be successfully treated in the hemodynamically stable patient. In 1990, Knudson et al47 described 52 patients with blunt liver trauma who were observed carefully with serial CT. No failures of nonoperative management were reported. This was followed by a report48 from the group at the University of Tennessee at Memphis, documenting the safety of nonoperative management of even the most severe liver injuries. The authors found transfusion requirements were markedly lower than for patients with severe liver injury who underwent surgery. The only criterion for initial nonoperative management was hemodynamic stability. As with splenic trauma, early identification of contrast extravasation on helical CT scans of the liver with subsequent angiographic embolization of the bleeding vessel has improved the success rate of nonoperative management.49

Recovery Phase of the Trauma Patient The time between operative or nonoperative treatment of a patient who has sustained trauma until discharge from acute care may be the period in which transfusion can be decreased the most without compromising the patient’s condition. During this period, there is no ongoing blood loss, so dramatic changes in hematocrit are not expected. The decision to admininster RBC transfusion during this period should be based not on

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maintaining an arbitrary hematocrit but on results of assessment of the physiologic need for transfusion. Traditional teaching that all patients should have a hematocrit of 30% (or a hemoglobin level of 10 g/dL) for optimal oxygen delivery has not been borne out in recent studies. Persons with normal blood volume and normal cardiopulmonary function tolerate hemoglobin levels as low as 5 g/dL.50 A prospective, randomized controlled trial in the care of critically ill patients demonstrated patients younger than 55 years or patients with Acute Physiology and Chronic Health Evaluation II (APACHE II) scores less than 20, may have a reduced mortality if a restrictive transfusion strategy is adopted and hemoglobin levels are maintained at 7 to 9 g/dL.51 The same reduction in mortality is not seen in a subgroup analysis of resuscitated critically ill trauma patients or brain-injured patients. In fact, those transfused to maintain the hemoglobin level of 7 to 9 g/dL vs 10 to 12 g/dL did not have any difference in outcomes.52,53 However, further prospective trials should be performed to confirm these findings, especially in a high-risk population such as brain-injured patients. Moderate to severely brain-injured patients may warrant earlier operative management of solid organ injury, especially stable patients with splenic injuries, because of the association of hypotension with increased mortality. If the mortality from the brain injury is worse with hypotension, nonoperative management of moderate splenic injuries in the stable patient may not be worth the risk of failure and exposure to hypotension. Hypothermia is associated with mortality in patients with brain injury and is also a result of severe hemorrhage and transfusion in the trauma patient.54 Decisions to administer RBC transfusion should be based on evidence of symptomatic anemia, including chest pain, dyspnea, fatigue, and significant tachycardia. In the absence of symptoms, RBC transfusion is not warranted. A more liberal transfusion trigger is probably warranted in the care of those with underlying severe cardiovascular disease.

Complications of Massive Transfusion Therapy Massive transfusion has been defined as replacement of a patient’s entire blood volume in 24 hours, transfusion of more than 10 RBC units, or replacement of more than 50% of the circulating blood volume within 3 hours.55,56 Massive resuscitation with fluids and blood components after hemorrhagic shock is associated with complex metabolic derangements and high morbidity and mortality rates. The clinical hallmark of severe hemorrhagic shock is the lethal triad of acidosis, hypothermia, and coagulopathy (Fig 39-2). Banked blood undergoes a number of metabolic and structural changes.56 When it is transfused in large volumes, the blood can theoretically cause severe derangements in physiologic values. It appears, however, that the severity and duration of shock rather than the volume of transfused blood are the primary determinants of physiologic derangement and ultimately patient outcome.57,58 It is vital that patients receiving massive transfusion also receive adequate fluid resuscitation such that oxygen delivery and organ perfusion are maintained. Survival after

Chapter 39: Transfusion Therapy in the Care of Trauma and Burn Patients

Acidosis

Hypothermia

Coagulopathy

Figure 39-2. The lethal triad.

massive transfusion is no longer uncommon. One patient received 186 units of blood and blood components within 12 hours and had a successful outcome.59,60 One problem associated with massive resuscitation is the occurrence of microvascular bleeding, or so-called medical bleeding. This is to be clearly differentiated from surgical bleeding. Medical bleeding is associated with mucous membrane bleeding, oozing from surgical wounds and raw tissue surfaces, oozing from catheter sites after application of direct pressure, and generalized petechiae and enlarging ecchymoses.61 It has been recommended that prophylactic transfusion of platelets and Fresh Frozen Plasma (FFP) be administered according to the volume of RBCs transfused.61-63 However, this practice pattern may not decrease the risk of microvascular bleeding and may markedly increase the number of donor units transfused. Several studies have evaluated changes in levels of coagulation factors and platelet count and their role in microvascular bleeding after trauma and massive transfusion.62-64 These studies document a decrease in both coagulation factors and platelet counts. One explanation is the development of a washout phenomenon, or dilutional coagulopathy, resulting from the use of stored blood deficient in both platelets and coagulation factors.64 Although dilution appears to play a role in thrombocytopenia and coagulopathy after massive resuscitation, it does not account for all changes after massive transfusion. In a regression analysis of thrombocytopenia after massive transfusion, Reed et al61 found that only 35% of the decrease in platelet count could be attributed to dilution. They found further that 50% of patients with microvascular bleeding needed large volumes of platelet transfusions well after the period of massive resuscitation. This finding suggested that factors in addition to dilution must play a role in persistent thrombocytopenia in these patients. In a related study,64 investigators hypothesized that platelet consumption accounted for this persistent thrombocytopenia, which was related to the pathophysiologic response accompanying lung injury, brain injury, massive tissue injury, sepsis, and endothelial damage. Several authors have examined the concentration of coagulation factors to determine whether dilution plays a contributing role. Martin et al65 found that although levels of coagulation factors decreased significantly at the end of a shock insult, the levels remained much higher than those considered adequate for normal hemostasis. Reed et al61,62 evaluated coagulation studies

in the treatment of patients who received massive transfusions. They found mild to moderate prolongation of prothrombin time (PT) and partial thromboplastin time (PTT) were common in this patient population but were not predictive of microvascular bleeding. Regression analysis showed that 15% to 35% of the elevation of the PT and PTT could not be attributed to coagulation factor deficiencies. This finding indicated that other, unidentifiable factors had a role in the elevation of the PT and PTT. Although no one study alone can confirm which patients at risk of microvascular bleeding will benefit from transfusions of platelets and FFP, certain tests are more useful than others. A PT less than 1.3 times control value has a 94% predictive value in indicating the absence of microvascular bleeding. Although the sensitivity of PT or PTT in identifying microvascular bleeding is only 50%, values greater than 1.8 times control value are 96% specific for microvascular bleeding. Bleeding time is of no value in determining which patients will have microvascular bleeding. The most sensitive laboratory predictors are a platelet count less than 50,000/µL and a fibrinogen level less than 0.5 g/L, with a combined sensitivity of 87% and a negative predictive value of 96%. This has led to the following recommendations: platelet transfusion for patients with a platelet count less than 100,000/ µL and evidence of microvascular bleeding; prophylactic platelet transfusion for patients with a platelet count less than 50,000/µL; and administration of supplemental FFP or cryoprecipitate to patients with fibrinogen levels of 0.8 g/L or less.62 Hypothermia is a contributing factor to platelet and coagulation factor dysfunction after massive transfusion. Villalobos et al66 studied the effects of hypothermia on platelets in dogs and found that thrombocytopenia was caused by sequestration of platelets by the liver, spleen, and other sites in the portal circulation rather than by platelet destruction. No change in platelet production was found during either cooling or rewarming. Reed et al67-69 reported on coagulation factor dysfunction secondary to hypothermia. Standard coagulation testing in the laboratory typically is performed at 37ºC. This method detects clotting abnormalities only if the actual factor content is decreased. The function of the coagulation cascade, however, depends on enzymatic activity, which can be adversely affected by hypothermia. The result is prolongation of clotting times even in the face of normal factor concentrations. Investigators68 found that the PTT was prolonged at all temperatures less than 35ºC and that the PT was prolonged at temperatures less than 33ºC. They concluded that if clinical oozing exists in a patient with hypothermia and results of coagulation studies performed at 37ºC are within the normal range, rewarming the patient is more appropriate and more effective than empiric transfusion of FFP. Recognition of the contributions of hypothermia to the development of medical bleeding has revolutionized the field of trauma surgery. The concept of the “damage control” laparotomy has been reborn. In this procedure, all efforts are directed at control of surgical bleeding and containment of intra-abdominal contamination. However, the operation is truncated to minimize the “on-table” time. The patient often is closed with laparotomy packs in place

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in a temporary abdominal wall closure.34 The patient is returned to the surgical intensive care unit, where aggressive rewarming and resuscitation continue. Only when acidosis, coagulopathy, and hypothermia have been corrected is the patient returned to the operating room for definitive management of injuries. It is suggested that storage of blood longer than 14 days is related to the side effects, and in many trauma centers with a large demand for blood components, many of the older units of blood in circulation are sent to high-volume centers where they will more likely be used before their expiration. In other words, trauma patients in busy trauma and medical centers tend to receive older blood. Transfused leukocytes have been implicated as the cause of many transfusion reactions, but older blood also has red cell morphology changes, adenosine triphosphate depletion, 2,3-diphosphoglycerale depletion, and other biochemical changes (see Chapter 4). Use of prestorage leukocyte-reduced blood components should in theory reduce the immunomodulatory side effects of blood transfusion, but there are many other contributing factors that alter blood in storage.70 It has been demonstrated that trauma patients with multiple organ failure who were transfused in the first 12 hours of admission received more blood that was greater than 14 days old.71 In addition, blood greater than 14 days old was an independent predictor of serious infection.72 Currently, the use of whole blood is limited to the military and war-related trauma. Although it is possible to move refrigerators and freezers close to areas of combat, storing platelets at the proper temperature and working within the expiration date are challenging. Thus, the military uses whole blood transfusion or their “walking donor” program. Whole blood transfusion is utilized as a lifesaving therapy in combat with otherwise untreatable coagulopathy in the absence of appropriate blood components.73 Whole blood is not used in the civilian trauma center. A retrospective review of patients who received whole blood transfusion from January 2001 to December 2004 revealed that of the 5294 transfusions given to 3287 patients, 545 units of whole blood were transfused to 87 (3%) patients.74 The United States military advocates the use of whole blood transfusion only for victims with life-threatening injuries and hemorrhage.

The Burn Patient Initial Resuscitation of the Burn Patient The large number of resuscitation formulas available for use in the treatment of thermally injured patients is a tribute to the fact that no one formula is an accurate predictor of the fluid requirements of every patient. No formula replaces the role of the physician, who must continually assess the adequacy of resuscitation. Ongoing controversies about resuscitation involve the use of colloid, the differences between adults and children, and the influence of inhalation injury on fluid requirements. In the first 24 hours of resuscitation, most patients need 2 to 4 mL/kg of fluid per percentage of total body surface area (BSA) burned. This fluid is given as lactated Ringer’s solution. The first

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half of the calculated requirement is given over the first 8 hours, measured from time of injury, not from the beginning of resuscitation. The rest is given over the next 16 hours (Parkland formula).75,76 Children also need maintenance fluids containing dextrose in addition to the resuscitative fluid. The presence of inhalation injury increases the volume of fluid needed to achieve adequate resuscitation. Whether colloid is used in the first 24 hours is determined on a case-by-case basis; administration of colloid should not be routine, but is utilized in the severely burned patient who is not responsive to resuscitation. Regardless of the volume of resuscitative fluid initially chosen, ongoing resuscitation should be dictated by the patient’s response to resuscitation. Urinary output of 1 mL/kg/hour is an adequate measure of resuscitation. Release of vasoactive mediators from injured tissue results in a diffuse capillary leak starting soon after injury. This loss of microvascular integrity results in extravasation of crystalloid and colloid solutions for the first 18 to 24 hours after thermal injury. This pathophysiologic process explains the enormous fluid requirements of burn patients for the first 24 hours. This is the reason that most burn resuscitation formulas incorporate the use of colloids after 24 hours—microvascular integrity apparently is restored at that time. After 24 hours, colloid remains largely intravascular. Colloid, generally 5% albumin, is infused at a dose based on burn size (generally 0.3 to 0.5 mL/kg per percentage of total BSA burned over 24 hours). During this period, crystalloid requirements decrease markedly.

Transfusion Therapy in the Care of Thermally Injured Patients As with severely injured trauma patients, thermally injured patients often have anemia throughout the hospital course. The cause of this anemia is very different from that of the anemia of trauma.77-81 The factors that predominate as the cause of anemia vary with respect to time from injury; they must be considered in evaluation for transfusion.78,79 Soon after burn injury, anemia is caused by direct destruction of erythrocytes within the cutaneous circulation and by hemorrhage into the burn wound. As time progresses, the factors resulting in anemia include hemolysis of injured erythrocytes and blood loss during dressing changes. Further hemolysis also occurs as blood flows through injured tissue and burn eschar. Additional blood loss occurs during burn wound débridement, split-thickness skin grafting, and postoperative dressing changes. Because of the stress associated with thermal injury, blood loss can occur in the gastrointestinal tract, as well as from unnecessary “routine” blood work. The response of burn patients to anemia is different from that of trauma patients.78-81 Several, but not all, studies have documented an increase in erythropoietin level after thermal injury as well as a paradoxical decrease in reticulocyte numbers.80,81 In a study with 27 patients, erythropoietin levels were inversely related to hemoglobin levels, but patients had persistent reticulocytopenia.81 The results of this study suggested a relative resistance

Chapter 39: Transfusion Therapy in the Care of Trauma and Burn Patients

to erythropoietin at the marrow level. An elevated level of circulating erythropoietin did not result in an expected increase in erythrocyte production. It appears that the erythropoietic response to thermal injury is inversely proportional to the size of the burn.80 Results of studies of the use of recombinant erythropoietin in the management of postburn anemia have not been encouraging. A study with pediatric burn patients did not show a statistically significant increase in hematocrit for either burn patients or healthy volunteers, although increases in reticulocyte count occurred.77 With no effect in either group, it is questionable if the dose or schedule of the drug was adequate. Similar studies in adult patients with 25% to 65% total BSA burns did not show a significant difference in reticulocyte counts, transfusion requirements, hemoglobin, hematocrit, or iron binding. There was, however, a significant increase in retuculocyte counts in patients with burns from 25% to 35%.82 More studies with perhaps higher doses need to be performed to determine if erythropoietin is effective in this population at different doses or administration regimens, or if the benefit is not in the increase in the hemoglobin, but other properties of erythropoietin. The relative resistance of the marrow of thermally injured patients to erythropoietin may be caused by an inhibitory factor in the serum. A study was performed in which serum from patients with more than 20% total BSA burned was cultured with mouse marrow. The authors found the number of erythroid colonies was significantly less in the presence of sera from burn patients even when erythropoietin was added to the system. When erythropoietin activity was assayed in vivo, no decrease in activity was detected. This finding suggests the inhibition did not occur at the level of the erythropoietin.79 Another study showed that erythroid colony-forming cells appeared to return to normal as healing occurred. This study also showed that granulocytopoiesis and thrombocytopoiesis were unaffected and proceeded at an accelerated rate.78 These authors postulated that redirection of the pluripotent stem cells away from the erythroid line to the granulocyte and monocyte cell lines may offer a survival advantage to burned patients because infection and sepsis are major sources of morbidity and mortality in this population. As in the care of trauma patients, the safety of lower hemoglobin and hematocrit levels in the care of burn patients has been demonstrated.83,84 Mann et al84 retrospectively examined the transfusion requirements of patients with burns of more than 10% total BSA who needed at least one operation. They found a fivefold decrease from 1980 to 1990 in the volume of blood transfused per percentage total BSA without adverse cardiac complications or increased morbidity. Because of their findings, the authors recommended that healthy patients do not need transfusion unless the hematocrit decreases to less than 15% to 20% in patients who need one operation, or 25% in patients who need more than one operative procedure. As with trauma patients, thermally injured patients with underlying cardiovascular disease should be treated with a more liberalized transfusion policy. Sittig and Deitch83 confirmed the foregoing findings and found a greater than a threefold decrease in transfusion

requirements when a more selective transfusion policy was implemented whereby 86% of blood transfused was given at the time of an operative procedure. Intraoperative use of hemostatic agents can further decrease transfusion requirements. It is well known that intraoperative blood loss is the limiting factor of the extent of excision and grafting performed. Thrombin spray and laparotomy pads saturated with thrombin and epinephrine have been used extensively to limit intraoperative blood loss.83,85 The use of tourniquets for excision and grafting of extremities has further decreased blood loss.86 Fibrin sealants may prove effective. Results of preliminary studies of the use of intraoperative blood recovery techniques show recovery and reinfusion of approximately 40% of shed blood without adverse inflammatory or infectious complications.87 As in the care of trauma patients, the importance of normothermia in preventing coagulopathy is recognized, and patients are aggressively warmed at the time of surgery.

Adjuncts in Transfusion Therapy for Trauma and Burn Patients Although crystalloid and blood are the standards in initial resuscitation of trauma patients, there is active investigational interest in developing alternative resuscitative materials. While it is clear that blood is an effective resuscitative fluid that rapidly expands circulatory volume and improves oxygen transportation, it has many disadvantages. It must be crossmatched; it has a short shelf life; it must be stored under closely monitored conditions; it is associated with a variety of immunologic and infectious complications; and its supply relies on the altruism of the donor population.

Blood Substitutes (Oxygen Carriers) There have been numerous attempts at developing synthetic products that will aid in oxygen-carrying capacity, mostly based on the use of solutions of hemoglobin-based oxygen carriers or perfluorocarbon emulsions. All of them offer the advantages of long shelf life, permissive storage conditions, absence of the need for compatibility testing, and virtual absence of infectious risks. Most oxygen carriers under development are based on either human or animal hemoglobin. The source of human hemoglobin is outdated RBCs. The disadvantage of human hemoglobin relates to a limitation in the availability of outdated RBCs; less than 3% of the 15.1 million units of blood donated in the United States are being discarded. Animal hemoglobin, particularly bovine hemoglobin, is potentially inexpensive and is readily available, although uncertainty regarding the purification of animal pathogens remains a concern as does the antigenicity of the components.88,89 Several companies are conducting efficacy trials of hemoglobin-based oxygen carriers. One company, Baxter Healthcare (Deerfield, IL) terminated work with a diaspirin cross-linked hemoglobin when multicenter trials with trauma patients with hypotension and stroke patients showed increased

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multiple organ failure, acute respiratory distress syndrome, and mortality during interim review.90 PolyHeme (Northfield Laboratories, Evanston, IL) has completed two Phase III trials and appears to lack the marked vasopressive effects of diaspirin cross-linked hemoglobin. A randomized, Phase II clinical trial of PolyHeme vs RBCs in the treatment of trauma patients showed a lower overall RBC transfusion requirement; there was no difference in circulating hemoglobin level, and there were no adverse events.91 Comparison of patients who received PolyHeme during three different studies, and a historical control group composed of consecutive surgical patients who declined blood transfusion for religious reasons, showed a mortality rate of 25% in the PolyHeme group vs 65% in the group that did not receive the oxygen carrier.92 A Phase III trial using PolyHeme as a resuscitative component in prehospital trauma patients has been conducted but the data have not yet been published. A bovine hemoglobin-based solution, Hemopure (Biopure Corporation, Cambridge MA), appears to have some vasopressor effects. One Phase III trial has been completed; plans for another Phase III trial are under discussion with the Food and Drug Administration. Hemopure has been licensed for use in clinical practice in South Africa since 2001. Sangart (San Diego, CA) has developed a PEGylated human hemoglobin product with minimal vasoactivity, which has entered Phase II clinical trials in Europe and the United States.93 Perfluorocarbons are synthetic molecules with high solubility for oxygen and carbon dioxide. Compared to blood however, their oxygen-carrying capacity is quite low, so patients must receive supplemental oxygen. The compounds are not derived from other animal products and, therefore, do not have the antigenic properties that accompany bioproducts. However, they can cause activation of complement, malaise, and flu-like symptoms; other side effects include thrombocytopenia, splenomegaly, and hepatomegaly. The perfluorocarbon emulsions are cleared by the reticulo-endothelial system relatively quickly with a half life of 12 to 24 hours. Currently, there are no perfluorocarbons in clinical trials mostly because of adverse effects found early in clinical trials and the lack of efficacy.89

patients revealed higher mortality in patients who received albumin, especially in those with more severe brain injury.95 There is little doubt that smaller volumes of colloid than of crystalloid are needed to restore intravascular volume and this is demonstrated in the studies described above. Meta-analysis from the Cochrane database has not found any mortality benefit to the use of colloids from the combined results of 63 eligible trials.96 To clarify the colloid vs crystalloid debate, several studies have examined outcomes, including survival and generation of extravascular lung water. Investigators in favor of crystalloid resuscitation argue that after severe trauma, there is depletion of interstitial volume as well as intravascular volume, a deficit not adequately addressed with colloid resuscitation. Crystalloid has been proposed as the best resuscitation fluid because it has been shown to restore the volume of both the intravascular and interstitial spaces without worsening pulmonary function.97 Pulmonary lymphatic vessels increase their flow significantly in animals, and the increase likely occurs in humans, minimizing the development of pulmonary edema after crystalloid resuscitation. Proponents of colloid resuscitation, however, believe that resuscitation of the intravascular component alone is beneficial and that use of colloid may decrease the incidence of pulmonary edema.98 Although this debate has been subjected to several meta-analyses, the results are conflicting because of highly heterogeneous patient populations and study designs. Advocates of crystalloid use note the lower cost of crystalloids than of colloids, although larger volumes are needed to reach similar endpoints. Although colloids remain in the intravascular space, in states of increased vascular permeability, colloids can leak into the interstitium, increasing the colloid oncotic pressure of the extravascular space. This increased oncotic pressure can hamper mobilization of third-space fluids after the acute resuscitative phase has been completed. Velanovich99 reviewed eight randomized, prospective trials comparing crystalloid to colloid resuscitation. He found a 5.7% relative decrease in mortality in the group receiving crystalloid resuscitation. When nontrauma trials were excluded, a 12.3% decrease in mortality favored crystalloid resuscitation.

Hypertonic Saline Solution Colloid and Crystalloid Solutions Although blood substitutes remain in clinical trials, other solutions have been evaluated for use in immediate resuscitation of trauma patients. It must be emphasized that colloid solutions are not effective in improving oxygen delivery in the hemorrhaging trauma patient. Colloid solutions are theoretically more efficient than crystalloid at restoring intravascular volume. The colloidcrystalloid debate is at least 50 years old. A potential indication for colloid administration is ongoing resuscitation of critically injured patients. The SAFE study94 published in 2004 examined the choice of resuscitation with 4% albumin vs normal saline in intensive care units (burns were excluded) and found no difference in mortality at 28 days. The subgroup analysis of trauma patients was small, but mortality trended toward worsened mortality in the albumin group. Post-hoc analysis of the brain-injured

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Several studies have examined use of hypertonic saline solution in the resuscitation of animals and humans from shock. Hypertonic saline solution has the advantage of requiring smaller volumes to reach endpoints similar to those of standard crystalloid resuscitation. This makes hypertonic saline solution particularly attractive for use in the prehospital setting, but it does not aid in oxygen-carrying capacity. Administration of hypertonic saline solution has been shown to elevate mean arterial pressure and cardiac output and to increase renal, mesenteric, total splanchnic, and coronary blood flow. Hypertonic saline solution also causes a small and transient increase in circulatory volume by means of transcapillary refill. These effects are well established after controlled hemorrhage. However, the utility of hypertonic saline solution in the management of uncontrolled hemorrhage has yet to be firmly established.

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Gross et al,100 using a model of uncontrolled hemorrhage in rats, found increased blood loss and mortality after administration of hypertonic saline solution. They concluded that hypertonic saline resuscitation must be delayed until after definitive control of hemorrhage.100 This study was criticized for its use of an anesthetic agent that may have exacerbated hypotension and affected the outcome. Coimbra et al101 compared hypertonic saline and shed blood resuscitation with crystalloid and shed blood resuscitation in a model of hemorrhage and cecal ligation and puncture in rats. They found improved survival among animals resuscitated with hypertonic saline solution and shed blood, improvement in pulmonary histologic features, and decreased local peritoneal sepsis. They hypothesized that resuscitation with hypertonic saline solution caused less impairment in cellular immune function, possibly contributing to the improvement in survival that they documented. In a prospective, randomized trial with 105 patients in hypovolemic shock, Younes et al102 found that infusion of 250 mL of hypertonic saline solution was not associated with any complications and did not affect mortality. It did improve mean arterial blood pressure significantly, acutely expanded plasma volume 24%, and significantly reduced the volume of crystalloid and blood needed for resuscitation. A multicenter trial evaluated the use of hypertonic saline and dextran in the prehospital treatment of trauma patients with hypotension.103 The investigators found improved survival among patients undergoing surgery who received 7.5% sodium chloride in 6% dextran 70 compared to those receiving the same volume of normal crystalloid solution. The difference reached statistical significance, although there was no overall improvement in survival rate. Use of hypertonic saline solution and dextran also appeared to be associated with a lower incidence of acute respiratory distress syndrome, renal failure, and coagulopathy. Reports by others have described a similar survival benefit among patients receiving hypertonic saline solution vs conventional resuscitative fluids in prehospital therapy for hypotension associated with trauma.104 Two studies have looked at the immunomodulating effects of hypertonic saline (7.5%)/ dextran (6%) in the resuscitation of trauma patients in hemorrhagic shock. Theses studies demonstrated the anti-inflammatory modulation of cytokines and cellular components that occur with the administration of this solution compared to normal crystalloid solutions (lactated Ringer’s and 0.9% sodium chloride).105,106

Recombinant Human Erythropoietin Recombinant human erythropoietin (rHuEPO) does not have a role in the actively bleeding trauma patient, but has been studied in the treatment of critically ill patients with a prospective, randomized control study.107 This study demonstrated an overall reduction in RBC transfusions for the patients receiving rHuEPO 40,000U weekly for three doses vs placebo. This effect was not observed until about 1 week following the administration of rHuEPO. Post-hoc subgroup analysis looked at trauma patients; nontrauma surgical patients; and nontrauma, nonsurgical medical patients. There was a trend in the trauma patients

receiving rHuEPO to have a lower mortality (4.8% receiving rHuEPO vs 10.4% receiving placebo), but this was not statistically significant.107 The cost of rHuEPO balanced against the benefit of its use (avoidance of transfusion-related complications) does not justify its use in the critically ill patient if there is no mortality difference.108 Corwin et al109 repeated this study of rHuEPO in critically ill patients in 2007. The repeat study addressed the trend toward a mortality difference seen in the first study published in 2002. The results showed a difference in 29day mortality in critically ill patients receiving rHuEPO (8.5% vs 11.4%, p ⫽ 0.02), and specifically in trauma patients admitted to the intensive care unit for more than 48 hours (3.5% vs 6.6%, p ⫽ 0.04). This difference in mortality was maintained at 140 days, but not seen in medical patients or nontrauma surgical patients. Unlike the previous study, there was no difference in the number of transfusions given between the treatment and placebo groups, which may be a reflection of the adoption of restrictive transfusion strategies in critically ill patients. In addition, post-hoc analysis showed there was a significant increase (20.3% vs 12.8%, p ⫽ 0.008) in the treatment group for thromboembolic events. This increase in events was not demonstrated with patients who received early administration of heparin.109 Independent of the effects of rHuEPO on blood transfusion and hematocrit, there have been multiple animal studies demonstrating what are thought to be anti-inflammatory, neurogenic, and anti-apoptotic characteristics of the drug in brain-injured animal models. In many of these studies, prevention of postinjury brain atrophy, behavioral abnormalities, and cognitive dysfunction have been demonstrated.110,111 What remains to be proven is the effect, if any, in the brain-injured patient in local and long-term outcomes.

Recombinant Activated Factor VIII Another agent that has been studied as an adjunct to transfusion in the trauma and burn patient is recombinant activated Factor VII (rFVIIa). This drug is licensed for the treatment of patients with Factor VII deficiency or Factor VIII inhibitors. It has been studied in hemorrhage associated with surgical procedures, trauma, and burns. The “lethal triad” of coagulopathy, hypothermia and acidosis is a well-known clinical scenario and resuscitation is often targeted to correct these variables. Administration of rFVIIa aims at correcting the coagulopathy limb and is known for being very effective in the nontrauma patient with bleeding disorders. For this reason, there is interest in finding a utility in the coagulopathic trauma patient. A multicenter and multinational study was conducted to look at the effectiveness of rFVIIa in reducing the number of blood transfusions in severely injured blunt and penetrating trauma patients. There was a significant reduction in the number of transfusions required (reduction of 2.6 RBC units) and the need for massive transfusion was reduced from 33% to 14% (massive transfusion defined as ⬎20 RBC units) in the severely injured blunt trauma population.112 Adverse events, mortality, and other clinical endpoints were the same in each of the treatment groups;

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however, there was a trend toward improvement in the incidence of acute respiratory distress syndrome in the blunt trauma treatment group and multiple organ failure in the penetrating trauma treatment group. The lack of effect on the number of RBC transfused in the penetrating trauma group could be related to the difference between surgical and medical bleeding. The administration to patients in combat with penetrating injuries appeared to decrease the number of transfusions by 20% in those requiring massive transfusion if it was received early (before transfusion of 8 RBC units) vs late (after 8 RBC units were given).113 Of concern with the use of rFVIIa is the risk for thromboembolic complications, especially when arterial injury is present. The incidence of thromboembolic complications with use of rFVIIa is from 3% to 9.4%112,114 and can be a cause of severe morbidity and mortality. Dose-dependent or temporal relationships to thrombotic events have not yet been elucidated, but as this drug is used more as an adjunct to transfusion therapy in the severely injured patient, these details may be determined. Although the potential exists for therapeutic benefit, rFVIIa has not been studied in a prospective fashion in brain-injured trauma patients and there no recommendation for its use in this setting. Use of rFVIIa has also been described in the burn patient. There is often major blood loss with the excision of burned skin and grafting. A single center pilot study of 18 patients115 showed a significant decrease in the number of blood components transfused (0.9 vs 2.2, p ⫽ 0.0013) and more specifically the number of RBC units transfused (0.5 vs 1.1, p ⫽ 0.004) when rFVIIa (40 µg/kg) was used at skin incision and redosed intraoperatively at 90 minutes. There was also a trend toward improved graft survival and reduction in multiple organ failure in the group receiving rFVIIa. This was, however, a pilot study and a study with better power should be conducted to confirm these results.

Summary Massive hemorrhage and its consequences of hypothermia, acidosis, and coagulopathy continue to be the leading cause of death during resuscitation of severely injured trauma patients. The early use of blood and blood components combined with aggressive crystalloid resuscitation and early surgical intervention has significantly decreased mortality. However, the risk of disease transmission has prompted reevaluation of the transfusion practices of both trauma and burn surgeons. Although it is recognized that these patients tolerate a lower hemoglobin level than was originally thought, the lower limit of safe hemoglobin level is not known. Although data from other surgical populations have helped to demonstrate that previously accepted transfusion triggers are not appropriate, the unique characteristics of injured patients do not allow universal application of these findings. It is possible to optimize transfusion practices once it is realized that injured patients experience distinct clinical phases based on time from injury. Although blood transfusion continues to be a lifesaving therapy, judicious use of blood and blood

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components can provide maximal benefit and minimize unnecessary risk to the patient after injury.

Disclaimer The authors have disclosed no conflicts of interest.

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19. Moore FA, Haenel JB, Moore EE, Whitehill TA. Incommensurate oxygen consumption in response to maximal oxygen availability predicts post-injury multiple organ failure. J Trauma 1992;33:58-66. 20. Velmahos GC, Demetriades D, Shoemaker WC, et al. Endpoints of resuscitation of critically injured patients: Normal or supranormal? A prospective randomized control trial. Ann Surg 2000;232: 409-18. 21. Durham RM, Neunaber K, Mazuski JE, et al. The use of oxygen consumption and delivery as endpoints for resuscitation in critically ill patients. J Trauma 1996;41:32-40. 22. Hayes MA, Timmins AC, Yau E, et al. Elevation of systemic oxygen delivery in the treatment of critically ill patients. N Engl J Med 1994;330:1717-22. 23. Gramm J, Smith S, Gamelli RL, et al. Effect of transfusion on oxygen transport in critically ill patients. Shock 1996;5:190-3. 24. Drucker WR, Chadwick CDJ, Gann DS. Transcapillary refill in hemorrhage and shock. Arch Surg 1981;116:1344-53. 25. Barrientos T, Hillman N, Peoples JB. The effects of dehydration on the dynamics of transcapillary refill. Am Surg 1982;48:412-16. 26. Prist R, Rocha-e-Silva M, Scalabrini A, et al. A quantitative analysis of transcapillary refill in severe hemorrhagic hypotension in dogs. Shock 1994;1:188-95. 27. American College of Surgeons Committee on Trauma. ACS advanced trauma life support for doctors, 7th ed. Chicago, IL: American College of Surgeons, 2004. 28. Knottenbelt JD. Low initial hemoglobin levels in trauma patients: An important indicator of ongoing hemorrhage. J Trauma 1991;31:1396-9. 29. Davis J, Shackford S, Mackersie R, et al. Base deficit as a guide to volume resuscitation. J Trauma 1988;28:1464-7. 30. Abramson D, Scalea T, Hitchcock R, et al. Lactate clearance and survival following trauma. J Trauma 1993;35:584-8. 31. Ivatury R, Simon R, Islam S, et al. A prospective randomized study of endpoints of resuscitation after major trauma: Global oxygen transport indices versus organ-specific gastric mucosal pH. J Am Coll Surg 1996;183:145-54. 32. Baron BJ, Sinert R, Zehtabchi S, et al. Diagnostic utility of sublingual PCO2 for detecting hemorrhage in penetrating trauma patients. J Trauma 2004;57:69-74. 33. Baron BJ, Dutton RP, Zehtabchi S, et al. Sublingual capnometry for rapid determination of the severity of hemorrhagic shock. J Trauma 2007;62:120-4. 34. Ikossi DG, Knudson MM, Morabito, et al. Contuinous muscle tissue oxygenation in critically injured patients: A prospective observational study. J Trauma 2006;61:780-8. 35. Cohn SM, Nathens AB, Moore FA, et al. Tissue oxygen saturation predicts the development of organ dysfunction during traumatic shock resuscitation. J Trauma 2007;62:44-54. 36. Shapiro MB, Jenkins DH, Schwab CW, et al. Damage control: Collective review. J Trauma 2000;49:969-78. 37. Lloyd EL. Hypothermia and cold stress. Rockville, MD: Aspen, 1986:42-8. 38. Peng RY, Bongard FS. Hypothermia in trauma patients. J Am Coll Surg 1999;188:685-96. 39. Spence RK, Costabile JP, Young GS, et al. Is hemoglobin level alone a reliable predictor of outcome in the severely anemic surgical patient? Am Surg 1992;58:92-5. 40. Czer LSC, Shoemaker WC. Optimal hematocrit value in critically ill postoperative patients. Surg Gynecol Obstet 1978;147:363-8.

41. King H, Shumacker HB. Splenic studies: I. Susceptibility to infection after splenectomy performed in infancy. Ann Surg 1952;136:239-42. 42. Knudson MM, Maull KI. Nonoperative management of solid organ injuries: Past, present and future. Surg Clin North Am 1999;79:1357-71. 43. Harbrecht BG. Is anything new in adult blunt splenic trauma? Am J Surg 2005;190:273-8. 44. Harbrecht BG, Zenati MS, Ochoa JB, et al. Management of adult blunt splenic injuries: Comparison between level I and level II trauma centers. J Am Coll Surg 2004;198:232-9. 45. Thompson SR, Holland AJA. Evolution of non-operative management for blunt splenic trauma in children. J Paediatr Child Health 2006;42:231-4. 46. Schurr MJ, Fabian TC, Gavant M, et al. Management of blunt splenic trauma: Computed tomographic contrast blush predicts failure of nonoperative management. J Trauma 1995;39:507-13. 47. Knudson MM, Lim RC Jr, Oakes DD, et al. Nonoperative management of blunt liver injuries in adults: The need for continued surveillance. J Trauma 1990;30:1494-500. 48. Croce MA, Fabian TC, Menke PG, et al. Nonoperative management of blunt hepatic trauma is the treatment of choice for hemodynamically stable patients, results of a prospective trail. Ann Surg 1995;221:744-55. 49. Carrillo EH, Spain DA, Wohltmann CD, et al. Interventional techniques are useful adjuncts in nonoperative management of hepatic injuries. J Trauma 1999;46:619-24. 50. Weiskopf RB, Viele MK, Feiner J, et al. Human cardiovascular and metabolic response to acute, isovolemic anemia. JAMA 1997;279:217-21. 51. Hébert PC, Wells G, Blajchman MA, et al. A multicenter, randomized, controlled clinical trial of transfusion requirements in critical care. N Engl J Med 1999;340:409-17. 52. McIntyre L, Hebert PC, Wells G, et al. Is a restrictive transfusion strategy safe for resuscitated and critically ill trauma patients? J Trauma 2004;57:563-8. 53. McIntyre LA, Fergusson DA, Hutchison JS, et al. Effect of a liberal versus restrictive transfusion strategy on mortality in patients with moderate to severe head injury. Neurocritical Care 2006;5:4-9. 54. Jeremitsky E, Omert L, Dunham CM, et al. Harbingers of poor outcome the day after severe brain injury: Hypothermia, hypoxia and hypoperfusion. J Trauma 2003;54:312-19. 55. Rutledge R, Sheldon GF, Collins ML. Massive transfusion. Crit Care Clin 1986;2:791-805. 56. Lovric V. Alterations in blood components during storage and their clinical significance. Anaesth Intensive Care 1984;12:246-51. 57. Waxman K, Shoemaker WC. Physiologic responses to massive intraoperative hemorrhage. Arch Surg 1982;117:470-5. 58. Canizaro PC, Pessa ME. Management of massive hemorrhage associated with abdominal trauma. Surg Clin North Am 1990;70:621-34. 59. Kivioja A, Myllynen P, Rokkanen P. Survival after massive transfusions exceeding four blood volumes in patients with blunt injuries. Am Surg 1991;57:398-401. 60. Michelsen T, Salmela L, Tigerstedt I, et al. Massive blood transfusion: Is there a limit? Crit Care Med 1989;17:699-700. 61. Reed RL, Ciavarella D, Heimback DM, et al. Prophylactic platelet administration during massive transfusion. Ann Surg 1986;230:40-8. 62. Ciavarella D, Reed RL, Counts RB, et al. Clotting factor levels and the risk of diffuse microvascular bleeding in the massively transfused patient. Br J Haematol 1987;67:365-8.

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85. Prasad JK, Taddonio TE, Thomson PD. Prospective comparison of a bovine collagen dressing to bovine spray thrombin for control of haemorrhage of skin graft donor sites. Burns 1991;17:70-1. 86. Housinger TA, Lang D, Warden GD. A prospective study of blood loss with excisional therapy in pediatric burn patients. J Trauma 1993;34:262-3. 87. Jeng JC, Boyd TM, Jablonski KA, et al. Intraoperative blood salvage in excisional burn surgery: An analysis of yield, bacteriology and inflammatory mediators. J Burn Care Rehabil 1998;19:305-11. 88. Cohn SM. Blood substitutes in surgery. Surgery 2000;127:599-602. 89. Stollings JL, Oyen LJ. Oxygen therapeutics: Oxygen delivery without blood. Pharmacotherapy 2006;26:1453-64. 90. Sloan E, Koenigsberg M, Gens D, et al. Diaspirin cross-linked hemoglobin (DCLHb) in the treatment of severe traumatic hemorrhagic shock: A randomized controlled efficacy trial. JAMA 1999;282:1857-64. 91. Gould SA, Moore EE, Hoyt DB, et al. The first randomized trial of human polymerized hemoglobin as a blood substitute in acute trauma and emergent surgery. J Am Coll Surg 1998;187:113-22. 92. Gould SA, Moore EE, Hoyt DB, et al. The life-sustaining capacity of human polymerized hemoglobin when red cells might be unavailable. J Am Coll Surg 2002;195:445-55. 93. Oloffson C, Ahl T, Johansson T, et al. A multicenter clinical study of the safety and activity of maleimide-polyethylene glycol-modified Hemoglobin (Hemospan) in patients undergoing major orthopedic surgery. Anesthesiology 2006;105:1153-63. 94. The SAFE Study Investigators. A comparison of albumin and saline for fluid resuscitation in the intensive care unit. N Engl J Med 2004;350:2247-56. 95. The SAFE Study Investigators. Saline or albumin for fluid resuscitation in patients with traumatic brain injury. N Engl J Med 2007;357:874-84. 96. Perel P, Roberts I. Colloids versus crystalloids for fluid resuscitation in critically ill patients. Cochrane Database Syst Rev 2007;(4): CD000567. 97. Shires GT, Barber AE, Illner HP. Current status of resuscitation: Solutions including hypertonic saline. Adv Surg 1995;28:113-70. 98. Henry S, Scalea TM. Resuscitation in the new millennium. Surg Clin North Am 1999;79(6):1259-67. 99. Velanovich V. Crystalloid versus colloid fluid resuscitation: A metaanalysis of mortality. Surgery 1989;105:65-71. 100. Gross D, Landau EH, Klin B, et al. Treatment of uncontrolled hemorrhagic shock with hypertonic saline solution. Surg Gynecol Obstet 1990;170:106-12. 101. Coimbra R, Hoyt D, Junger W, et al. Hypertonic saline resuscitation decreases susceptibility to sepsis after hemorrhagic shock. J Trauma 1997;42:602-7. 102. Younes RN, Aun F, Accioly CQ, et al. Hypertonic solutions in the treatment of hypovolemic shock: A prospective, randomized study in patients admitted to the emergency room. Surgery 1992;111:380-5. 103. Mattox KL, Maningas PA, Moore EE, et al. Prehospital hypertonic saline/dextran infusion for post-traumatic hypotension. Ann Surg 1991;213:482-91. 104. Vassar MJ, Perry CA, Holcroft JW. Prehospital resuscitation of hypotensive trauma patients with 7.5% NaCl versus 7.5% NaCl with added dextran: A controlled trial. J Trauma 1993;34:622-32. 105. Rizoli SB, Rhind SG, Shek PN, et al. The immunomodulatory effects of hypertonic saline resuscitation in patients sustaining

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106.

107.

108.

109. 110.

traumatic hemorrhagic shock—A randomized, controlled, doubleblinded trial. Ann Surg 2006;243:47-57. Bulger EM, Cuschieri J, Warner K, Maier RV. Hypertonic resuscitation modulates the inflammatory response in patients with traumatic hemorrhagic shock. Ann Surg 2007;245:635-41. Corwin HL, Gettinger A, Pearl RG, et al. Efficacy of recombinant human erythropoietin in critically ill patients—a randomized controlled trial. JAMA 2002;288:2827-35. Shermock KM, Horn E, Lipsett PA, et al. Number needed to treat and cost of recombinant human erythropoietin to avoid one transfusion-related adverse even in critically ill patients. Crit Care Med 2005;33:497-503. Corwin HL, Gettinger A, Fabian TC, et al. Efficacy and safety of epoetin alpha in critically ill patients. N Engl J Med 2007;357:965-76. Siren AL, Radyushkin K, Boretius S, et al. Global brain atrophy after unilateral parietal lesion and its prevention by erythropoietin. Brain 2006;129:480-9.

111. Lu D, Mahmood A, Qu C, et al. Erythropoietin enhances neurogenesis and restores spatial memory in rats after traumatic brain injury. J Neurotrauma 2005;22:1011-17. 112. Boffard KD, Riou B, Warren B, et al. Recombinant Factor VIIa as adjunctive therapy for bleeding control in severely injured trauma patients: Two parallel randomized, placebo-controlled, doubleblind clinical trials. J Trauma 2005;59:8-18. 113. Perkins JG, Schreiber MA, Wade CE, Holcomb JB. Early versus late recombinant factor VIIa in combat trauma patients requiring massive transfusion. J Trauma 2007;62:1095-9. 114. Thomas GO, Dutton RP, Hemlock B, et al. Thromboembolic complications associated with factor VIIa administration. J Trauma 2007;62:564-9. 115. Johansson PI, Eriksen K, Nielsen SL, et al. Recombinant FVIIa decreases perioperative blood transfusion requirement in burn patients undergoing excision and grafting—results of a single center pilot study. Burns 2007;33:435-40.

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Transfusion Therapy in Solid-Organ Transplantation Glenn Ramsey Professor of Pathology, Northwestern University School of Medicine, Chicago, Illinois, USA

Recent clinical progress in organ transplantation has been tempered by the limited supply of organs.1 The blood bank has a vital place on the hospital-wide team that makes transplantation possible for severely ill patients. Approximately 1% of all Red Blood Cell (RBC) and platelet transfusions and approximately 5% of all plasma and cryoprecipitate transfusions are used in organ transplantation surgery.2 Blood banks also play an increasing role in recent efforts to prevent ABO- and HLA-mediated graft rejection. In the United States, 257 programs perform organ transplantation. Table 40-1 shows the number of transplants of each organ performed in the United States in 2006.3 Also shown are the 3-year graft and patient survival percentages for transplantation procedures performed on adults in the United States in 2001 with follow-up data through 2004. Pediatric results are similar.

Organ Procurement In the United States, organ procurement and allocation are regulated by the Health Resources and Services Administration (HRSA) of the Department of Health and Human Services. Under the authority of the National Organ Transplant Act [Title 42 US Code (USC), Section 273-274], HRSA oversees the Organ Procurement Transplant Network (OPTN) [Title 42 Code of Federal Regulations (CFR), Section 121].4,5 The law for Medicare participation (42 USC 1320b-8) requires membership in the OPTN for all transplant centers (42 CFR 482.45) and organ procurement organizations (42 CFR 486.301-348). The OPTN in turn formulates policies required for its members.6 The OPTN is administered by the United Network for Organ Sharing (UNOS) under contract with HRSA. Medicare also requires that hospitals

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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Table 40-1. US Organ Transplants, Waiting Lists, and Adult 3-Year Graft and Patient Survival Rates* Organ

2006 Transplants

2007 Waiting List

Kidney DD LD Kidney and pancreas Liver Heart Lung Heart-lung Pancreas Intestine

17,090 10,659 6,431 924

73,233

6,650 2,192 1,405 31 463 175

16,714 2,627 2,335 115 1,644 215

2,321

Graft Survival Rate (%)

Patient Survival Rate (%)

79 88 85 kidney 79 pancreas 74 79 64 53 63 51

88 95 91 79 80 66 54 91 62

*Waiting lists are as of September 2007. Survival rates are for transplantation procedures performed in 2001 with 3 years of follow-up evaluation.3 Pancreas rates are averages of pancreas alone and pancreas after kidney. DD ⫽ deceased donor; LD ⫽ living donor.

report deaths to the 58 organ procurement organizations for evaluation of organ donation eligibility. As in transfusion medicine, blood group compatibility and disease transmission are of high concern for organ transplants. To reduce the risk of error, OPTN/UNOS policies specify that organ donors and transplant candidates each have two separate ABO typings performed, and the entry of these blood types into the national matching registry must be verified by a second person (OPTN Policies 3.1 and 3.2.4).6 Medicare regulations also require these measures for organ donors (42 CFR 486.344). ABO errors in labeling or distribution are included in the OPTN/UNOS evaluation plan for assessing compliance with requirements.7,8 Organ transplants have transmitted not only bloodborne, transfusion-risk agents, including West Nile virus and Trypanosoma cruzi (Chagas’ disease), but also other donor infections such as tuberculosis, methicillin-resistant Staphylococcus

Chapter 40: Transfusion Therapy in Solid-Organ Transplantation

aureus, rabies, lymphocytic choriomeningitis virus, and toxoplasmosis.9-11 Donor-transmitted cancers occurred in 1 in 12,000 deceased-donor organ transplants in the United States. Although a history of cancer in an organ donor is not necessarily disqualifying, certain tumor types prone to occult metastasis are of extra concern, such as melanoma, lymphoma, and carcinoma of the breast, lung, colon, and kidney.12,13 OPTN/UNOS is authorized by federal regulation to establish policies for organ donor screening and testing. Their policies provide a list of infections and medical conditions that, if present, must be communicated to the transplant center. Donors must be tested for hepatitis B surface antigen (HBsAg); antibodies to human immunodeficiency virus (anti-HIV-1/2), hepatitis B core antigen (anti-HBc), hepatitis C virus (anti-HCV), human T-cell lymphotropic virus (anti-HTLV-I/II), cytomegalovirus (anti-CMV), and Epstein-Barr virus (anti-EBV); and a serologic test for syphilis (VDRL or RPR) (OPTN Policies 2.2 and 4.1).6 The Food and Drug Administration (FDA) maintains a Web page with current information on infectious-disease tests cleared for organ and cadaver (non-heart-beating) donors.14 These include several nucleic acid tests, although viral nucleic acid testing is not required by OPTN/UNOS. West Nile virus testing is not required, although HRSA issued a notice in 2004 that encouraged screening of living donors near the time of donation.15 Cadaver donations providing tissues are also subject to separate FDA tissue transplant safety regulations that focus on reducing the risk of relevant communicable diseases (21 CFR 1271). FDA-cleared cadaveric tests must be used for tissue donors when appropriate and available. Donors are ineligible to contribute organs or tissues if their infectious-disease tests are invalidated by excess hemodilution from recent transfusions or infusions, as defined in organ donor risk factors from the US Centers for Disease Control and Prevention (CDC) (OPTN Policy 4.1.1) and by tissue regulation (21 CFR 1271).5,6 OPTN/UNOS policies require prompt investigation of possible organ-borne infections and medical conditions, including look-back to other recipients from suspect donors (OPTN Policy 4.7).6 A nationwide transplant safety sentinel network with a standardized donor identification system is under development in the United States. The circulation of a brain-dead donor is maintained until the organs are harvested by transplantation surgeons. Sometimes blood component transfusions are needed for oxygenation and hemostasis. After cadaveric organs are removed, hearts and lungs must be used within 4 to 6 hours, livers within 12 hours, pancreata within 18 hours, and kidneys within 18 to 30 hours.1 Preservative solutions are used during cold ischemia until transplantation.

within a region, the patient waiting lists are compiled according to ABO blood group. For each transplant procedure, two to four persons are on the waiting lists for the most commonly transplanted organs (Table 40-1). Organs and organ donors are still scarce. The need for organs has stimulated efforts to expand the supply by using organs from living donors and by splitting livers. Over one-third of US kidney transplants are from living donors.16 The graft and patient survival rates are better than for renal transplantation from cadavers. A small number of liver transplants (4%) are from living donors, from whom the right lobe is removed. Both the graft split liver and the living donor’s liver regenerate to normal size in a few weeks. Cadaveric livers sometimes are split (4%). The right lobe is given to one patient and the rest of the liver to another patient.

Immunologic Barriers The fundamental test for organ donation and transplantation is the ABO group, because solid organs almost always must be ABO compatible. Anti-A and anti-B bind to endothelial cells, setting off a cycle of complement fixation, vascular damage, and thrombosis that leads to ischemia and rejection. ABO-incompatible liver transplants are less susceptible to hyperacute rejection than are other organs but the risk of eventual rejection is still high. As group O RBCs are sometimes in shortest supply in relation to demand, so too are group O organs. Group O patients wait longer on average than other patients for nonemergency cadaveric transplantation. HLA compatibility is also important for most organs, except the liver (see Chapter 57). Nonhepatic transplant candidates are assessed for HLA antibodies and their history of HLA exposure through pregnancy, transfusion, or prior transplant.6 Recipient HLA antibodies against nonhepatic grafts lead to a much higher risk of acute and chronic rejection. After ABO compatibility, deceased-donor kidneys are first prioritized to recipients matched for all six HLA-A, -B, and -DR antigens, because these matches have slightly better graft survival. Kidneys can also be stored longer than other organs, permitting transport to a matched recipient. Kidney and pancreas transplants, together or separate, generally must be HLA Class I (T lymphocyte) crossmatch-compatible before transplantation. OPTN/UNOS policy requires that heart and lung grafts also be prospectively HLAcompatible if the recipient has HLA antibodies or has been exposed. Livers do not require HLA matching or crossmatching. Leukocyte-reduced blood components are discussed below.

Immunohematology Organ Transplants

Patient Alloantibodies

The systems for ranking the priorities of patients are specific for each organ and incorporate severity of illness. For each organ

In the 1980s, 6% to 8% of adult recipients of kidney or liver transplants had clinically significant red cell alloantibodies. Another 8% of liver recipients developed antibodies after the

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large number of transfusions given during surgery.17,18 Both of these patient groups now receive fewer transfusions before and during surgery than in the past. Most patients with renal failure receive erythropoietin. Patients with cirrhosis often are treated to reduce variceal bleeding from portal hypertension with medications to reduce venous pressure (β-blockers, octreotide), sclerotherapy or band ligation of varices, and if necessary, transjugular intrahepatic portosystemic shunting. Consequently, pretransplantation red cell antibody problems are less frequent. Advance screening for the presence of antibodies to non-ABO antigens is still advisable for candidates for liver transplantation. Some liver transplantation patients have multiple alloantibodies, or alloantibodies to high-frequency antigens; hence, the supply of antigen-negative units is limited. The following strategy has been successful in some programs: 1) give at least the first blood volume of RBCs as compatible, antigen-negative units; 2) after this degree of hemodilution of the antibodies, give unscreened or antigen-positive units; and 3) reserve additional antigen-negative units for after surgery, when transfusions return to small volumes and in anticipation of a possible anamnestic response after immune stimulation.19 When Rh-negative patients need large amounts of RBCs during liver or other organ transplantation, it may be necessary to give Rh-positive RBCs. Follow-up tests show that these patients usually do not make anti-D.20,21 Transplantation immunosuppression greatly reduces the expected high rate of Rh sensitization among patients with normal immune function. HLA alloantibodies against the donor can cause prompt rejection of renal and cardiac grafts. Candidates for these transplants undergo periodic HLA antibody tests, and then HLA crossmatching against donor lymphocytes to confirm compatibility.

antibodies are of donor origin and are not patient autoantibodies. This phenomenon emerged after azathioprine for immunosuppression was replaced by cyclosporine, which is more permissive for secondary antibody responses, and it has occurred with tacrolimus as well. Non-ABO red cell antibodies can develop in a similar manner if the organ donor has been alloimmunized against an antigen in the recipient. Rh and other antibodies have been reported, and some have caused temporary hemolysis.22,23 Women donating kidneys to their children or their partner sometimes transmit pregnancy-induced antibodies. When an organ donor is found to have a clinically significant red cell alloantibody it is prudent to notify the organ procurement agency or in the case of a living donor, the recipient’s physician. Then patients may receive RBC transfusions that are negative for the target antigen regardless of whether the patient is antigen positive or antigen negative. Platelet antibodies have been transferred from organs of a donor who died of cerebral hemorrhage caused by immune thrombocytopenic purpura.24 Perhaps any antibody-producing cells in the organ donor can be transferred temporarily to the recipient.

Blood Transfusion Needs Table 40-2 shows recent data for mean blood component usage in nonhepatic organ transplantation. Primary cardiac transplantation without previous cardiac surgery is similar to routine cardiac surgery, but the use of components increases in operations for patients who have undergone previous sternotomy. The use

Antibodies from Passenger Lymphocytes As with hematopoietic stem cell transplants, organ transplants contain lymphocytes that sometimes can make antibodies against antigens in the recipient.22,23 This is most common when group O organs are placed into non-group-O patients. OPTN/UNOS policies restrict the use of group O deceased-donor organs for non-group-O patients (OPTN Policy 3).6 In the United States in 2005 there were about 500 excess group O deceased liver donors over the number of group O deceased-donor recipients, indicating that about 9% of deceased-donor liver transplants were from group O donors to non-group-O recipients.16 In living-donor liver transplants, comparable statistics showed that about 20% of them (60 cases) were group O to non-group-O recipients. In approximately 40% of liver transplant recipients and 10% of kidney recipients receiving an ABO-unmatched organ, the patient develops IgG antibodies from graft lymphocytes that react against his or her own A or B antigens. A few cases have been caused by group A or B organs in group AB patients. The patient’s direct antiglobulin test becomes reactive 1 to 2 weeks after transplantation, and some of these antibodies produce sudden marked hemolysis. The antibodies typically persist for a few weeks. Immunoglobulin allotyping has proved that these

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Table 40-2. Mean Blood Component Usage in Nonhepatic Organ Transplantation Organ

Cardiac Primary Repeated sternotomy After support device (with aprotinin) Lung Single Double Pancreas Kidney

Red Blood Cells

Plasma

Platelets

Reference

1

1-3

1-4

1-2

1-6

1-8

8

13

12

Prendergast25 Wegner26 Prendergast25 Wegner26 Wegner26

2 6 2 1

1 4 0 0

2 6 0 0

Triulzi27 Triulzi27 Nyman28 Danielson29

Units are rounded to the nearest integer. Platelets are expressed as platelet concentrates, with apheresis units counted as 6 units. The cardiac transplantation range is from two centers. In one center, aprotinin reduced overall component transfusions in repeated sternotomy cases in a randomized study.25

Chapter 40: Transfusion Therapy in Solid-Organ Transplantation

of aprotinin in the care of patients undergoing second operations reduced overall blood component needs in a randomized study.25 Patients undergoing transplantation after use of a cardiac support device need large amounts of blood, even when treated with aprotinin, in part because of anticoagulation before transplantation.26 Double-lung transplantation uses more blood than does single-lung transplantation. Uncomplicated renal transplantation usually is a type-and-screen procedure, as is the living-donor nephrectomy.

Liver Transplantation The liver transplantation operation is simple in concept, but serious complications can develop at any time.30 In the preanhepatic first stage, the vascular and biliary connections of the liver are dissected and exposed—portal vein, bile duct, hepatic artery, and inferior vena cava. The liver is separated from the diaphragm and the retroperitoneum, often exposing a raw, bare surface prone to bleeding. In the typical procedure, the major vessels are clamped, the vascular and bile duct connections are transected, and the liver is removed. The first stage usually takes 4 to 8 hours. In the anhepatic second stage, the graft and recipient suprahepatic and infrahepatic vena cava and portal vein are anastomosed end to end. The liver is flushed to remove most of its preservative solution. This second stage usually takes 1 to 1.5 hours. At the beginning of the third stage, the post-anhepatic stage, the large vessels are unclamped to restore portal inflow, and the hepatic artery and bile duct are connected. If all goes well, the third stage is completed in 2 to 4 hours. Liver transplantation presents a unique set of challenges for transfusion and hemostasis needs. Patients with acute liver failure have low levels of clotting factors. Patients with cirrhosis also usually have thrombocytopenia caused by splenomegaly and low levels of thrombopoietin, much of which is made by the liver. Platelet function may be reduced by liver failure, uremia in hepatorenal syndrome, or anemia. Conversely, some patients have a hypercoagulability syndrome because of high levels of Factor VIII and decreased levels of fibrinolytic proteins. During the operation, certain problems can arise in each stage. During dissection, portal hypertension can contribute to venous bleeding from the areas around the liver. Before and during the anhepatic stage, fibrinolysis sometimes develops from imbalance of its inhibitors and promoters. The graft is flushed, but when the new liver is unclamped, the patient often is partially anticoagulated by heparin present from the donor’s anticoagulation and by heparin-like substances from injured endothelium released into the circulation. To counteract all the possible problems, the liver transplantation, anesthesia, and laboratory team uses an array of tactics to meet the transfusion and hemostasis needs of the patient. Surgeons use an argon-beam laser to coagulate bleeding surfaces. Two surgical techniques are used in the care of selected patients to improve hemodynamics. If the anatomic features of the graft

are favorable, the liver can be sewn on to the inferior vena cava in a so-called “piggyback” manner to maintain partial blood flow through the vena cava during the operation. Some surgeons use venovenous bypass during the anhepatic stage, either routinely or if interruption of vena caval flow causes too much hypotension. This perfusion technique shunts venous return from the inferior vena cava and the portal vein up to the subclavian vein and improves cardiac output if needed. To monitor coagulation status and to anticipate transfusion needs, hemoglobin, platelet count, prothrombin time, activated partial thromboplastin time, and fibrinogen are measured regularly. Many programs use thromboelastography (TEG) to supplement routine clotting tests.31 The TEG instrument measures the initiation, rate, and strength of whole-blood clotting, and displays the results in the operating room as they develop over 20 to 60 minutes. TEG also has the following three functions not available in routine coagulation testing: 1) fibrinolysis can be readily detected; 2) the patient’s blood can be tested in vitro with protamine or ε-aminocaproic acid to assess whether these medications would help correct heparinization or fibrinolysis; and 3) hypercoagulability is also observed in some TEG results, but is not well correlated with clinical evidence of thrombosis. TEG testing can be employed in an algorithm for transfusion decision-making.32 To meet the patient’s transfusion needs, intraoperative blood recovery and reinfusion is helpful.33 One-third or more of the patient’s total RBC transfusions can be provided by recovering shed blood during the operation. The anesthesiologist can give large volumes of warmed blood and fluids through a rapid-infusion pump that delivers more than 1 L per minute if necessary. Plasma usage usually closely parallels RBC use in operations with large transfusion needs, in effect reconstituting whole blood for the transfusions. If available, whole blood is suitable for the first blood volume or so, because these patients usually have normal levels of Factor VIII. Solvent/detergent-treated (SD) plasma has been used successfully, although it may be deficient in some antifibrinolytic proteins.34 The SD plasma product in the United States, which is no longer available, was associated with thrombotic or hemorrhagic problems during liver transplant. However, a European product has been used successfully in this setting. Several programs have reported a range of factors correlating with perioperative transfusion needs. The laboratory variables that appear most often in their conclusions are preoperative anemia, platelet count, prothrombin time (or international normalized ratio), and creatinine.35 The latter two values, along with bilirubin, are the elements for calculating the model of end-stage liver disease (MELD) score for predicting mortality risk and prioritizing adult transplant candidates in the United States. Higher MELD scores have also been associated with more blood use.32 Published blood component usage varies greatly among programs, with recent examples of mean RBC usage ranging from 3 units to 11 to 16 units (Table 40-3). Although there may be some publication bias toward favorable results, patient populations and intraoperative transfusion and surgical practices

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Table 40-3. Blood Component Usage in Liver Transplants Location

North America Los Angeles Toronto Scottsdale Montreal Europe Budapest Groningen France Barcelona

Value

RBC

FFP

Platelets (doses)

Comments

Ref.

Mean Mean Median Mean Mean Mean

16 11 5 3 3 0.4

22 15 6 3 4 0

1 1 0.3 1 0.4 0

MELD ⬎30 MELD ⭐30 TEG-guided, aprotinin Aprotinin Hypovolemia, aprotinin

Xia36 Xia36 McCluskey35 Frasco32 Massicotte37 Massicotte38

Median Median Median Median

12 5 5 2

14 6 6 NA

1 0.3 1 NA

Aprotinin, selected cases Aprotinin Eight centers Temporary portacaval shunt, 52%

Nemes39 Nemes39 Ozier40 Ramos41

Median shown when available. Units rounded to nearest integer, except platelets. RBCs ⫽ red blood cells; FFP ⫽ fresh frozen plasma; MELD ⫽ model of end-stage liver disease score; TEG ⫽ thromboelastography; NA ⫽ not available.

account for some of the reported variance. In one study, the anesthesiologist was a major determinant. In France the main variable in a national comparison was the institution. Two European centers compared their practices to determine why one had higher blood usage and lower graft and patient survival. They found, as did previous workers, that more bleeding and higher RBC usage correlated with higher mortality. The center with less blood use took more time during surgery for hemostasis, and was more likely to do “piggyback” transplants or venovenous bypass, both of which preserve circulatory hemodynamics when needed. Jehovah’s Witnesses have undergone liver transplantation without blood component transfusions, with careful patient selection and the use of multiple blood conservation strategies.42 Liver transplants still require massive transfusion in some cases. Although some of these patients have preoperative coagulopathy, this usually can be corrected with appropriate transfusions in the first stage of the transplantation procedure. The underlying factors in these large hemorrhages usually are intraoperative problems such as unusually severe portal hypertension, cardiopulmonary instability, and anatomic and technical complications causing surgical blood loss. These problems often are not predictable from the patient’s transplantation evaluation, and significant blood use frequently is unexpected. The role of antifibrinolytic agents in liver transplants has been mixed. When fibrinolysis develops and causes bleeding, tranexamic acid or ε-aminocaproic acid is often utilized. However, opinions vary on whether these drugs or aprotinin should be used as regular prophylaxis, in view of their potential to cause unintended clotting complications. Aprotinin has been used widely in Europe. A meta-analysis of controlled trials concluded that tranexamic acid and aprotinin reduced blood loss without increasing adverse thrombotic events.43 However, other

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reviewers found that aprotinin’s benefits were mainly in patient groups with higher blood loss, and did not recommend routine use.44,45 Concern has emerged recently about aprotinin and excess mortality after cardiac surgery, and the drug has been suspended by the manufacturer. Recombinant Factor VIIa was tested in two double-blind randomized trials in liver transplants. In a single-bolus trial, patients received one 20, 40, or 80 µg/kg bolus just before surgery.46 In the second trial, doses of 60 or 120 µg/kg were repeated every 2 hours.47 The median transfusion needs were not different from placebo in either study. The role of the drug in selected patients at high risk of bleeding or with refractory coagulopathy is under study. Even without medications, thrombosis (either systemic or localized to the hepatic artery) is an occasional complication during liver transplant operations. In some patients, this complication is associated with hypercoagulability found during TEG, and moderate heparinization may be advisable. Aggressive correction of coagulopathy should be avoided when clotting would present special difficulties, as during venovenous bypass, or when the hepatic artery is small or not flowing well.

Special Needs Prevention of Cytomegalovirus Infection Cytomegalovirus infection is a major infectious complication of organ transplantation because of the cellular immunosuppression needed. If a candidate for solid-organ transplantation is anti-CMV-negative, CMV-reduced-risk cellular blood components should be provided (ie, obtained from anti-CMV-negative donors or leukocyte reduced to remove the bulk of CMV viral load).48 When an anti-CMV-negative transplant recipient

Chapter 40: Transfusion Therapy in Solid-Organ Transplantation

receives an anti-CMV-negative organ, CMV-reduced-risk transfusions are recommended when possible to prevent third-party CMV infection from the blood donor. The growing availability of leukocyte-reduced components has increased the supply of CMV-reduced-risk components for liver transplantation. For patients who are already anti-CMV-positive or who receive an anti-CMV-positive graft, CMV-reduced-risk transfusions generally are not necessary. The risk of CMV infection comes from the new organ or from reactivation of the original infection. CMV-reduced-risk components do not provide appreciable clinical benefit in these patients (see Chapter 47).

Leukocyte Reduction The leukocyte reduction of blood components for organ transplantation presents a paradox. This is one clinical setting in which transfused white cells were historically beneficial.49 In the 1970s and early 1980s, transfusions were commonly given to candidates for renal transplantation to achieve better graft survival. RBC transfusions leukocyte reduced by means of freezing and thawing did not confer the same advantage. The effect was attributed to immunosuppression by transfused white cells. When immunosuppression improved, the transfusion advantage decreased. However, even with cyclosporine, donorspecific transfusions can impart some benefit for engraftment and survival.50 The other side of the paradox is that if the transplant recipient has previous HLA antibodies against the prospective graft kidney, transplantation is contraindicated because rejection of these organs may ensue. Donor-specific transfusions pose a risk of immunization against the donor, preventing the planned transplant. From this perspective, leukocyte reduction of pretransplant transfusions would be desirable. HLA alloimmunization also adversely affects survival of heart and lung grafts. The balance of current opinion probably falls on the side of leukocyte reduction to avoid development of HLA antibodies in candidates for nonhepatic organ transplantation. Research continues on transfusion immunomodulation in transplantation (see Chapter 45).

Blood Irradiation Graft-vs-host disease (GVHD) has occurred from passenger leukocytes in transplanted organs. However, GVHD from transfusions in these patients is rare.51 Solid-organ transplantation usually is not included in recommended indications for blood irradiation (see Chapter 54).

employed several methods to evade the ABO barrier for selected patients: 1) exchanges of directed organ donors, 2) neonatal tolerance, 3) A2 organs, and 4) humoral immunosuppression to permit accommodation of fully ABO-incompatible organs.52,53 Blood bank support is vital to these efforts (Table 40-4). The majority of kidney transplants are from living donors, but some patients may not have an ABO-compatible donor. In order to obtain ABO-compatible organs, some transplant centers arrange for willing donors to exchange with another patient.53 Extended chains of such exchanges have been performed. Another strategy is for a donor to contribute a nondirected kidney to the general organ supply, and then the donor’s patient of concern receives priority for an ABO-compatible deceased-donor organ. Infants do not make their own circulating antibodies for several months after birth. This immunologic “loophole” has been successfully exploited for the transplantation of ABOincompatible hearts in young children.54 Graft survival is similar to ABO-compatible transplants, and fewer infants die waiting for a heart when ABO is not a limitation. This has been adopted in OPTN/UNOS organ allocation policy, including upper limits on antibody titers in infants 1 to 2 years old (OPTN Policy 3.7.8) 6 The presence of the incompatible ABO antigen at a very young age leads to long-term suppression of antibody production, with absence of donor-specific anti-A or anti-B peripheral B lymphocytes, and low or no anti-A or anti-B against the heart’s antigens. The tissues of persons with the A2 blood group have weaker group A expression than in persons with the A1 blood group, about 20% of the normal quantity. Kidneys from group A2 donors have been successfully transplanted to group O and group B patients, as have group A2B kidneys to group B patients.55,56 In some centers, candidates’ anti-A titers are prescreened, and those with high titers are excluded from receiving a group A2 or A2B kidney. However, transplants to eligible low-titer patients have been performed with regular immunosuppression, and results are similar to ABO-compatible grafts. Fully ABO-incompatible kidney transplants have been performed using protocols with extra immunosuppression, removal of preexisting ABO antibodies by plasmapheresis, blockage of antibody production, and frequent monitoring of ABO antibody levels. These programs are most often established in livingdonor transplants, when the date of transplant can be scheduled in advance, in contrast to unscheduled deceased-donor organ Table 40-4. Roles for the Blood Bank in ABO-Incompatible Organ Transplant Protocols ●

Breaching Immunologic Barriers ABO Because there are several times more transplant candidates than available annual organ donations, transplant centers have

●

● ●

Seek A2 organ donors with A1 lectin or ABH gene typing. Select or process blood components to avoid giving passive graft ABO antibodies to recipients before and after receipt of transplant. Carefully titrate IgM and IgG ABO antibodies. Perform plasmapheresis to remove and control ABO antibody levels in pre- and perioperative periods.

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transplants, which may not permit extended pretransplant preparations. Japan, which has very few deceased or group A2 organ donors, has gleaned the most experience. A national registry of 564 ABO-incompatible transplants in 1989-2003 comprised 14% of all living-donor kidneys.57 With the use of apheresis and splenectomy, the overall 5-year graft survival was 74%, compared to 81% in ABO-compatible controls. Outcomes improved over time, and the most recent 2-year graft survival was 94% for ABO-incompatible transplants. Recent studies in Japan and other developed countries have sought to replace splenectomy with other means to suppress antibody production, such as rituximab (anti-CD20) and intravenous immune globulin (IVIG) or cytomegalovirus immune globulin (CMVIG) for immunomodulation, and the administration of mycophenolate mofetil to suppress both B and T lymphocytes.58-60 In all protocols, plasmapheresis to reduce and control anti-A or -B titers is a central feature. In the current guidelines for therapeutic apheresis from the American Society for Apheresis (ASFA), ABO-incompatible kidney and neonatal heart transplants are Category II indications, generally accepted adjuncts to other treatments, and incompatible liver transplants are in Category III, with suggestion of benefit, but efficacy not established.61 Several European renal transplant centers have performed plasmapheresis with an in-line column containing A or B antigen to remove ABO antibody by immunoadsorption.62 As a corollary to these efforts, recipients or potential recipients of ABO-incompatible organs should not receive transfusions of large quantities of recipient-type plasma containing graft ABO antibody. The transfusion service selects components of appropriate ABO type, or processes them to remove graft antibody before transfusion. ABO antibody titration is a critical laboratory test for physicians and their patients in these protocols. IgM antibody mediates complement fixation and endothelial damage in acute transplant rejection of ABO-incompatible organs, and IgM is more readily removed by plasmapheresis than IgG. However, many transplant series emphasize IgG titers for patient eligibility, rejection risk, and plasmapheresis guidance. A working group from several US centers recommended reporting both IgM and IgG titers.63 Antibody titers routinely performed in the blood bank are subject to some degree of imprecision, with a range of ⫾1 dilution (a fourfold difference). However, many transplant protocols set more precise titer targets for eligibility or antibody removal. In Japan, broad inter-institutional variation in titer results on survey specimens was improved with a more standardized method.64 Future studies may illuminate which titration methods are most clinically useful and reproducible, and determine the role of more quantitative methods for antibody levels. In the United States, the UNOS registry was examined for the outcomes of ABO-incompatible kidney transplants between 1995 and 2003.65 With deceased-donor transplants (n ⫽ 201), the 5-year graft survivals for fully incompatible transplants were the same as ABO-compatible cases (66-67%). For living-donor

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kidneys (n ⫽ 191), the ABO-incompatible grafts had lower overall long-term survival (66% vs 80% at 5 years), although the difference was mainly in the first year. ABO-incompatible transplants are most vulnerable to rejection in the first month after surgery, and antibody reduction measures focus on this period. However, after the initial risk phase, most grafts survive despite the continuing presence of graft ABO antibody in the plasma and donor-type ABO antigens in the graft endothelium. This survival in the face of humoral immunity has been termed accommodation. Several mediators of rejection might be involved in this phenomenon.66 Graft ABO antibodies sometimes remain at lower levels than before the transplant procedure, and may change in specificity. In ABOincompatible kidneys after 3 to 12 months of graft survival, microarray gene expressions of several regulators of inflammation, apoptosis, signal transduction, and cell interaction were significantly altered in ways that suggested injury protection, when compared to ABO-compatible control grafts.67 Other factors for further investigation include: 1) the effects of graft-derived A and B antigens in plasma, such as those on endothelial-derived von Willebrand factor; 2) complement pathway components and their immunomodulation of the local immune response; and 3) nitric oxide release from ABO-incompatible endothelial cells, which could dampen apoptosis and platelet adhesion.66

HLA Similar to the ABO blood group, the HLA system poses a significant barrier to transplantation in patients with preexisting antibodies, but immunologic manipulations are making some headway for these patients. Broadly alloimmunized patients are identified by a high percentage of positive lymphocytotoxic crossmatches to Class I antigens on donor or panel T cells. In the United States, 14% of renal transplant candidates have antibodies against ⭓80% of potential donors (panel-reactive antibody, PRA).16 These patients have great difficulty receiving a compatible living or deceased-donor kidney. Protocols to suppress HLA antibodies have been termed desensitization. The two main alternate strategies currently used are 1) high-dose IVIG, and 2) plasmapheresis and low-dose IVIG or CMVIG.68 How IVIG works in these protocols is uncertain, but possibilities include receptor blockade, anti-idiotypic antibody content, and inhibition of the activation of lymphocytes and complement.69 High-dose IVIG is given at up to four monthly intervals before receipt of living-donor or deceased-donor transplants, until crossmatches are negative, and then once, 1 month after surgery. In-vitro testing to identify candidates in advance has been performed by assessing whether the presence of IVIG inhibits a patient’s antibody in lymphocytotoxicity testing. Of 77 sensitized patients passing this prescreening, 87% received living or deceased-donor organs.69 Rejection episodes occurred in 28%, but the 3-year graft survival was 87%. Plasmapheresis and low-dose IVIG have been used mainly before living-donor kidney transplants. Multiple plasmapheresis

Chapter 40: Transfusion Therapy in Solid-Organ Transplantation

procedures are performed every other day before and after the transplant procedure (the number of procedures in proportion to the preexisting antibody titer) and IVIG is given after each procedure. The center with the most experience used CMVIG, converted 63% of patients’ crossmatches to negative before transplantation, and had approximately 85% 3-year graft survival.70,71 A randomized double-blind clinical trial of desensitization compared high-dose IVIG to placebo in 101 renal transplant candidates with PRA ⭓50%.72 The mean PRA was reduced from about 75% to 60% in the treatment group during four monthly infusions, and 35% of treatment patients received a kidney, compared to 17% of controls. For the transplants, the 2-year graft survival rates of 75% to 80% were similar in the two groups. One center reported comparative data from periods when either high-dose IVIG or plasmapheresis/IVIG plus rituximab were used. The plasmapheresis/IVIG patients were desensitized in 85% of 48 cases, compared to 38% of 13 high-dose IVIG cases.73 Antibody-mediated rejection has also been treated with either of these two approaches, as well as rituximab alone.74 In the ASFA guidelines, plasmapheresis for HLA desensitization and antibody-mediated rejection are both Category II indications, generally accepted adjunctive therapy.61

Other Applications of Therapeutic Apheresis Photopheresis is under study for the prevention and treatment of heart and lung transplant rejection.75 Leukocytes are collected, exposed to psoralen and ultraviolet A light ex vivo, and then reinfused. This is thought to induce immunomodulation of T cells regulating or mediating organ rejection. ASFA classifies this therapy in Category III (suggestion of benefit, insufficient evidence of efficacy) for treating lung rejection manifesting as bronchiolitis obliterans.61 Also under Category III is plasmapheresis in acute liver failure, as a supportive therapy until hepatocellular recovery or transplantation.61

Artificial Organs Several types of cardiac assist devices are in use or under development for the care of patients with severe cardiac failure.76 Some are for temporary use or as a bridge to cardiac transplantation. Others are intended for permanent implantation. While using these devices, patients often develop coagulopathy from platelet dysfunction or anticoagulation. Transfusions during these treatments can cause HLA alloimmunization, and leukocyte-reduced components should be used if transplantation is planned. Hepatic assist devices are perfused with the patient’s blood or plasma to remove toxins.77 These systems are in clinical trials to support patients until their liver recovers, as in poisoning, or until a liver transplant is available. One category of these systems passes the blood through various absorbent materials such as charcoal, resins, or albumin. The other type metabolizes toxins in the blood with immobilized porcine or human hepatocytes in a perfused bioreactor.

Summary As organ transplant graft and patient survivals continue to gradually improve, the limited supply of organs has stimulated many creative ways to hurdle traditional immunologic barriers of ABO and HLA tissue incompatibility. The role of transfusion medicine has expanded beyond transfusions to include the laboratory and clinical support of therapies to prevent and reverse graft rejection, making transplants more available and more durable. Progress in this field may lead to new advances in other diseases when immune reactions are foe, not friend.

Disclaimer The author has disclosed no conflicts of interest.

References 1. Stuart FP, Abecassis MM, Kaufman DB, eds. Organ transplantation. 2nd ed. Georgetown, TX: Landes Bioscience, 2003. 2. Ramsey G, Sherman LA. The new age of organic blood banking. Transfusion 1998;38:9-11. 3. UNOS Transplant Patient DataSource. Richmond, VA: United Network for Organ Sharing, 2007. [Available at: http://www.unos. org (accessed October 8, 2007).] 4. United States code. Washington, DC: Government Printing Office, 2000 (supplement 4, 2005). [Available at http://www.gpoaccess.gov/ uscode/index.html (accessed October 2, 2007).] 5. Electronic code of federal regulations (e-CFR). Washington, DC: Office of the Federal Register, National Archives and Records Administration, 2008. [Available at http://ecfr.gpoaccess.gov (accessed May 6, 2008).] 6. Policies. Richmond, VA: Organ Procurement and Transplant Network, 2007. [Available at http://www.optn.org/policiesAndBylaws/policies.asp (accessed October 2, 2007).] 7. Friedman AL, Lee KC, Lee GD. Errors in ABO labeling of deceased donor kidneys: Case reports and approach to ensuring patient safety. Am J Transplant 2007;7:480-3. 8. Evaluation plan. Richmond, VA: Organ Procurement and Transplantation Network, 2007. [Available at http://www.optn.org/ content/policiesAndBylaws/evaluation_plan.asp (accessed October 8, 2007).] 9. Kotton CN. Zoonoses in solid-organ and hematopoietic stem cell transplant recipients. Clin Infect Dis 2007;44:857-66. 10. Obed A, Schnitzbauer AA, Bein T, et al. Fatal pneumonia caused by Panton-Valentine Leucocidine-positive methicillin-resistant Staphylococcus aureus (PVL-MRSA) transmitted from a healthy donor in living-donor liver transplantation. Transplantation 2006;81:121-4. 11. Bronnert J, Wilde H, Tepsumethanon V, et al. Organ transplantation and rabies transmission. J Travel Med 2007;14:177-80. 12. Kauffman HM, McBride MA, Cherikh WS, et al. Transplant tumor registry: Donor related malignancies. Transplantation 2002;74:358-62. 13. Kauffman HM, Cherikh WS, McBride MA, et al. Deceased donors with a past history of malignancy: An Organ Procurement and

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14.

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Transplantation Network/United Network for Organ Sharing update. Transplantation 2007;84:272-4. Food and Drug Administration. Donor screening assays for infectious agents and HIV diagnostic assays. (March 7, 2008) Rockville, MD: CBER Office of Communication, Training, and Manufacturers Assistance, 2008. [Available at http://www.fda.gov/cber/products/ testkits.htm (accessed May 6, 2008).] US Health Resources and Services Administration. A special announcement from HRSA regarding West Nile virus. (January 9, 2004) [Available at http://www.optn.org/news/newsDetail .asp?id=303 (accessed October 3, 2007).] 2006 annual report of the US Organ Procurement and Transplantation Network and the Scientific Registry of Transplant Recipients: Transplant data 1996-2005. Rockville, MD: Health Resources and Services Administration, 2006. [Available at http:// www.optn.org (accessed September 27, 2007).] Brantley SG, Ramsey G. Red cell alloimmunization in multitransfused HLA-typed patients. Transfusion 1988;28:496-8. Ramsey G, Cornell FW, Hahn LF, et al. Red cell antibody problems in 1000 liver transplants. Transfusion 1989;29:396-400. Ramsey G, Cornell FW, Hahn LF, et al. Incompatible blood transfusions in liver transplant patients with significant red cell alloantibodies. Transplant Proc 1989;21:3531. Ramsey G, Hahn LF, Cornell FW, et al. Low rate of Rhesus immunization from Rh-incompatible blood transfusions during liver and heart transplant surgery. Transplantation 1989;47:993-5. Casanueva M, Valdes MD, Ribera MC. Lack of alloimmunization to D antigen in D-negative immunosuppressed liver transplant recipients. Transfusion 1994;34:570-2. Ramsey G. Red cell antibodies arising from solid organ transplants. Transfusion 1991;31:76-86. Yazer MH, Triulzi DJ. Immune hemolysis following ABO-mismatched stem cell or solid organ transplantation. Curr Opin Hematol 2007;14:664-70. Friend PJ, McCarthy LJ, Filo RS, et al. Transmission of idiopathic (autoimmune) thrombocytopenic purpura by liver transplantation. N Engl J Med 1990;323:807-11. Prendergast TW, Furukawa S, Beyer III AJ, et al. Defining the role of aprotinin in heart transplantation. Ann Thorac Surg 1996;62:670-4. Wegner JA, DiNardo JA, Arabia FA, et al. Blood loss and transfusion requirements in patients implanted with a mechanical circulatory support device undergoing cardiac transplantation. J Heart Lung Transplant 2000;19:504-6. Triulzi DJ, Griffith BP. Blood usage in lung transplantation. Transfusion 1998;38:12-15. Nyman T, Elmer DS, Shokouh-Amiri MH, et al. Improved outcome of patients with portal-enteric pancreas transplantation. Transplant Proc 1997;29:637-8. Danielson CFM, Filo RS, O’Donnell JA, et al. Institutional variation in hemotherapy for solid organ transplantation. Transfusion 1996;36:263-7. Ozier Y, Albi A. Liver transplant surgery and transfusion. Int Anesthesiol Clin 2004;42:147-62. Kang Y. Thromboelastography in liver transplantation. Semin Thromb Hemost 1995;21(Suppl 4):34-44. Frasco PE, Poterack KA, Hentz JG, et al. A comparison of transfusion requirements between living donation and cadaveric donation liver transplantation: Relationship to model of end-stage liver disease score and baseline coagulation status. Anesth Analg 2005;101:30-7.

33. Philips SD, Maguire D, Deshpande R, et al. A prospective study investigating the cost effectiveness of intraoperative blood salvage during liver transplantation. Transplantation 2006;81:536-40. 34. Ramsey G. Treating coagulopathy in liver disease with plasma transfusions or recombinant factor VIIa: An evidence-based review. Best Pract Res Clin Haematol 2006;19:113-26. 35. McCluskey SA, Karkouti K, Wijeysundera DN, et al. Derivation of a risk index for the prediction of massive blood transfusion in liver transplantation. Liver Transpl 2006;12:1584-93. 36. Xia VW, Du B, Braunfield M, et al. Preoperative characteristics and intraoperative transfusion and vasopressor requirements in patients with low vs. high MELD scores. Liver Transpl 2006;12:614-20. 37. Massicotte L, Sassine M, Lenis S, et al. Transfusion predictors in liver transplant. Anesth Analg 2004;98:1245-51. 38. Massicotte L, Lenis S, Thibeault L, et al. Effect of low central venous pressure and phlebotomy on blood product transfusion requirements during liver transplantations. Liver Transpl 2006;12:117-23. 39. Nemes B, Polak W, Ther G, et al. Analysis of differences in outcome of two European liver transplant centers. Transplant Int 2006;19:372-80. 40. Ozier Y, Pessione F, Samain E, et al. Institutional variability in transfusion practice for liver transplantation. Anesth Analg 2003;97:671-9. 41. Ramos E, Dalmau A, Sabate A, et al. Intraoperative red blood cell transfusion in liver transplantation: Influence on patient outcome, prediction of requirements, and measures to reduce them. Liver Transpl 2003;9:1320-7. 42. Jabbour N, Gagandeep S, Mateo R, et al. Transfusion free surgery: Single institution experience of 27 consecutive liver transplants in Jehovah’s Witnesses. J Am Coll Surg 2005;201:412-17. 43. Molenaar IQ, Warnaar N, Groen H, et al. Efficacy and safety of antifibrinolytic drugs in liver transplantation: A systematic review and meta-analysis. Am J Transplant 2007;7:185-94. 44. Lentschnener C, Roche K, Ozier Y. A review of aprotinin in orthotopic liver transplantation: Can its harmful effects offset its beneficial effects? Anesth Analg 2005;100:1248-55. 45. Xia VW, Steadman RH. Antifibrinolytics in orthotopic liver transplantation: Current status and controversies. Liver Transpl 2005;11:10-18. 46. Planinsic RM, van der Meer J, Testa G, et al. Safety and efficacy of a single bolus administration of recombinant factor VIIa in liver transplantation due to chronic liver disease. Liver Transpl 2005;11:895-900. 47. Lodge JPA, Jonas S, Jones RM, et al. Efficacy and safety of repeated perioperative doses of recombinant factor VIIa in liver transplantation. Liver Transpl 2005;11:973-9. 48. Update on provision of CMV-reduced-risk cellular blood components. Association bulletin #02-4. Bethesda, MD: AABB, 2002. 49. Leukocyte reduction. Association bulletin 99-7. Bethesda, MD: AABB, 1999. 50. Marti H, Henschkowski J, Laux G, et al. Effect of donor-specific transfusions on the outcome of renal allografts in the cyclosporine era. Transplant Int 2006;19:19-26. 51. Triulzi DJ, Nalesnik MA. Microchimerism, GVHD, and tolerance in solid organ transplantation. Transfusion 2001;41:419-26. 52. Magee CC. Transplantation across previously incompatible immunological barriers. Transplant Int 2006;19:87-97. 53. Stegall MD, Dean PG, Gloor JM. ABO-incompatible kidney transplantation. Transplantation 2004;78:635-40.

Chapter 40: Transfusion Therapy in Solid-Organ Transplantation

54. West LJ. B-cell tolerance following ABO-incompatible infant heart transplantation. Transplantation 2006;81:301-7. 55. Bryan CF, Nelson PW, Shield CF, et al. Long-term survival of kidneys transplanted from live A2 donors to O and B recipients. Am J Transplant 2007;7:1181-4. 56. Bryan CF, Winklhofer FT, Murillo D, et al. Improving access to kidney transplantation without decreasing graft survival: Long-term outcomes of blood group A2/A2B deceased donor kidneys in B recipients. Transplantation 2005;80:75-80. 57. Takahashi K, Saito K. Present status of ABO-incompatible kidney transplantation in Japan. Xenotransplantation 2006;13:118-22. 58. Mannami M, Mitsuhata N. Improved outcomes after ABOincompatible living-donor kidney transplantation after 4 weeks of treatment with mycophenolate mofetil. Transplantation 2005;79:1756-8. 59. Gloor JM, Lager DJ, Fidler ME, et al. A comparison of splenectomy versus intensive posttransplant antidonor blood group antibody monitoring without splenectomy in ABO-incompatible kidney transplantation. Transplantation 2005;80:1572-7. 60. Segev DL, Simpkins CE, Warren DS, et al. ABO-incompatible hightiter renal transplantation without splenectomy or anti-CD20 treatment. Am J Transplant 2005;5:2570-5. 61. Szczepiorkowski ZM, Bandarenko N, Kim HC, et al. Guidelines on the use of therapeutic apheresis in clinical practice—evidencebased approach from the Apheresis Applications Committee of the American Society for Apheresis. J Clin Apher 2007;22:106-75. 62. Tyden G, Donauer J, Wadstrom J, et al. Implementation of a protocol for ABO-incompatible kidney transplantation—a three-center experience with 60 consecutive transplantations. Transplantation 2007;83:1153-5. 63. Montgomery RA, Hardy MA, Jordan SC, et al. Consensus opinion from the Antibody Working Group on the diagnosis, reporting, and risk assessment for antibody-mediated rejection and desensitization protocols. Transplantation 2004;78:181-5. 64. Kobayashi T, Saito K. A series of surveys on assay for anti-A/B antibody by Japanese ABO-incompatible Transplantation Committee. Xenotransplantation 2006;13:136-40.

65. Futagawa Y, Terasaki PI. ABO incompatible kidney transplantation— an analysis of UNOS Registry data. Clin Transplant 2006;20:122-6. 66. King KE, Warren DS, Samaniego-Picota M, et al. Antibody, complement and accommodation in ABO-incompatible transplants. Curr Opin Immunol 2004;16:545-9. 67. Park WD, Grande JP, Ninova D, et al. Accommodation in ABOincompatible kidney allografts, a novel mechanism of self-protection against antibody-mediated injury. Am J Transplant 2003;3:952-60. 68. Beimler JHM, Susal C, Zeier M. Desensitization strategies enabling successful renal transplantation in highly sensitized patients. Clin Transplant 2006;20(suppl 17):7-12. 69. Jordan SC, Vo AA, Peng A, et al. Intravenous gammaglobulin (IVIG): A novel approach to improve transplant rates and outcomes in highly HLA-sensitized patients. Am J Transplant 2006;6:459-66. 70. Zachary AA, Montgomery RA, Ratner LE, et al. Specific and durable elimination of antibody to donor HLA antigens in renal-transplant patients. Transplantation 2003;76:1519-25. 71. Montgomery RA, Zachary AA. Transplanting patients with a positive donor-specific crossmatch: A single center’s perspective. Pediatr Transplant 2004;8:535-42. 72. Jordan SC, Tyan D, Stablein D, et al. Evaluation of intravenous immunoglobulin as an agent to lower allosensitization and improve transplantation in highly sensitized adult patients with end-stage renal disease: Report of the NIH IG02 trial. J Am Soc Nephrol 2004;15:3256-62. 73. Stegall MD, Gloor J, Winters JL, et al. A comparison of plasmapheresis versus high-dose IVIG desensitization in renal allograft recipients with high levels of donor specific alloantibody. Am J Transplant 2006;6:346-51. 74. Jordan SC, Vo AA, Tyan D, et al. Current approaches to treatment of antibody-mediated rejection. Pediatr Transplant 2005;9:408-15. 75. Marques MB, Tuncer HH. Photopheresis in solid organ transplant rejection. J Clin Apher 2006;21:72-7. 76. Baughman KL, Jarcho JA. Bridge to life—cardiac mechanical support. N Engl J Med 2007;357:846-9. 77. Rozga J. Liver support technology—an update. Xenotransplantation 2006;13:380-9.

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Apheresis

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41

Apheresis: Principles and Technology of Hemapheresis Ronald O. Gilcher1 & James W. Smith2 1 2

Medical Director Emeritus, Oklahoma Blood Institute, Oklahoma City, Oklahoma, USA Medical Director, Oklahoma Blood Institute, Oklahoma City, Oklahoma, USA

Apheresis, a word of Greek derivation, means “removal” in its broadest sense. In this chapter, it refers to any procedure during which blood is withdrawn from the donor or patient and separated ex vivo into some or all of its components. Some of these components are retained for donation or therapeutic purposes. The others are returned, usually “on line,” to the person. The word pheresis once was used synonymously with the word apheresis; however, apheresis is the preferred word. Hemapheresis is frequently used synonymously with apheresis.

Historical Background The first experimental apheresis procedure was performed in 1660 by Richard Lower of Oxford, England, who performed a manual procedure on dogs. Plasmapheresis (removal of plasma with return of red cells was first performed in France in 1902 and in Russia in 1914.1 In 1914 at Johns Hopkins University, Roundtree and Turner used plasmapheresis in artificial kidney research.1 In 1960, Soloman and Fahey used manual plasmapheresis therapeutically to reduce elevated globulin levels in a patient with a hyperviscosity syndrome, and thus began the era of therapeutic apheresis.1,2 Apheresis became a practical reality with technologic advances, which included development of plastic bags, integrally connected tubing, and ex-vivo centrifugation. Initially, apheresis involved blood separation as an “off-line” disconnected process in laboratories with freestanding component separation centrifuges. Now, apheresis is routinely performed as an “on-line” procedure with fully automated blood cell separators at the donor’s or patient’s bedside. Manual donor plasmapheresis with paid plasma donors was the major method of collecting Source Plasma for fractionation into plasma derivatives (albumin, immunoglobulins, Factor VIII,

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

and Factor IX) between 1950 and 1980. Because the process was “off line,” requiring separation of the donor from the unit of whole blood during plasma separation, the possibility of returning the red cells to the wrong donor existed and occasionally did occur. The long time of 1.5 to 2 hours to collect 500 to 700 mL of plasma led to the use of paid donors and ultimately to the contamination of many plasma derivatives with hepatitis B virus (HBV) and hepatitis C virus (HCV), and human immunodeficiency virus (HIV). In the 1980s, automated on-line cell separation devices were developed by Haemonetics (Braintree, MA), Baxter Fenwal (Deerfield, IL), and Organon Teknika (Boxtel, The Netherlands) for collection of Source Plasma in the United States and Europe. By 1985, an estimated 120,000 to 150,000 automated donor plasmapheresis procedures had been performed in the United States; however, 15 years later that number was more than 12 million a year and now is estimated to exceed 17 million donations per year worldwide.3 Granulocytes, platelets, and Fresh Frozen Plasma (FFP) were the original components collected by means of apheresis technology. Today, numerous blood components are collected from a single donor by means of automated apheresis technology, including the following: 1. Platelets. 2. Plasma. 3. Red Blood Cells (RBCs). 4. RBCs plus plasma (RBCP). 5. 2-unit RBCs (2RBCs). 6. Platelets and RBCs. 7. Platelets and plasma. 8. Granulocytes stimulated with steroids or colony-stimulating factors. 9. Hematopoietic progenitor cells (HPCs) stimulated with granulocyte colony-stimulating factor (G-CSF) or granulocytemacrophage colony-stimulating factor (GM-CSF). In the past few decades, plateletpheresis procedures have increased dramatically—from 171,200 in 1989 to 1,167,000 in 2006.4 RBC apheresis procedures, especially 2RBC collections, are rapidly increasing. Nationally, the collection of apheresis RBCs increased from

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824,000 in 2004 to 1,619,000 in 2006.4 Of the 149,763 collections of allogeneic RBCs at the Oklahoma Blood Institute (OBI) in 2000, 13,944 (9.3%) resulted from RBC apheresis procedures. In 2006, the apheresis RBCP or 2RBC procedures at OBI produced 24,685 units (13% of total RBC units). Therapeutic apheresis procedures (therapeutic plasma exchange and therapeutic cytapheresis) have increased as well—from an estimated 60,000 to 80,000 in 1985 to more than 112,000 in 2006.4 One reason for the increase is the use of hemapheresis technology to remove red cells provides a new approach for patients with hemochromatosis. Another reason is the expanding role of photopheresis in managing conditions such as graftvs-host disease (GVHD) and graft rejection.

Current Technology Overview Edwin J. Cohn, originator of plasma fractionation processes to produce plasma derivatives such as albumin and γ-globulin, developed a prototype machine for centrifugal separation of the cellular elements of blood from plasma. Allen “Jack” Latham, cofounder of Haemonetics, working with Cohn’s prototype, developed a disposable polycarbonate plastic bowl with a rotary seal, which was first used to collect platelets in 1971. Concurrently, George Judson, an IBM engineer on loan to the National Cancer Institute, developed another machine to facilitate white cell (granulocyte) collection.1 Plasmapheresis and eventually therapeutic plasma exchange (TPE) and therapeutic

cytapheresis were natural developments in the use of these early apheresis devices to manage human diseases known or thought to be mediated by plasma or cellular factors. The apheresis devices currently approved by the US Food and Drug Administration (FDA) for donor and patient (therapeutic) use are outlined in Table 41-1. These devices are either discontinuous flow (Haemonetics) or modified continuous flow [Fenwal (Lake Zurich, IL), Fresenius (Bad Homburg, Germany), CaridianBCT (Lakewood, CO)] systems that can be used for oneor two-arm venous access for apheresis procedures. The spinning membrane technology used in the Autopheresis-C (Fenwal) and the flexible bowl technology of Transfusion Technologies (acquired by Haemonetics in 2000) are unique. The latter device has an expandable membrane over a flat disk. Fluid pressure controls the shape and volume of the collecting apheresis chamber and offers unique options for future blood component collection and separation. This flexible bowl is being used in an intraoperative and postoperative autotransfusion device (Haemonetics OrthoPAT System) and in the Haemonetics Cymbal Automated Blood Collection System currently approved for 2RBC collection.

Fenwal Technology Fenwal uses continuous-flow technology and a sealless system in the CS3000 blood cell separator, originally introduced in the early 1980s. The device is fully automated and computer controlled. The CS3000 concentrates platelets during collection into a final volume of approximately 200 mL. An enhancement of the CS3000 with a platelet-collection chamber called the TNX-6

Table 41-1. Apheresis Devices Used in the United States for Donor Collection and Patient Therapeutics Manufacturer/Device Fenwal CS-3000 Amicus ALYX Autopheresis-C Fresenius AS104 CaridianBCT Spectra Trima/Trima Accel Optia Haemonetics MCS
(LN9000) MCS
(LN8150) Cymbal PCS-2 Therakos UVAR-XTS

Type

Product/Procedure

Venous Access

Intended Use

C/CF C/CF C/CF SM/DF

PLAP, HPC, TPE PLAP, AFFP 2RBC, RBCP AFFP, SP

1 or 2 arms 1 or 2 arms 1 arm 1 arm

Donor or patient Donor Donor Donor

C/CF

PLAP, HPC, TPE

1 or 2 arms

Donor or patient

C/CF C/CF C/CF

PLAP, HPC, TPE PLAP, AFFP, RBC, 2RBC TPE, HPC

1 or 2 arms 1 arm 1 or 2 arms

Donor or patient Donor Patient

C/DF C/DF C/DF C/DF

PLAP, AFFP, TPE RBCP, 2RBC 2RBC AFFP, SP

1 or 2 arms 1 arm 1 arm 1 arm

Donor or patient Donor Donor Donor

C/DF

PCWBC

1 arm

Patient

C  centrifugal; CF  continuous flow; DF  discontinuous flow; SM  spinning membrane; PLAP  platelet by apheresis; AFFP  apheresis fresh frozen plasma; RBC  red blood cell; 2RBC  2-unit red blood cells; TPE  therapeutic plasma exchange; SP  source plasma; HPC  hematopoietic progenitor cells; PCWBC  photochemically modified white blood cells.

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Chapter 41: Apheresis: Principles and Technology of Hemapheresis

separation chamber has decreased the white cell content in the platelet product. The CS3000 Plus can be used to collect granulocyte-platelet concentrates and HPCs. These devices are being slowly removed from the market. Fenwal has developed a newer cell separator called the Amicus, which operates in a manner similar to the CS3000, but has a smaller footprint and operates as a one-arm venous access device for platelet collection. The high extraction coefficient for platelets allows frequent collection of double products, such as two apheresis platelet products each containing at least 3.0  1011 platelets and less than 1.0  106 residual leukocytes without the use of a leukocyte reduction filter. In donors with platelet counts of 250,000/µL before donation, double plateletpheresis products are obtained more than 75% of the time. The ALYX is a relatively new and compact mobile automated blood collection system that was initially developed as a 2RBC apheresis collection device. It has been approved for collection of concurrent plasma. Following collection in the rigid, cylindrical centrifuge chamber, red cells are pumped through an on-line leukocyte reduction filter into the final storage bag(s). Fenwal also uses a device called the Autopheresis-C with a spinning membrane, thus incorporating both membrane and centrifugation technology to collect plasma. In the United States, this device is commonly used to collect Source Plasma as well as apheresis FFP. This is a discontinuous system in which only a single venipuncture is performed. Fenwal attempted to change the porosity of the membrane to collect platelet-rich plasma,5 but the procedure has been abandoned.

Fresenius Technology Fresenius introduced a cell separator in Europe in 1987 primarily for the collection of platelets. The Fresenius AS104 is currently licensed in the United States for the collection of apheresis platelets and is licensed for TPE and collection of HPCs. It is a continuous-flow, double-venipuncture system. Fresenius has introduced an upgraded version called the AS204 in Europe but not in the United States. Further development led to the COM.TEC, the latest commercialized version in Europe and Canada. It is not yet available in the United States.6

CaridianBCT Technology CaridianBCT (formerly Gambro and COBE) acquired the IBM biomedical services technology (the IBM-2997 cell separator) and then developed the COBE Spectra as a sealless system based on the original IBM-2997 rotating-channel belt. The Spectra is a continuous-flow system in which one- or two-arm venous access is used for platelet collection. The Spectra uses a leukocyte reduction system to collect leukocyte-reduced platelet units. The Spectra device indicates when units contain excessive numbers of leukocytes and thus when counting is needed to document leukocyte reduction status. The Spectra device also is used to collect HPCs and to perform therapeutic apheresis procedures. The newest CaridianBCT cell separators are the Elutra Cell Separation System (not currently FDA-approved) and the

Spectra Optia Apheresis System (recently FDA-approved). The Elutra Cell Separation System is an “off-line” system that uses counterflow centrifugal elutriation to separate cell populations. The Spectra Optia Apheresis System is based on the Spectra but uses a Trima platform and is aimed at the therapeutic apheresis and cell therapy markets. This instrument will eventually incorporate the multiple programs from the Spectra, but in the more portable size of the Trima platform. At the present time it is approved only for TPE. The Trima system has evolved into the Trima Accel Collection System and is also a relatively new device. The original Trima device is fully automated; the technology is similar to that of the Spectra device. The Trima is a one-arm venous access device, has no rotating seal, and has a smaller footprint than does the Spectra. The Trima is capable of collecting double units (6.0  1011 platelets) that are leukocyte reduced to 1.0  106 or fewer residual white cells. The Trima has been designed and is FDA approved to collect a single or double RBC unit along with the apheresis platelet product. To date it has not been used for therapeutic procedures. Collection of 1 or 2 RBC units with a platelet unit (3.0  1011 platelets) that is leukocyte reduced is a novel idea and has the advantage of increasing the RBC supply of a blood center. The disadvantage is the possibility of deferring a donor on the next visit if the donor’s hematocrit decreases to less than 38%. Because of the low extracorporeal red cell mass in the Trima device, a donor giving red cells and a plateletpheresis product can return within 72 hours to donate another plateletpheresis product with the Trima device—provided the donor’s hematocrit is 38% or higher.7 For donors with a predonation platelet count of 250,000/µL, double plateletpheresis products can be collected more than 75% to 80% of the time. The Trima Accel Collection System collects platelets, red cells, plasma, and combinations of these blood components. It is unique in that it uses the single-channel belt, whereas the original Trima used the dual-channel belt for the collection of platelets. By using the single-channel belt coupled to an internal leukocyte reduction system, the Trima Accel has an increased platelet extraction coefficient and still maintains a low residual white cell count of less than 106 per component. The advantages of the Trima Accel are 1) it can collect the same number of platelets as the “standard” Trima but in a shorter time, 2) it can collect more platelets in the same time as the “standard” Trima, or 3) it can collect the same number of platelets in the same time as the “standard” Trima, but do it by processing less of the donor’s blood, which means delivering less citrate to the donor and reducing citrate-induced donor reactions. Obsolete CaridianBCT technologies include a continuousflow device with a flat membrane for blood separation and the COBE/IBM 2997.

Haemonetics Technology The discontinuous-flow cell separators (MCS
LN9000; MCS
LN8150; PCS-2) produced by Haemonetics operate with a fixed centrifuge speed and a variable centrifugal force to separate a

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donor’s or patient’s blood into components. A disposable hardshelled rotating bowl separates the incoming blood in such a way that red cells move to the periphery and plasma to the inside of the rotating bowl. The buffy coat, which contains the white cells and platelets, forms a layer between the red cells and plasma. Platelets or mononuclear cells are collected with the aid of optical detectors by means of a fluid-surge elutriation process. The remaining red cells and plasma are returned to the donor, and the platelets are retained until the desired yield is obtained with multiple passes or cycles. This discontinuous technology is amenable to a one- or two-arm protocol, although the one-arm venous access technique is used more often because of donor preference for a single needlestick.8 The Cymbal Automated Blood Collection System uses the Dynamic Disk, which is a flexible expandable variable volume bowl for the collection of 2RBC as currently FDA approved. It is also a discontinuous flow system. The Haemonetics plasma collection system-2 (PCS-2), which currently does not have a functionally closed rotating seal, has a disposable bowl specifically designed to collect plasma. This has become the most commonly used automated cell separator for collection of Source Plasma in the United States and the world. The PCS-2 also is used to collect transfusable plasma in clinically useful volumes of 500 to 600 mL (apheresis FFP). The MCS
cell separators are fairly lightweight [60 to 70 lb (27 to 31.5 kg)], extremely portable and mobile, fully automated, and flexible in terms of blood components collected. The MCS
LN9000 is used strictly to collect platelets with or without concurrent plasma. Unlike the Fenwal Amicus and Caridian Trima or Spectra leukocyte reduction system, the initial platelet product is not leukocyte reduced to an acceptable level (1.0  106 residual white cells) and must undergo leukocyte reduction via filtration, which is now accomplished with an on-line process that produces a final transfusable product that contains 1.0  106 residual white cells in 3.0  1011 or more platelets. The MCS
LN8150 has been developed for collection of either RBCP or 2RBC.9-11 Each RBC unit contains approximately 180 to 200 mL of absolute red cell mass with the machine preprogrammed for the desired amount of red cell mass. Both MCS
devices (LN9000 and LN8150) have a functionally closed rotating seal to maximize product shelf life. The MCS
LN8150 operates with an anticoagulant to anticoagulated whole blood (AC:ACWB) collection ratio of 1:16 with citrate-phosphatedextrose-dextrose (CP2D), which further reduces the amount of citrate in the RBCP or 2RBC units but markedly reduces the citrate in the plasma collected. The plasma of the RBCP collection contains one-half the citrate of whole-blood-derived (WBD) FFP. The red cells from the 2RBC procedure can be filtered on line using a single filter to yield 2 leukocyte-reduced RBC units. These systems return saline to minimize the chance of donor hypovolemia. The newest instrument/system available from Haemonetics is the Cymbal Automated Blood Collection System, a small, lightweight, portable, discontinuous flow device intended for the collection of 2 RBC units. This device uses a variable volume

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bowl that expands as blood fills it, with separation occurring based on density in the centrifugal field. The system uses CP2D with AS-3 and filters the red cell on line to achieve leukocyte reduction.

Therakos (Johnson & Johnson) Technology The newest photopheresis device (UVAR-XTS system) is a discontinuous-flow cell separator that collects buffy coat containing mononuclear cells from patients with a variety of conditions such as cutaneous T-cell lymphoma, GVHD resulting from marrow transplantation, or solid-organ rejection. Methoxsalen (Uvadex) is injected into the buffy coat product, the cells are incubated and irradiated with ultraviolet light, and the component is transfused back to the patient. Amelioration of the pathologic process through immunologic effects on clones of mononuclear cells is the desired outcome. The older Therakos UVAR photopheresis system used oral administration of methoxypsoralen and is no longer supported.

Other Technology Other devices, both centrifugal and membrane based, are used outside the United States. Dideco (Mirandola, Italy) makes an automated blood cell separator (Excel) for the collection of apheresis platelets. Organon Teknika developed a hollow-fiber membrane (Plasmapur) plasma collection system, which was previously used in Europe to collect source plasma. Several Japanese and European manufacturers make hollow-fiber separators and filters for donor and therapeutic use. Immunoadsorption and cellular adsorption technologies continue to be tried for removal of pathologic antibodies and to modify cell-mediated immunity respectively, although most have been withdrawn from, or are not readily available in, the US market.12 Several methods have been developed for removal of lipid fractions from patients with homozygous type II hypercholesterolemia. These technologies (see Chapter 43) are expensive and for the most part rather complicated, but effective in a select group of patients.13,14

Donor Apheresis General Information The key forces driving transfusion medicine in the early 21st century are donor and recipient safety enhancement, improved product quality, error reduction, blood availability, legal and regulatory issues, and cost. The issues of safety, quality, and error reduction with regard to blood components are enhanced with the use of apheresis-derived products. Metered anticoagulation, automation during collection, reduced variables, labeling techniques, and fewer donor exposures all enhance safety, quality, and error reduction. The issues of blood availability (especially RBC units) and increasing cost (especially for testing for infectious and noninfectious agents and additional processes such as leukocyte reduction) have become critical issues. Apheresis components that have been more expensive to produce in the past are now being produced as

Chapter 41: Apheresis: Principles and Technology of Hemapheresis

double- and triple-yield products or as multiple types of products (RBCP and RBCs plus platelets). This reduces costs while increasing availability. The advantages of apheresis-derived blood components are listed in Table 41-2. Blood components that can be obtained are presented in Table 41-3.

Donor Care Donors who undergo automated apheresis must go through routine screening and testing procedures. At minimum, apheresis Table 41-2. Advantages of Apheresis-Derived Blood Components ● ● ● ● ● ● ● ● ●

Reduced donor exposure: full transfusion dose Frequent repeat donor: “pedigreed” donors Higher quality products: more quality control per component collected Consistent and standardized products (yields) Matching donors to patients Reduced donor reactions High donor acceptance Double yield or multiple full-dose blood component collections Safety enhancement: for the patient

Table 41-3. Apheresis-Derived Blood Components from a Single Procedure Procedure Primary PLAP PLAP
Plasma PLAP
RBC 2RBC RBCP Plasma Granulocytes HPC Secondary Cryoprecipitate Cryoreduced Plasma

Instrument

MCS
(LN9000), Spectra, Trima/TrimaAccel, CS3000, Amicus, AS104 MCS
(LN9000), Spectra, Trima/TrimaAccel, CS3000, Amicus AS104 Trima/Trima Accel MCS
(LN8150), Cymbal, ALYX MCS
(LN8150), ALYX PCS-2, Autopheresis-C MCS
(LN9000), Spectra, CS3000, Spectra, Optia, CS3000, AS104 PCS-2, Autopheresis-C PCS-2, Autopheresis-C

PLAP  plateletpheresis; RBC  red blood cells; 2RBC  2-unit red blood cells; RBCP  red blood cells plus plasma; HPC  hematopoietic progenitor cells.

component donors must meet all the requirements of wholeblood donors. The main difference is that the “needle in to needle out” time for a 500-mL whole-blood donation is only 8 to 12 minutes, whereas the time for plasmapheresis and red cell apheresis is 35 to 45 minutes, and the time for plateletpheresis is 60 to 120 minutes. The longer total time for apheresis donation requires more precise scheduling and a more dedicated donor. When 2 RBC units are donated, the interval between donations is 112 days, for a maximum of three donations per year. For whole blood donors, the maximum is six donations per year. Plateletpheresis donors can give up to 24 donations per year. Donors of transfusable apheresis plasma can make up to 13 donations per year (every 28 days) if the blood center obtains a variance from the FDA with regard to regulations for Source Plasma donors, who donate more frequently. The blood center plasma donors are known as infrequent donors under this variance. In the United States, Source Plasma (for fractionation into albumin, intravenous immune globulin, Factor VIII, and Factor IX) is obtained almost exclusively from compensated donors who donate at commercially operated plasma centers. These donors may donate up to 800 mL of absolute plasma per donation as frequently as twice a week, depending on the donor’s weight. The donor must be examined at defined intervals by a physician and must have total protein and serum protein electrophoresis determinations made at FDA-required intervals as specified in the Code of Federal Regulations.15 Apheresis donors tend to have lower reaction rates than do donors of whole blood (Table 41-4).16 Vasovagal reactions are most common among first-time blood donors; apheresis donors usually are repeat donors.17 Hypovolemia is less common among apheresis donors than among whole-blood donors for two reasons. First, the total volume removed in apheresis donation procedures is less than that in whole-blood donation because the component has a lower volume, as in plateletpheresis, or volume is returned to the donor as crystalloid solution in the anticoagulant and saline solution. Second, the relatively long time at rest during apheresis donation allows transcapillary refilling of the intravascular compartment from the interstitial space. Allergic reactions to iodine skin preparations—the reaction is to the organic radical attached to the iodine—are very rare. However, a mild citrate

Table 41-4. Donor Reactions Reaction

Whole Blood

Apheresis FFP

Apheresis Platelets

2RBC, RBCP

Vasovagal Hypovolemia Allergic Citrate effect toxicity

Occasionally Occasionally Very rare

Rare Rare Very rare

Rare Rare Very rare

Rare Rare Very rare

None None

Rare Very rare

Frequent Occasional

Rare Very rare

FFP  fresh frozen plasma; 2RBC  2-unit red blood cells; RBCP  red blood cells plus plasma; Occasional  0.5% to 2.5%; Rare  0.5%; Very rare  0.01%; Frequent  5% to 20%.

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effect (circumoral paresthesia, feelings of vibration or buzzing, tingling, or coldness) is not uncommon for most plateletpheresis donors. More severe citrate toxicity (muscle cramping, total body shivering, nausea, vomiting, and tetany) is uncommon but is potentially serious and necessitates slowing the return rate of citrate-containing plasma.18,19 The intravenous administration of calcium-containing solutions is generally not recommended, unless the reactions are unusually severe or prolonged. Hemolysis occurs rarely and when it does, the cause is almost always mechanical, such as occlusion or partial occlusion of redcell-containing tubing. On rare occasions (1 in 3000 to 5000 donations), hyperlipemic plasma in platelet donors causes elevated and visible free hemoglobin levels in the collected component. The mechanism of this hemolysis remains unclear. Venous access is an important consideration in donor apheresis procedures because of the need for 1) long “needle in to needle out” time, 2) a prolonged high flow rate, 3) increased frequency of donation, and 4) the occasional need for two venipunctures with continuous-flow equipment. Possible venous access injuries include blood infiltration and bruising and, rarely, venous thrombosis. Cutaneous nerve injuries are rare, and deep nerve injuries (median, ulnar, and radial nerves) are almost nonexistent. The development of smaller-needle technology for venous access will help reduce these problems. Extracorporeal blood volume (ECBV) in apheresis procedures is greatly reduced with use of newer devices, such as the Amicus and Trima. With the older devices, especially the discontinuous-flow devices, ECBV tends to be greater but generally is not a problem except in therapeutic apheresis involving pediatric patients. The supine position of donors and access to oral fluids during the procedure eliminate most problems. However, when the older, higher ECBV devices are used, malfunction of a machine can lead to loss of ECBV, resulting in a 56-day donor deferral. The low ECBV of the newer devices allows another plateletpheresis procedure within 3 days if necessary. If the ECBV of the apheresis device is 100 mL or less, plateletpheresis may be performed less than 8 weeks after a whole-blood donation.20(p24) In the early years of donor apheresis, lymphocyte depletion with loss of immunologic memory was a concern because of high white cell levels in apheresis components, especially in platelets. This has proved not to be a problem, and with the current focus on leukocyte-reduced apheresis components, it is no longer a concern even for very frequent apheresis donors.21 Frequent plateletpheresis does not clinically decrease platelet counts in donors.22

Specific Products and Procedures Plateletpheresis donations are limited to 24 per year by the AABB and the FDA. Plateletpheresis donation can occur every 72 hours but not more than twice a week. If the interval between plateletpheresis donation is 4 weeks or more, a predonation platelet count is not required. If the interval is less than 4 weeks, a predonation platelet count must be obtained and must be 150,000/µL for the procedure to be performed. If red cell loss during the procedure exceeds 200 mL, the waiting period is 8 weeks before the next

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donation. Donors with predonation platelet counts of 300,000/ µL usually can donate a double-dose platelet product (6.0  1011 platelets). Almost all plateletpheresis products in the United States are produced as leukocyte reduced (meeting AABB standards20(p37) for 5.0  106 or fewer residual leukocytes). However, the European standard is 1.0  106 or fewer residual leukocytes per product. Prestorage leukocyte reduction can be accomplished “in process” (nonfiltration leukocyte reduction, CaridianBCT and Fenwal) or immediately after (filtration, Haemonetics) for virtually all plateletpheresis procedures. Prestorage leukocyte reduction has clear advantages over poststorage or bedside leukocyte reduction filtration in reducing febrile reactions and other leukocyte-related complications by preventing accumulation of cytokines and leukocyte fragments during storage.23-25 The intravascular volume deficit during the procedure should not exceed 10.5 mL/kilogram of the donor’s weight. Plateletpheresis units must contain 3.0  1011 or more platelets per unit in at least 75% of the units tested at maximal storage time. The physician in charge of the donor apheresis unit can make a medical decision to accept a donation from someone not eligible at the time if the benefits to the intended recipient outweigh the risks to the donor. This can occur when the donor is an HLA match or is HPA-1a (PlA1) negative and no other donor is available. Use of single venous access and the high platelet extraction coefficients in the newer plateletpheresis devices (Amicus, Trima Accel) allow shorter collection times or higher product yields in normal collection times. Automated red cell collection with apheresis technology can be performed using the Haemonetics MCS
LN8150, which can collect 2 units of RBCs (2RBC) or 1 RBC unit and 1 largevolume plasma unit (RBCP), or the Caridian Trima, which can collect 1 plateletpheresis product and 1 or 2 RBC units.10,11,26 The Fenwal ALYX and Haemonetics Cymbal have also been designed for collection of 2 RBC units. The 2RBC procedure is becoming increasingly popular in the United States to collect group O, Rh-negative RBC units with the intent of increasing RBC availability. The advantages are 1) a standardized red cell mass (180 to 200 mL per unit), 2) metered anticoagulation, which obviates mixing and reduces clot formation, 3) the ability to return fluids to the donor and reduce any risk of hypovolemia, 4) on-line separation of RBCs and plasma as either a 2RBC or RBCP product, eliminating secondary separation procedures, and 5) the use of smaller needles. For the collection of 1 RBC unit along with another component (plasma or platelets) the standard hematocrit criteria apply. However, for 2RBC collection, FDA has imposed specific donor hematocrit, height, and weight requirements.11 Table 41-5 outlines the current criteria for allogeneic 2RBC collection as defined by the FDA. The use of 2RBC collection from female donors is limited. After 2RBC apheresis donation, donor deferral for any procedure (whole blood or apheresis) is 16 weeks. The RBCP criteria for collection of 1 unit of RBCs and 1 unit of jumbo plasma are outlined in Table 41-6. The anticoagulant used in RBCP and 2RBC procedures with the Haemonetics MCS
LN8150 is CP2D at an

Chapter 41: Apheresis: Principles and Technology of Hemapheresis

AC: ACWB ratio of 1:16. This markedly reduces (50%) the citrate returned to the donor during these procedures as well as the citrate in the plasma collected. The absolute volume of plasma collected (about 90%) is higher than that derived from a wholeblood collection (approximately 80% of which is absolute). Automated collection of plasma can be accomplished with all of the apheresis devices, either as a sole collection product (Fenwal Autopheresis-C and Haemonetics PCS-2) or as a concurrent product with apheresis platelets or as part of an RBC apheresis procedure (RBCP). Table 41-6 shows the volume of plasma that can be Table 41-5. Criteria for Allogeneic Donation of 2-Unit Red Blood Cells by Apheresis Donor Weight (lb)

Donor Height (in)

Donor Hematocrit (%)

Maximum Absolute Red Cell Volume (mL)

Men 130-149 150-174 175

61 61 61

40 40 40

180  2 200  2 210  2

Women 150-174 175

65 65

40 40

180  2 200  2

Table 41-6. Criteria for Allogeneic Donation of Red Blood Cells and Plasma by Apheresis Donor Weight (lb)

Donor Hematocrit (%)

Max Absolute Red Cell Volume (mL)

Max Absolute Plasma Volume (mL)

Men 110-129 130-149 150

38 38 38

185 195 210

450 500 550

Women 110-129 130-149 150-174 175

38 38 38 38

180 190 190 200

450 450 500 550

collected during the RBCP procedure according to donor weight and hematocrit. Plasma collected with the Autopheresis-C or PCS-2 is collected at a 1:16 AC:ACWB ratio, as is the plasma of the RBCP. Apheresis FFP is a unique component and differs substantially from WBD FFP (Table 41-7). There is more absolute plasma per milliliter of anticoagulated plasma (90% in apheresis FFP vs 80% in WBD FFP), much less glucose, and only onehalf to two-thirds of the citrate. A truly transfusable dose can be obtained that markedly affects the clotting factor status of an adult patient depending on patient size and the severity of the clotting deficiency. This component meets the criteria for a source of FFP espoused at the 1984 National Institutes of Health consensus conference on the use of plasma.27 The number of residual platelets and white cells in apheresis FFP also is less than in WBD FFP. This may be important in reducing the recipient reactions to plasma transfusion caused by the release of cytokines or leukocyte fragments by passenger leukocytes into the plasma.23,24 Apheresis cryoprecipitate can be derived by means of cryoprecipitation of apheresis FFP. This product was not recognized by the FDA and has generally been abandoned. The Oklahoma Blood Institute previously produced apheresis cryoprecipitate from a volume of absolute plasma equivalent to 3 units of WBD FFP. The apheresis cryoprecipitate had a volume of approximately 100 mL (wet cryo) and did not require pooling or dilution but could be simply thawed and infused. This is in contrast to WBD cryoprecipitate, which is prepared as a dry cryo (volume, 15 to 20 mL) and requires dilution and pooling before administration.11 The plasma remaining after removal of cryoprecipitate had a volume of approximately 400 mL and was used as replacement fluid for plasma exchange in the care of patients with thrombotic thrombocytopenic purpura (TTP). Granulocyte donations are relatively infrequent because of the development of better antibiotics to prevent and manage the infections associated with granulocytopenia and because of the availability of cytokines such as GM-CSF and G-CSF, which help reduce the duration of the granulocytopenic period in patients with marrow failure. When granulocytes are needed, as in the management of neutropenia with bacterial or fungal sepsis not

Table 41-7. Comparison of Plasma Collected by Apheresis and Whole Blood Donation Collection Technique MCS
LN8150

ALYX

PCS-2/Auto-C

Concurrent

WBD FFP

Total volume (mL) Absolute plasma (%) AC (% citrate)

450-550 90 CP2D (3%)

450-550 87 CPD (3%)

500-800 90 Na Citrate (4%)

250-450 80-87 ACD (3%)

AC:ACWB ratio Grams of citrate (per 100 mL of plasma)

1:16 0.3

1:12 0.4

1:16 0.4

1:8-1:12 0.6-0.4

200-250 80 CPDA-1, CPD, CP2D, (3%) 1:8 0.6

WBD  whole blood derived; FFP  fresh frozen plasma; AC = anticoagulant; CP2D  citrate-phosphate-dextrose-dextrose; ACD  acid-citrate-dextrose; CPDA-1  citrate-phosphate-dextrose-adenine; CPD  citrate-phosphate-dextrose.

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responding to antibiotics, collection of granulocytes poses problems for the donor and the patient. To collect an adequate number of granulocytes (see Chapter 15), the donor must be stimulated before donation with steroids and G-CSF to increase the circulating pool of granulocytes. Then hydroxyethyl starch (HES), available in low- and high-molecular-weight forms, must be infused during collection to increase red cell sedimentation and to facilitate separation and collection of granulocytes.28,29 The use of GCSF, steroids, and HES (pharmacomanipulation of the donor) can cause problems such as musculoskeletal pain, weight gain, fluid overload, allergic reaction to the starch, and reactivation of peptic ulcer disease by the steroids. However, only rarely are these problems severe. HPCs can be collected with a variety of cell separators, but the Spectra, CS3000, and AS-104 are the most commonly used cell separators for HPC collection in the United States.30-33 Circulating HPCs are collected by harvesting the buffy-coat-rich portion of the blood and specifically extracting the mononuclearcell-rich portion of the buffy coat, which contains the CD34positive progenitor or stem cells.33 The number of CD34
cells needed to repopulate the marrow varies from 4 to 10  106/kg of body weight. The HPCs usually are cryopreserved in autologous plasma with a 10% final concentration of dimethyl sulfoxide and are frozen in a controlled-rate freezer and stored at temperatures colder than 135ºC. Collection of autologous HPCs by apheresis declined 26.1% between 2001 and 2004. Allogeneic HPC collections by apheresis decreased by 21.7% in the same period. Probable changes responsible for this decrease in total HPC collections by apheresis include: 1) marrow transplant protocols are no longer recommended for treatment of breast cancer patients, 2) new drugs such as rituximab and imatinib are used as first-line treatment rather than HPCs collected by apheresis, 3) newer nonmyeloablative protocols involve less processing and thus fewer collections, and 4) collection efficiency is increased by improved stem cell mobilization regimens. However, apheresis collection of allogeneic HPCs stimulated with G-CSF or GM-CSF as a replacement for classic allogeneic marrow HPC collection for hematopoietic reconstitution (see Chapter 33) is increasing in frequency. This may account for the rebound of allogeneic HPC apheresis collections in 2006, by an increase of 25.2%.4 Autologous HPC apheresis collections also increased in 2006 — by 24.9%.4

Adverse Effects on Donors and Recipients Donor apheresis procedures are as safe or safer than donation of whole blood because of the ability to return fluids to donors and because the longer collection times allow better fluid equilibration and a lower risk of hypovolemia.34,35 Hematomas with the use of single venous access devices (MCS
, Amicus, Trima) are no more frequent than in donation of whole blood. The major “expected” adverse consequence is related to the effects of the citrate anticoagulant returned to the donor. Donors of lower body weight are more likely to experience citrate symptoms. Almost all plateletpheresis donors and 2RBC donors experience some citrate effects

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(circumoral paresthesia, buzzing sensation, and nasal stuffiness). These effects are not worrisome but they should trigger a slowing of the procedure before more serious toxic effects occur, such as muscle tightening, chills, nausea, vomiting, or tetany. Administering oral antacids, warming donors, and slowing the flow rate reduce the citrate effects. Calcium gluconate (10 mL of 10% solution given slowly intravenously) rarely is needed.36,37 A single plateletpheresis procedure can remove 25% to 50% of circulating platelets, but the release of platelets that normally are sequestered in the spleen almost always prevents significant thrombocytopenia in the donor. In frequent plateletpheresis procedures, it is not uncommon for the donor’s platelet count to decrease and then rebound after 1 to 2 weeks. Adverse effects in recipients of plateletpheresis products are equal to or less than the reactions that occur with administration of whole-blood-derived platelet products. The use of leukocyte reduction with almost all plateletpheresis products has markedly reduced but not eliminated transfusion-associated febrile reactions and appears to be reducing the incidence of transfusion- associated GVHD and posttransfusion purpura.38 Transfusion-related acute lung injury (TRALI) is now recognized as one of the greatest noninfectious risks of transfusion. The greatest transfusionassociated infectious disease risk today is bacterial contamination and occurs almost exclusively in platelets. Because plateletpheresis products are from a single donor and a single collection event, the risk of bacterial contamination may be less with plateletpheresis products than with platelets from several whole-blood donations. However, bacterial contamination data from Europe showed no difference between pooled buffy coat whole-blood-derived platelets and apheresis platelet concentrates.39

Therapeutic Apheresis Overview Therapeutic apheresis has been classified traditionally as therapeutic cytapheresis or TPE, but must now include plasma immunoadsorption and cellular adsorption. Strictly speaking, TPE implies removal of the patient’s plasma with replacement of a crystalloid or colloid solution to maintain a normovolemic state in the patient. Plasmapheresis generally implies removal of an aliquot of plasma without fluid replacement. Because these procedures are discussed completely in Chapters 42 and 43, only basic principles are mentioned here. Critical factors involved in therapeutic apheresis procedures include the following considerations: 1. What is to be removed and how best to remove it. What is to be removed from the patient depends on the diagnosis. How it is to be removed depends on the equipment available. For patients with disease mediated by a known plasma factor such as an autoantibody, immune complex, a protein-bound drug or toxin, and a high cholesterol or triglyceride level, it is clear what must be removed. 2. How much is to be removed with each procedure. How much plasma is to be removed depends on the plasma factor involved. The removal of one plasma volume results in a net

Chapter 41: Apheresis: Principles and Technology of Hemapheresis

overall exchange of 62% of the patient’s plasma volume because of progressive dilution by replacement solutions to maintain normovolemia during the procedure.40 This is true whether plasma or red cells are being removed (Table 41-8). The most effective TPEs remove 1.0 to 1.5 plasma volumes (62% to 78% overall exchange). If the plasma factor is an IgG antibody, only about 45% to 50% of the total body IgG is within the intravascular compartment, whereas the remainder is in the extravascular spaces. Because equilibration is rapid, it is difficult to reduce IgG antibodies with only one TPE; daily TPEs are necessary. Conversely, approximately 90% to 92% of IgM antibodies reside in the intravascular compartment and are more easily removed with one large-volume TPE. Some institutions limit TPE to 1.0 to 1.5 calculated plasma volumes per procedure in most instances because of increased efficiency and a lower rate of complications than with larger-volume exchanges (2.0 volumes or greater). A simple method for estimating the blood volume of adults is shown in Table 41-9. One of the authors (ROG) developed this modified rule of fives to quickly estimate blood and plasma volumes. It agrees with results of recent blood volume studies within 15%.41,42 Plasma volume is calculated by means of multiplying the obverse of the hematocrit (1.0 minus the decimal equivalent of the hematocrit) by the estimated blood volume. 3. How often the procedure should be performed. How often a therapeutic apheresis procedure should be performed depends on how ill the patient is and the rate of regeneration or redistribution of the substance being removed. Removal of IgG antibodies generally requires daily TPE because of regeneration and redistribution from the extravascular to the intravascular space.

Therapeutic plasma exchange occasionally must be performed every 12 hours to save a patient with florid liver failure awaiting a liver transplant. 4. How many procedures should be performed. The endpoint of therapeutic apheresis (TPE or therapeutic cytapheresis) with regard to the number of procedures needed depends on the patient’s clinical response and specific laboratory measurements. In disorders mediated by antibodies, only a limited number of procedures usually are needed. This is because TPE is generally a treatment that “buys time” during the wait for other therapeutic measures, such as immunosuppression, to take effect. Recurrence or exacerbation of some diseases, such as TTP or inflammatory neuropathy, frequently necessitates extending the therapeutic apheresis time frame to achieve durable remissions. 5. What the replacement solution should be, if any. Replacement solutions can be crystalloid or colloid (Table 41-10). Although they are less expensive, crystalloid solutions are less useful for large-volume daily plasma exchanges because of the rapid onset of hypoproteinemia and dilutional coagulopathy. In addition, the volume of crystalloid needed is at least two to three times the plasma removed because of the rapid movement of the crystalloid solution into the extravascular space. The most commonly used replacement solution in TPE is 5% albumin, a true natural colloid, which is slightly hyperoncotic. This form of albumin is stripped of its calcium and acts like a calcium sponge by binding calcium ions. It is a safe product, never having been associated with transmission of any known viral infection (HAV, HBV, HCV, or HIV). Because it is slightly hyperoncotic, albumin can be diluted with saline solution to 4.0% to 4.5% for plasma exchange. The other natural colloid is FFP, which

Table 41-10. Replacement Solutions for Plasma Exchange Table 41-8. Plasma Volumes Exchanged: Fraction Removed and Remaining Plasma Volume Removed

Fraction Removed (%)

Fraction Remaining (%)

0.5 1.0 1.5 2.0 2.5 3.0

40 62 78 85 91 94

60 38 22 15 9 6

Men Women

Body Mass and Build Fat

Thin

Normal

Muscular

60 55

65 60

70 65

75 70

Values are in milliliters of whole blood per kilogram of body weight.

Comment

Crystalloid Solutions Saline 0.9% Ringer solution Lactated Ringer’s solution Balanced electrolyte solution

Least expensive, high Na and Cl Inexpensive, less Na and Cl Inexpensive, less Na and Cl Most physiologic but more expensive

Colloids (Natural) Albumin 5% Plasma protein fraction 5% Fresh frozen plasma (FFP) Donor retested FFP Solvent/detergent-treated plasma

Table 41-9. Estimating Blood Volume: Modified Rule of Fives Gender

Solution

Colloids (Artificial) Hydroxethyl starch (high MW) Hydroxethyl starch (low MW) Dextran Modified fluid gelatin

Safe, hyperoncotic, and expensive Not available in United States Less safe, isooncotic, contains clotting factors Slightly safer than FFP, more costly than FFP Not available in United States, risk of non-lipidenveloped virus transmission, pooled — includes Octaplas and Uniplas 450,000 average MW 150,000 average MW Infequently used Not in United States, used in Europe

MW  molecular weight.

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can be derived from whole blood or apheresis donation. As a byproduct of the RBCP procedure, apheresis plasma contains only half of the citrate of WBD FFP and is 90% absolute plasma vs 80% for WBD FFP. This is the replacement fluid of choice for patients with fulminant liver failure who require plasma exchange to save their lives.43 However, because of an increased incidence of allergic reactions and slightly increased risk of transmission of infectious disease, FFP is generally used only when clotting factors or other proteins must be replaced or in the management of TTP, in which studies have established it as the replacement fluid of choice.44 The issue of whether cryoprecipitate-reduced plasma, solvent/detergent-treated (SD) plasma, or unaltered FFP is best in the management of TTP is still not resolved. Artificial colloid solutions (HES or dextran) are used relatively infrequently in the United States and offer no medical advantage over 5% albumin. Depletion of immunoglobulins when using nonimmunoglobulin replacement solutions can put the patient at risk of pneumonia if the IgG levels drop too low. Plasma or IVIG can be given to prevent iatrogenic hypogammaglobulinemia from occurring. Two new SD plasma products are available (Octaplas) or in development (Uniplas) both of which contain normal levels of ADAMTS13 and Factor H, which are beneficial in treating TTP and atypical hemolytic uremic syndrome, respectively.45 In addition, the use of Octaplas has resulted in a lower number of allergic and citrate reactions than the use of cryosupernatant plasma in acute TTP.46 SD plasma is currently not available in the United States. Solvent/detergent treatment does not inactivate viruses that do not have a lipid envelope, such as hepatitis A or human parvovirus B19. SD plasma is a pooled product and has normal amounts of clotting factors but is deficient in some of the anticlotting factors (protein C, protein S, and antithrombin) and plasminogen. Another plasma product with reduced infectious disease risk is donor retested plasma, which is rarely used in the United States. The donor is retested at least 110 days after the initial donation. The unit is not transfused until the retest has been performed and the results are negative for all viral markers tested. The development of pathogen-inactivated plasma will further reduce the risk of transmission of infectious agents but will not prevent the risk of prion transmission believed to be the etiologic agent for variant Creutzfeldt-Jakob disease. 6. What equipment should be used. The type of equipment used depends on the particular needs of the therapeutic apheresis service. The newest of the therapeutic devices in the United States is the Caridian Optia (see Table 41-1). Each of the different manufacturers’ equipment has certain advantages. Most TPE procedures are performed with centrifugal-based machines. Add-on devices such as staphylococcal protein A immunoadsorption columns can be used ex vivo to remove IgG selectively and then to return the immunoadsorbed plasma to the patient.47 Another special type of therapeutic apheresis, called photopheresis, is used to manage a hematologic malignant disease with cutaneous lesions called cutaneous T-cell lymphoma or Sézary syndrome. Photopheresis is the use of extracorporeal photochemotherapy to modify circulating malignant T cells. These

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ultraviolet irradiated and altered T cells in some way modulate an antitumor effect when reinfused.48,49 The new model of the Therakos Photopheresis System (UVAR-XTS) operates with a form of psoralen (methoxsalen) that is injected into the product collected with the device. This technique reduces the risks of systemic exposure to psoralen in the patient, which occurred in the original Therakos Photopheresis System (no longer available or supported). 7. What type of vascular access should be used. Vascular access can be through peripheral veins, central veins, or both. If many TPEs are planned, central venous catheters usually are necessary. Double- or triple-lumen dialysis catheters are ideal for central venous access. Strict catheter care is critical to prevent clotting of the catheter and infections at the catheter site. Flushing the catheter with a heparinized solution immediately after exchange is critical to prevent clotting. If catheter clotting should occur, injection of plasminogen-activating drugs into the clotted lumen frequently lyses the clot and restores patency. Placement of central venous catheters is critical so that the catheter tip is not in the heart (right atrium) but in the superior vena cava. Cardiac irritability with premature ventricular beats can occur if the catheter is in the right atrium. The irritation is caused directly by the catheter or indirectly by return of fluids that are cold, hypocalcemic, hypokalemic, or high in citrate ion. A chest radiograph should be obtained after placement of a subclavian central venous catheter, not only to locate the tip of the catheter within the superior vena cava but also to ensure that the catheter did not perforate the subclavian vein and cause a hemothorax or pneumothorax. For some patients, surgical placement of an arteriovenous shunt, such as an expanded polytetrafluoroethylene (Gore-Tex) graft tunneled under the anterior thigh to connect the femoral artery and vein, may be necessary for long-term TPE. 8. What anticoagulant should be used and how much. The use of anticoagulants in therapeutic apheresis usually is limited to citrate anticoagulants (ACD-A, ACD-B, or sodium citrate), heparin, or both. Acid-citrate-dextrose A (ACD-A) is a 3% citrate anticoagulant and ACD-B a 2% citrate anticoagulant. Sodium citrate is available as a 4% citrate anticoagulant. The advantage of citrate as an anticoagulant is that any amount returned to the patient during the red cell return in a TPE procedure is usually a small amount and is metabolized rapidly unless the patient is in severe liver failure. Citrate produces only local anticoagulation in the collected blood and never produces systemic anticoagulation. However, this can be a disadvantage when flow is low, because catheter clotting can occur. If the flow rate is low and heparin is not contraindicated, heparin can be used alone or in combination with citrate. Heparin may enhance systemic anticoagulation more than is expected because of the additional effect of dilution of clotting factors by the non-plasma replacement solutions. The half-life of heparin in the body is approximately 90 minutes. When heparin is used to anticoagulate blood in the draw line and is not used systemically, 17,500 U is added to 500 mL of saline solution (35 U of heparin per milliliter of saline solution). With

Chapter 41: Apheresis: Principles and Technology of Hemapheresis

an AC:ACWB ratio of 1:8, this formula delivers 5 U of heparin per milliliter of whole blood collected. At an AC:WB ratio of 1:10, it delivers 3.9 U of heparin per milliliter of whole blood collected (3.5 U of heparin per milliliter of anticoagulated blood collected). 9. Where the procedure should be performed, such as at the blood center or in the inpatient or outpatient areas of a hospital. Therapeutic apheresis procedures should be performed only when emergency care is readily available. If the procedure is performed at the blood center on an outpatient basis, the blood center must have a knowledgeable physician available. The nursing staff performing the procedure must be trained in resuscitation care, and a crash cart with a cardiac monitor and defibrillator must be available. In the hospital, the same requirements apply for outpatient procedures. Critically ill patients, such as those with acute TTP, should be treated in an intensive care setting where emergency care is readily available. 10. Whether the presence of a physician is needed throughout each procedure. A physician must be available to the nurses for each procedure, but the severity of the patient’s illness determines whether a physician is needed at the bedside for each therapeutic apheresis procedure. At the Oklahoma Blood Institute, a patient services department handles all therapeutic apheresis procedures, and all procedures are performed by knowledgeable registered nurses. 11. What complications or adverse reactions are to be expected, such as thrombocytopenia, bleeding, thrombosis, arrhythmia, plasma-induced immune reactions. Complications and adverse reactions can and do occur. Volume overload, venous access problems, bleeding, thrombosis, immunoglobulin depletion, citrate reactions, hypokalemia, hypocalcemia, hypotension, allergic reactions, infection, viral transmission from blood components, thrombocytopenia, anemia, TRALI, and death can occur. The mortality has been estimated at three deaths per 10,000 procedures.18

Summary Apheresis technology has rapidly advanced and is replacing some of the manual whole-blood collection techniques. Although more expensive and requiring personnel with greater skill levels to operate the equipment, the new techniques make it possible to collect two or more transfusion doses of blood components from a single donor, theoretically making safer and higherquality blood components available from a smaller donor base and reducing the chances of error. The ability to use smaller needles and to return fluids will make procedures safer for donors as well. Therapeutic apheresis will continue to advance as new agents such as immunoadsorbents are combined with cell and plasma separation devices, whether centrifugal or membrane. The development of HPCs for marrow repopulation, especially when allogeneic donors are routinely stimulated with cytokines (eg, G-CSF), is signaling an even newer era of apheresis advancement. The new era of pathogen inactivation technology

and its application to replacement fluids will improve safety in the use of FFP derivatives for therapeutic apheresis.

Disclaimer The authors have disclosed no conflicts of interest.

References 1. Huestis DW, Bove JR, Busch S. Practical blood transfusion. 3rd ed. Boston, MA: Little, Brown, 1981:315-72. 2. Barnes A. Hemapheresis perspectives. In: Kolins J, Jones JM, eds. Therapeutic apheresis. Bethesda, MD: AABB, 1983:1-13. 3. Robert P. International directory of plasma fractionators. Orange, CT: Marketing Research Bureau, 2007. 4. Department of Health and Human Services. The 2007 National Blood Collection and Utilization Survey Report. Washington, DC: DHHS, 2008. 5. Simon T, Lee EJ, Heaton A, et al. Storage and transfusion of platelets collected by an automated two-stage apheresis procedure. Transfusion 1992;32:624-8. 6. Zingsem J, Strasser E, Ringwald J, et al. Evaluation of a new apheresis system for the collection of leukoreduced single-donor platelets. Transfusion 2007;47:987-94. 7. Murphy MF, Seghatchian J, Krailadsiri P, et al. Evaluation of COBE Trima for the collection of blood components with particular reference to the in vitro characteristics of the red cell and platelet concentrates and the clinical responses to transfusion. Transfus Sci 2000;22:39-43. 8. Simon TL. The collection of platelets by apheresis procedures. Transfus Med Rev 1994;8:132-45. 9. Klein HG. It seemed a pity to throw away the red cells: Selective component collection (editorial). Transfusion 1993;33:788-90. 10. Meyer D, Bolgiano DC, Sayers M, et al. Red cell collection by apheresis technology. Transfusion 1993;33:819-24. 11. Smith JW, Gilcher RO. Red blood cells, plasma, and other new apheresis-derived blood products: Improving product quality and donor utilization. Transfus Med Rev 1999;13:118-23. 12. Minoz J, Clavo M, Garcia O, et al. Adsorptive monocyte and granulocyte apheresis in the chronic inflammatory illness: Ulcerative colitis, Crohn’s disease, rheumatoid arthritis and Behcet syndrome. ISBT Science Series 2007;2:96-101. 13. Bosch T. State of the art of lipid apheresis. Artif Organs 1996;20:292-5 14. Bosch T. Lipid apheresis: From a heroic treatment to routine clinical practice. Artif Organs 1996;20:414-9. 15. Code of federal regulations. Title 21 CFR 640.65. Washington, DC: US Government Printing Office, 2008 (revised annually). 16. Winters J. Complications of donor apheresis. J Clin Apher 2006;21:132-41. 17. Newman BH. Donor reactions and injuries from whole blood donation. Transfus Med Rev 1997;11:64-75. 18. Huestis DW. Adverse effects in donors and patients subjected to hemapheresis. J Clin Apher 1984;2:81-90. 19. Simon TL, Moore RC, Sierra ER, et al. Storage of platelets from a new cell separator in a citrate-plasticized container. In: Rock G, ed. Apheresis. New York: Wiley-Liss, 1990:11-14.

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20. Price TH, ed. Standards for blood banks and transfusion services, 25th ed. Bethesda, MD: AABB, 2008. 21. Strauss RG. Effects on donors of repeated leukocyte losses during plateletpheresis. J Clin Apher 1994;9:130-4. 22. Katz L, Palmer K, McDonnell E, Kabat A. Frequent plateletpheresis does not clinically significantly decrease platelet counts in donors. Transfusion 2007;47:1601-6. 23. Heddle NM, Klama L, Singer J, et al. The role of the plasma from platelet concentrates in transfusion reactions. N Engl J Med 1994;331:625-8. 24. Brand A. Passenger leukocytes, cytokines, and transfusion reactions (editorial). N Engl J Med 1994;331:670-1. 25. Dzik WH. Effects on recipients of exposure to allogeneic donor leukocytes. J Clin Apher 1994;9:135-8. 26. Shi PA, Ness PM. Two unit red cell apheresis and its potential advantages over traditional whole-blood donation. Transfusion 1999;39:218-25. 27. National Institutes of Health Consensus Conference. Fresh-frozen plasma: Indications and risks. JAMA 1985;253:551-3. 28. Herzig RH. Granulocyte transfusion therapy: Past, present, and future. In: Garratty G, ed. Concepts in transfusion therapy. Bethesda, MD: AABB, 1985:267-94. 29. Price TH. The current prospects for neutrophil transfusions for the treatment of granulocytopenic infected patients. Transfus Med Rev 2000;14:2-11. 30. Bandarenko N, Owen HG, Mair DC, et al. Apheresis: New opportunities. Clin Lab Med 1996;16:907-29. 31. Norol F, Scotto F, Duedari N, et al. Peripheral blood stem cell collection with a blood cell separator. Transfusion 1993;33:894-7. 32. Leibundgut K, Muff J, Hirt A, et al. Evaluation of the Fresenius cell separator AS 104 for harvesting peripheral blood stem cells in pediatric patients. Transfus Sci 1994;15:93-9. 33. Hester JP, Wallerstein RO. Peripheral blood stem cell transplantation for breast cancer patients with bone marrow metastases using GMCSF priming. Transfus Sci 1993;14:65-9. 34. Wiltbank TB, Giordano GF. The safety profile of automated collections: An analysis of more than 1 million donations. Transfusion 2007;47:1002-5. 35. Bueno JL. Do we really know the real risks of apheresis donations. ISBT Science Series 2007;2:68-74. 36. Hester JP, McCullough J, Mishler JM, et al. Dosage requirements for citrate anticoagulants. J Clin Apher 1983;1:149-57. 37. Olson PR, Cox C, McCullough J. Laboratory and clinical effects of the infusion of ACD solution during plateletpheresis. Vox Sang 1987;33:79-87. 38. Williamson LM, Stainsby D, Jones H, et al. The impact of universal leukodepletion of the blood supply on hemovigilance reports of posttransfusion purpura and transfusion-associated graft-versushost disease. Transfusion 2007;47:1455-67.

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39. Schrezenmeier H, Walther-Wenke G, Muller TH, et al. Bacterial contamination of platelet concentrates: Results of a prospective multicenter study comparing pooled whole-blood-derived platelets and apheresis platelets. Transfusion 2007;47:644-52. 40. Chopek M, McCullough J. Protein and biochemical changes during plasma exchange. In: Berkman EM, Umlas J, eds. Therapeutic hemapheresis. Bethesda, MD: AABB, 1980:13-52. 41. Heaton A, Holme S. Blood donation and red cell volume (RCV) regeneration in donors of different weights (abstract). Vox Sang 1994;67(Suppl):13. 42. Holme S, Heaton A. Red cell volume (RCV) distribution in volunteers: implications for eligibility for two-unit red cell donation (abstract). Transfusion 1994;34(Suppl):S58. 43. Agdokan M, Camci C, Gurakar A, et al. The effect of total plasma exchange on fulminant hepatic failure. Clin Apher 2006;21:96-9. 44. Rock GA, Shumak KH, Buskard NA, et al. Comparison of plasma exchange with plasma infusion in the treatment of thrombotic thrombocytopenic purpura. N Engl J Med 1991;325:393-7. 45. Heger A, Kannicht C, Romisch J, Svae TE. Normal levels of ADAMTS13 and factor H are present in the pharmaceutically licensed plasma for transfusion (Octaplas) and in the universally applicable plasma (Uniplas) in development. Vox Sang 2007;92:206-12. 46. Scully M, Longair I, Flynn M, et al. Cryosupernatant and solvent detergent fresh-frozen plasma (Octaplas) usage at a single centre in acute thrombotic thrombocytopenic purpura. Vox Sang 2007; 93:154-8. 47. Weinstein R, Sato PTS, Shelton K, et al. Successful management of paraprotein-associated peripheral polyneuropathies by immunoadsorption of plasma with staphylococcal protein A. J Clin Apher 1993;8:72-7. 48. Rook AH, Wolfe JT. Role of extracorporeal photopheresis in the treatment of cutaneous T-cell lymphoma, autoimmune disease, and allograft rejection. J Clin Apher 1994;9:28-30. 49. Edelson RL. Photopheresis. J Clin Apher 1990;5:77-9.

Appendix 41-1. Internet Resources on Apheresis AABB: www.aabb.org ASFA (American Society for Apheresis): www.apheresis.org WAA (World Apheresis Association): www.worldapheresis.org Fenwal: www.fenwalinc.com Fresenius: www.fresenius.com CaridianBCT: www.caridianbct.com Haemonetics: www.haemonetics.com Therakos: www.therakos.com

42

Therapeutic Plasma Exchange Bruce C. McLeod Professor of Medicine and Pathology, Rush Medical College, and Director, Blood Center, Rush University Medical Center, Chicago, Illinois, USA

Therapeutic plasma exchange (TPE) has been compared to the practice of bloodletting to remove evil humors. The notion of therapeutic removal has changed little since medieval times; however, the concept of an evil humor has been discarded in favor of an evidence-based understanding that some blood cells and plasma components can be harmful. Three subtypes that can be distinguished among plasma macromolecules that are candidates for therapeutic removal are as follows: 1) antibodies that are troublesome because of their binding specificity—these are often autoantibodies; 2) molecules that confer abnormal physical properties, such as hyperviscosity or cold insolubility, on plasma and hence on the blood—these, too, are usually antibodies, although they may be bound in immune complexes; and 3) molecules such as low-density lipoproteins (LDLs) that have nonimmune toxicity. From a theoretic point of view, it is easier to envision a significant therapeutic effect when the molecule to be removed is relatively large and has a relatively long half-life in the circulation, with a correspondingly low rate of synthesis. In practice, the majority of successfully treated disorders are caused by pathogenic immunoglobulin G (IgG), which has these properties. It has been suggested from time to time that removal of complement or coagulation proteins, or inflammatory mediators derived from immunocytes, might contribute to a therapeutic effect. Such proposals have seemed unlikely to the author because the candidate molecules are rapidly synthesized and are relatively short lived, and in fact no therapeutic effect involving such molecules has been proven to date. Therapeutic plasma exchange can be employed in a fourth way, not foreseen by medieval barbers or 18th century physicians, and that is to achieve relatively high levels of a normal plasma constituent that is deficient in patient plasma and is not available in a concentrated form for simple infusion.

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

General Principles of Therapeutic Plasma Exchange Mathematic Principles Patients often compare TPE to an automobile oil change, and it is instructive to consider the factors that make this analogy inapt. A 100%-efficient 5-liter oil change can be accomplished in 10 minutes, because the engine need not operate during the procedure. In TPE, limitations are imposed on rate and efficiency by the need to keep the heart pumping and the bloodstream nearly full, so that only a small portion of the total blood volume can be outside the body at any time. Because TPE must proceed gradually, either continuously or in small increments, an ever-increasing proportion of the material being removed is not patient plasma but rather replacement fluid infused earlier in the procedure. During this process, the level of an entirely intravascular substance that is absent in the replacement medium can be predicted by the formula: yx
y0ex, in which y0 is the starting concentration of the substance, e is the base natural logarithm, and yx is the concentration of the substance after x patient plasma volumes have been exchanged. If y0 is assigned a nominal value of 1.0, the function yields the smooth asymptotic middle curve plotted in Fig 42-1 for a continuous exchange. The flanking curves, describing small incremental discontinuous exchanges, are similar. This formula accurately forecasts the outcome of an exchange for macromolecules such as LDL and IgG that have a substantial extravascular reservoir, provided that equilibration between the intravascular and extravascular compartments is slow relative to the removal rate.2 Because the molecule targeted for removal by TPE is often an IgG antibody, which is approximately 50% extravascular,2 and because removal of accessible (that is, intravascular) IgG becomes progressively less efficient during a TPE procedure, most practitioners limit an exchange to 1 to 1.5 times the patient’s estimated plasma volume. An exchange of this magnitude will remove 60% to 75% of intravascular material while limiting side effects from depletion of normal plasma components. The intravascular IgG

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1.0

Continuous exchange y/y0
ex Discontinuous exchange 0.1 plasma volumes per step

Fraction remaining (y/y0 )

0.8

0.6

Replacement added before removal y
y0

0.4

0.2

Removal done before replacement y
v  x y0 v

(

1.0

n

)

2.0

% of total stores Extravascular

Intravascular

Plasma volumes exchanged (x)

3.0

Figure 42-1. Calculated fraction of intravascular substance remaining during a plasma exchange, assuming no equilibration with extravascular material. (Used with permission from Chopek and McCullough.1)

50

25 Hours 0 Days 25

50 100 75 50 25 Hours 0 Days

level will rise during the ensuing 1 to 2 days by equilibration with extravascular sources, and further removal by a subsequent exchange can then be undertaken more efficiently. The effects of a series of TPEs on extravascular, intravascular, and total IgG are shown schematically in Fig 42-2. Human serum albumin is the most common replacement fluid in TPE, for reasons that are given below. In such exchanges,

630

n

( v v x )

Figure 42-2. Computer-generated curve estimating amounts of intravascular and extravascular IgG (upper curves), and total IgG (lower curve) during a course of four one-plasma-volume therapeutic plasma exchanges with an IgG-free replacement medium. Published formulas were used for rates of removal during exchanges and reequilibration after exchanges. No correction was made for continuing synthesis.

all plasma constituents are removed while only albumin is replaced; thus plasma component depletion in an individual exchange is almost completely nonselective.2 However, because most other plasma constituents are synthesized much more rapidly than IgG, a series of such exchanges will result in a reasonably selective depletion of IgG over the course of treatment. This is shown in Fig 42-3.

6.0

1200

5.0

1000

4.0

800

3.0

600

2.0

400

1.0

200

Regulation of IgG Metabolism Because IgG removal is often the goal of TPE, it is worthwhile to consider certain aspects of IgG metabolism. The subclasses IgG1, IgG2, and IgG4 together constitute about 90% of total IgG. Their catabolic rates are proportional to total IgG level and their half-lives are therefore inversely proportional to concentration. This pattern has been taken to indicate the existence of a saturable receptor that protects IgG from catabolism. A receptor on endothelial cells having this property has been characterized, which appears to be identical to the FcRn receptor on neonatal intestinal epithelial cells that facilitates transport of intact maternal antibody in breast milk.4 It is difficult to measure the IgG synthetic rate in humans. Previous animal studies concerning levels of specific antibody were interpreted to show that the synthetic rate for IgG exhibits negative feedback, increasing when IgG or specific antibody levels or both are lower.5 More recently, Junghans6 has shown that “knock-out” mice genetically deficient for the FcRn receptor catabolize IgG quite rapidly and maintain very low IgG levels, but they have the same IgG synthetic rate as normal mice. This argues against negative feedback regulation of IgG synthesis and suggests that a reduction in antibody levels induced by TPE would not produce a meaningful “rebound” increase in IgG synthesis. Intravenous immune globulin (IVIG) has been reported to be effective in some of the same antibody-mediated diseases as TPE. Yu and Lennon7 recently proposed that exogenous immune globulin competes with endogenous IgG for FcRn receptors and thereby promotes accelerated catabolism of the latter, including any pathogenic antibodies. In this view, the beneficial effects of IVIG and TPE are essentially the same; both lead to lower levels of harmful antibodies. Therapeutic plasma exchange lowers levels quickly but may be followed by slower catabolism, whereas IVIG presumably has a slower onset of action but promotes rapid catabolism. This concept of the effect of IVIG could provide a

1

2

3

4

5

6

7

8

mg/dL

Albumin (mean ⴞ SD)

1400

IgG (mean ⴞ SD)

Figure 42-3. Total protein, albumin, and IgG levels before and after therapeutic plasma exchange with albumin/saline replacement. Exchanges were carried out three times per week for 3 weeks on seven patients. Points and ranges represent means and standard deviations. Note this disproportionate decrease in IgG levels. (Used with permission from McLeod et al.3)

7.0

g/dL

Total protein (mean ⴞ SD)

Chapter 42: Therapeutic Plasma Exchange

9

Exchange

Table 42-1. Colloid Replacement Fluids for TPE Fluid

Advantages

Disadvantages

5% Albumin

Virus inactivation Ease of use Reactions rare

High cost Most proteins not replaced

Single-donor plasma*

All proteins replaced

High cost Inconvenient† Citrate reactions Urticaria Viral infection risk

Solvent/detergent-treated All proteins partially plasma replaced‡ Lipid-coated viruses inactivated

Very high cost Inconvenient† Citrate reactions Urticaria Pooled product Unavailable in US

6% Hetastarch

No proteins replaced Hypotensive reactions Dosage limit

Low cost Viral safety Ease of use Slow catabolism

*Fresh Frozen Plasma or Plasma Cryoprecipitate Reduced. †

Must be thawed before use; must match patient ABO group.

‡

Coagulation factors 80% of normal levels.

TPE
therapeutic plasma exchange; US
United States.

compelling rationale for sequential treatment of autoantibody diseases with TPE followed by IVIG.

Replacement Fluids Saline replacement alone can suffice when only 500 to 1000 mL of plasma is removed in a manual plasmapheresis, but colloid replacement must be given in a multiliter plasma exchange.

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Available colloid replacement fluids are listed in Table 42-1, which also summarizes their relative advantages and disadvantages. For the majority of indications the standard replacement medium is 5% human serum albumin in normal saline; however, substitution of 25% to 50% of the total with saline has been shown to be well tolerated in certain patient groups. Although it is a pooled product, 5% albumin is preferred over plasma as a source of replacement colloid because 1) it can be pasteurized to inactivate all known blood-borne pathogens, 2) it can be given without regard to blood type, and 3) it does not require thawing or other preparation before use. Adverse reactions to albumin are rare.8 Exchanges of plasma for albumin produce temporary deficiencies of other plasma proteins, such as coagulation factors; however, these are usually subclinical and levels are rapidly restored by ongoing synthesis and reequilibration.2 There are a few circumstances in which replacement of patient plasma with donor plasma seems prudent. Prominent among these is the treatment of thrombotic microangiopathies. Affected patients customarily receive exchanges with Fresh Frozen Plasma (FFP) in light of abundant evidence that patients with thrombotic thrombocytopenic purpura (TTP) respond better to plasma than to albumin. Cryoprecipitatereduced plasma is also effective for this purpose.9 Some plasma may also be given toward the end of an exchange to patients with preexisting thrombocytopenia or humoral coagulopathy, who are considered to be at increased risk for bleeding complications when the dilutional coagulopathy of an albumin exchange is superimposed, or to patients with ongoing blood loss regardless of pretreatment coagulation status. Plasma replacement carries a higher risk of urticarial and hypocalcemic reactions.10 One group reported infusing a solution of hydroxyethyl starch (HES), a less costly volume expander, in the early part of an exchange.11 They reason that recommended dosage limitations for HES will not actually be exceeded, because much of the infused HES will be removed in exchange for albumin infused later. This group has seen successes, albeit with a higher incidence of side effects. HES is not recommended for patients with renal impairment, underlying coagulopathy, or a history of hypersensitivity to HES.

Selective Extraction of Plasma Components Practitioners of TPE have long recognized the inherent wastefulness in removing and discarding all plasma components in order to deplete just one. Selective extraction of a pathogenic component from separated plasma, with recovery and reinfusion of normal constituents, has been considered an attractive goal. Several applications and methods for on-line separation have been explored,12 but commercial availability and widespread acceptance have been slow to be realized. Obstacles to commercially practical devices include the high cost and limited capacity of truly selective, biocompatible sorbents and the high extracorporeal volume associated with

632

sorbent modules having a depletion capacity equivalent to TPE. One approach is to use pairs of small, low-capacity sorbent modules with alternating flow cycles such that one module absorbs material from patient plasma while the absorbent capacity of the other is restored by elution of bound material. This approach to selective extraction lowers the volume and cost of sorbent modules but requires an additional instrument to manage the cycling-elution-regeneration process.12 Selective extraction techniques and their applications are discussed in more detail in Chapter 41.

Indication and Treatment Intensity Categories To assist practitioners in assessing the value of apheresis in specific circumstances, the American Society for Apheresis (ASFA) has described indication categories for therapeutic apheresis.13 The definitions can be summarized as follows: Category I denotes diseases for which apheresis therapy is a standard firstline therapy, although it may not always be necessary; Category II denotes diseases in which apheresis is a valuable second-line therapy when first line measures fail or are poorly tolerated; Category III indicates uncertainty due to inadequate data or controversy due to conflicting reports; and Category IV is reserved for diseases in which there is negative data from controlled trials or anecdotal reports. The indication categories assigned by ASFA are provided in tabular form for the diseases in each of the following sections. In addition, these tables include the author’s perceptions of the customary intensity (frequency and duration) of TPE therapy for most of the diseases. The four categories used are: aggressive (A), which implies daily TPE until remission or improvement; routine (R), which implies five to seven treatments every other day (or three times per week); prolonged (P), which implies one to three treatments per week for 3 to 8 weeks; and chronic (C), which implies one treatment every 1 to 4 weeks, continuing indefinitely. These intensity categories, which need not be mutually exclusive, are summarized in Table 42-2. Note that more than one category may be applicable to a given disease, or even a given patient, depending on extant clinical circumstances. The references cited in the following discussions are not exhaustive. Readers seeking a more extensive bibliography may consult the chapters on TPE in a recent text devoted to apheresis2,12,14-16 and articles in recent journal issues devoted to clinical applications of therapeutic apheresis.13,17-20,21

Table 42-2. Intensity of Treatment Categories for TPE Level

Schedule

Duration

Aggressive (A) Routine (R) Prolonged (P) Chronic (C)

Daily 3 times a week 1-2 times a week Every 1-4 weeks

3 treatments to indefinite 5-7 treatments 3-8 weeks Indefinite

TPE
therapeutic plasma exchange.

Chapter 42: Therapeutic Plasma Exchange

Therapeutic Plasma Exchange in Neurologic Disorders Immune processes, especially formation of circulating antibody to structures in the nervous system, have been implicated in several neurologic diseases, and TPE has become an important therapy for some of them. The diseases to be considered in this section appear in Table 42-3 along with their respective indications and intensity categories.

Guillain-Barré Syndrome Guillain-Barré syndrome (GBS) affects the peripheral nervous system. It is the most common cause of acute paralysis with areflexia in the developed world, with an incidence of one to two cases per 100,000 population per year. A typical clinical course begins with symmetric distal paresthesias, which are followed by leg and arm weakness. Symptoms spread proximally and reach a peak of severity by 14 to 30 days after onset. About one-fourth of patients with GBS have mild illness and remain ambulatory throughout. The remainder are disabled by paralysis and may have oropharyngeal and respiratory weakness as well. One-fourth will require assisted ventilation at some point, and the worst cases are marked by quadriplegia, ophthalmoplegia, and prolonged ventilator dependence. A nerve conduction defect resulting from demyelination is found in most cases, but there are variants in which axonal damage is evident in motor fibers or both motor and sensory fibers. In another variant (Miller Fisher syndrome) symptoms are limited to ophthalmoplegia, ataxia, and areflexia. The spinal fluid usually contains few cells and only a moderately elevated concentration of protein. A conduction block indicating demyelination is the usual finding in electrophysiologic studies, although inexcitability may also be seen in the axonal variants.22

Demyelination is believed to be caused by circulating myelin antibodies. Early experiments showed that sera from patients with GBS produced demyelination in animals. Later studies have found antibodies to myelin glycolipids, such as GM1, GM1b, GD1a, and GD1b, in the sera of many patients. Antibodies to GQ1b and GT1a are relatively specific for the Miller Fisher variant. Guillain-Barré syndrome is often associated with a history of recent infection with Campylobacter jejuni, cytomegalovirus, Epstein-Barr virus, Mycoplasma pneumoniae, or other organisms. It has been postulated that antibodies are formed in response to a strain-specific lipopolysaccharide that is antigenically similar to one of the myelin gangliosides; eg, GM1 after C. jejuni infection.22 Epidemic Chinese acute motor neuropathy is an illness of rural Chinese children that has epidemiologic characteristics of an infectious disease. It is clinically similar to GBS and is also associated with evidence of C. jejuni infection and GM1 antibodies.23 Spontaneous recovery is the usual outcome of GBS and may be associated with the decline in antibody levels expected after recovery from infection. Patients with mild illness require no treatment, but more severely affected patients need careful observation so that appropriate supportive therapies, such as mechanical ventilation, can be implemented when needed. Neither oral nor pulse intravenous steroids are helpful in GBS; however, large randomized controlled trials have documented that TPE can shorten recovery time and reduce disability.24-27 The North American trial enrolled 245 disabled patients, 142 of whom received TPE.24 At 4 weeks, 59% of treated patients vs 39% of controls had improved by one grade in a clinical grading scale devised for the study. Mean improvements were 1.1 and 0.4 grades, respectively. The median time to improve one grade was 19 days in treated patients vs 40 days in controls, while median

Table 42-3. Indication and Intensity of Treatment Categories for TPE in Neurologic Disorders Disorders

Antibody Specificities

ASFA Indication Category*

Intensity Category†

Guillain-Barré syndrome Chronic inflammatory demyelinating polyneuropathy Peripheral neuropathy with monoclonal gammopathy Myasthenia gravis Lambert-Eaton myasthenic syndrome Neuromyotonia and limbic encephalitis Stiff-person syndrome Paraneoplastic neurologic syndromes Rasmussen encephalitis Sydenham chorea PANDAS Multiple sclerosis

Peripheral nerve myelin Peripheral nerve myelin Myelin-associated glycoprotein AChR Voltage-gated calcium channel Voltage-gated potassium channel GAD-65 Various Glu R3 Unknown Unknown Unknown

I I I, II I II NR III III II I I III

R P P A, R, P, C R R, C R R R R R R

*I
standard first-line therapy; II
second-line therapy; III
controversial; IV
no efficacy. †

A
aggressive; R
routine; P
prolonged; C
chronic (see Table 42-2).

TPE
therapeutic plasma exchange; ASFA
American Society for Apheresis; AChR
acetylcholine receptor molecule; NR
not rated; GAD-65
65-kD isoform of glutamic acid decarboxylase; Glu R3
glutamate receptor; PANDAS
pediatric autoimmune neuropsychiatric disorders associated with streptococcal (infection).

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times to walk unassisted were 53 and 85 days, respectively. In the subgroup of ventilated patients, the median times to weaning were 24 and 48 days, respectively, and the median times to walk unassisted were 97 vs 169 days. A similar shortening of recovery time in severely affected patients was shown in the French Cooperative Group trial involving 220 patients.25 In another study of 556 patients, this group showed benefit from TPE in mildly affected patients as well.26 A typical treatment schedule in these trials consisted of five to six exchanges of 1.0 to 1.5 plasma volumes over 7 to 14 days. Patients with GBS may need careful monitoring, perhaps in an intensive care unit, because they may have autonomic neuropathy and are more prone to hemodynamic instability during TPE. Later studies have shown that IVIG is also beneficial in GBS. A large multicenter trial compared IVIG, TPE, and TPE followed by IVIG, with 121 to 130 patients in each treatment group.28 Mean disability grades at 4 weeks improved by 0.8, 0.9, and 1.1 respectively, while the median times to walk unassisted were 51, 49, and 40 days. The trends favored TPE plus IVIG, although none of the differences was statistically significant. Some authorities now favor IVIG for initial treatment, citing its relative simplicity, wider availability, and lower incidence of adverse effects.22

Chronic Inflammatory Demyelinating Polyneuropathy Chronic inflammatory demyelinating polyneuropathy (CIDP) is an acquired neuropathy that may follow either a continuously progressive or an intermittent, relapsing course. Both weakness and sensory loss are usually present, and both distal and proximal sites may be affected. Proximal weakness can help to differentiate CIDP from other chronic neuropathies, while progression for more than 2 months helps to distinguish it from GBS. Nerve conduction studies should suggest demyelination, which may be apparent in nerve biopsy tissue if this is obtained. Patchy inflammatory infiltrates may be seen in nerve root biopsies. The cerebrospinal fluid usually has a moderately elevated protein concentration and a cell count less than 10/µL.29 If certain exclusionary criteria are met, the diagnosis of CIDP is deemed “typical” or “atypical” on the basis of clinical findings and “definite,” “probable,” or “possible” based on electrophysiologic studies in one recent classification scheme.30 Treatment is recommended for all groups, and the results of treatment are similar in all.31 Although it is usually idiopathic, CIDP may also occur in the context of an associated condition, such as inflammatory bowel disease, chronic active hepatitis, connective tissue disease, Hodgkin disease, human immunodeficiency virus (HIV) infection, or monoclonal gammopathy.29 The precise cause of CIDP remains unknown; however, the disease associations, the clinical similarities to GBS, and the histopathology suggest an immune process. The presence of a monoclonal protein in some cases points to an antibodymediated disorder, as does the finding that an animal model of CIDP (experimental allergic neuritis) can be passively transferred with serum. Recent studies have demonstrated antibody to myelin protein antigens such as P0, P2, and PMP22 in the sera

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of some patients with CIDP, although a definite cause-and-effect relationship has not been established.32 Most patients with CIDP will respond to moderately high doses of glucocorticoids.29 Early trials of TPE were conducted in patients with CIDP who had failed to improve with steroids or were unable to tolerate them. A double-blind, sham-controlled crossover trial reported by Dyck et al33 showed improvement in five of 15 patients treated with TPE for 3 weeks; after crossover a similar proportion responded in the sham group. However, many patients worsened after TPE was stopped. A similar study in 18 previously untreated patients was reported by Hahn et al.34 Of the 15 patients who completed the trial, 12 improved during the TPE portion. These patients took prednisone for 6 months after completing TPE and, following recovery from a brief relapse when TPE was stopped, many maintained good function. IVIG has also been investigated in CIDP. Hahn et al35 published a double-blind, placebo-controlled crossover trial in 30 patients. Study patients were permitted to take prednisone at stable low doses. Overall 63% (19 patients) responded to IVIG, while 17% (five patients) improved on placebo. Two studies have compared IVIG to TPE. In a retrospective study by Choudhary and Hughes,36 21 of 33 patients (64%) improved after TPE treatment, while a response to IVIG was seen in 14 of 21 patients (67%) treated at the same institution. A prospective observerblinded crossover study of 20 patients at the Mayo Clinic suggested that both therapies were able to produce rapid, statistically significant improvement; the authors concluded that either was appropriate as a primary treatment.37 Some authorities recommend using IVIG first, based on its relative simplicity, wide availability, and lower incidence of adverse effects.30 The TPE protocols used in CIDP have tended to specify relatively prolonged treatment.21(p121) A proposed schedule is three oneplasma-volume exchanges each week for 2 weeks, followed by two exchanges each week for another 4 weeks.33

Peripheral Neuropathy and Monoclonal Gammopathy The background incidence of a circulating monoclonal immunoglobulin is about 1% in adults over 50 and rises to about 3% in adults over 70, whereas about 10% of patients with polyneuropathy have such a protein.38 Thus, principles of epidemiology suggest a causal relationship in at least some cases. This idea is supported by studies showing that myelin antibody activity is expressed by the monoclonal proteins of many patients with neuropathy. Specificity for a carbohydrate epitope on myelin-associated glycoprotein (MAG) can be identified in a majority of IgM-associated neuropathies. The same epitope is found on myelin glycoprotein P0 and on other gangliosides in nerve cell membranes. Injection of anti-MAG into experimental animals is known to produce demyelination. Additional patients have antibody activity against myelin sheath sulfatides, membrane-associated chondroitin sulfate C moieties, or various myelin gangliosides.20,39 Most clinical features of neuropathies associated with a monoclonal gammopathy are similar to CIDP29; however, sensory

Chapter 42: Therapeutic Plasma Exchange

abnormalities tend to be more prominent and, while progression may be slower overall, it is also more relentless, so that instances of spontaneous improvement are uncommon. The prevalence of neuropathy is higher in patients with IgM paraproteins than in those with IgG or IgA except in osteosclerotic myeloma, where the prevalence of neuropathy with IgG or IgA proteins is quite high, sometimes as a part of the POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, skin changes). Nerve biopsies show demyelination, and immunofluorescence may demonstrate IgM and complement in IgM-associated cases.38,39 Patients with multiple myeloma or Waldenström’s macroglobulinemia should be treated with appropriate chemotherapy and may experience improvement in the neuropathy as a result. However, many patients have neuropathy in addition to a monoclonal gammopathy of undetermined significance (MGUS) and are usually treated with immunosuppressive regimens similar to those used in CIDP.38 Therapeutic plasma exchange can be helpful in MGUSassociated neuropathy. In a sham-controlled trial in 39 patients, twice-weekly exchange led to improvement in disability scores, weakness scores, and electrodiagnostic parameters in the blinded portion of the trial. Scores also improved in sham-treated patients who received true TPE in an open follow-up study.40 In this and other studies, a response was noted more frequently in patients with IgG and IgA paraproteins than in those with IgM.14,21(p146)

Myasthenia Gravis Myasthenia gravis is a disease of the neuromuscular junction that is characterized clinically by fatigability, with or without weakness in skeletal muscle. Ocular myasthenia, with diplopia and ptosis, may be the initial presentation, but other muscles are eventually affected in most patients. Involvement of muscles enervated by lower cranial nerves leads to the most serious symptoms, including dysphagia and respiratory insufficiency.41 About 85% of patients with myasthenia have circulating antibody to a portion of the α-subunit of the acetylcholine (ACh) receptor molecule (AChR) on the motor end plate of muscle cells. Up to 41% of those lacking anti-AChR have circulating antibody to a muscle-specific kinase (MuSK) that is also found in the motor end plate. A few others have antibody to the muscle cytoplasmic proteins titin or RyR.41 There are two kinds of drug therapy for myasthenia. Acetylcholinesterase inhibitors, such as neostigmine and pyridostigmine, slow the degradation of ACh at the neuromuscular junction, enhancing its action on remaining receptors. Immunosuppressive drugs, such as prednisone and azathioprine, are also useful. These agents reduce damage to receptors through general antiinflammatory properties or decreased antibody synthesis or both.42 Surgical treatment is also employed. Malignant thymoma is sometimes associated with myasthenia in younger patients. Strength may improve in such patients after surgical resection of the thymus. This observation led to trials of thymectomy in younger patients without tumors, who may also experience disease remission.41,42

Because of the relationships between circulating antibody, pathology, and symptoms, TPE has seemed a very reasonable approach to treatment for myasthenia. Although a controlled trial has never been published, numerous open trials have suggested that TPE can lead to rapid symptomatic improvement in concert with lower levels of circulating AChR antibody. Therapeutic plasma exchange has also been effective in patients who test negative for antibody, suggesting that some pathogenic antibodies may not be detected by current assays.20,41 As a result of favorable experience, TPE is a widely accepted therapy for myasthenia; however, it is not recommended for all patients. It is instead reserved for those with severe disease and those who are intolerant of, or unresponsive to, other therapies. Patients whose breathing, swallowing or walking is inadequate are good candidates for the rapid improvement brought about by TPE, even as an initial treatment.42 Therapeutic plasma exchange can also be useful to optimize muscle function before thymectomy or other surgery. An occasional patient will need regular TPE at 2- to 4-week intervals in addition to maintenance drug therapy for optimal function. Myasthenia may respond to IVIG. No randomized trials have been conducted to prove efficacy; however, in a controlled study comparing IVIG to TPE in 87 patients, either three or five infusions of 0.4 g/kg IVIG were equivalent to three TPE treatments. The median time to response was shorter for TPEtreated patients (9 days vs 15 days), but not significantly so (p
0.14).43 A multicenter retrospective chart review found that ventilatory status at 2 weeks and functional status at 1 month were significantly better in patients treated with TPE.44

Lambert-Eaton Myasthenic Syndrome Lambert-Eaton myasthenic syndrome (LEMS) is also characterized by weakness and fatigue. Neither bulbar nor oculomotor symptoms are likely to be prominent in LEMS, but signs of dysautonomia, such as dry mucous membranes and orthostatic hypotension, are common. The syndrome is most often seen in patients with cancer, with almost half of cases being in patients with small cell lung cancer. Neuromuscular symptoms may precede any other sign of tumor.45 The pathophysiology of LEMS also involves the neuromuscular junction, but the defect is in the nerve cell ending instead of the muscle cell. Symptoms are caused by circulating antibody against “active zones” in the nerve terminus, which house the voltage-gated calcium channels that mediate electrical events in neuromuscular impulse transmission. These antibodies reduce the amount of ACh released during depolarization events, causing weakness in affected skeletal muscles as well as dysfunction in autonomic nerves.45 Curiously, cholinesterase inhibitors are not as effective in LEMS as they are in myasthenia gravis.46 More useful are agents that prolong nerve action potentials by blocking voltage-gated potassium channels (VGKCs). These drugs may improve muscle strength in patients with LEMS, presumably by enhancing ACh release; 3,4-diaminopyridine is the most useful agent of this type.

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Immunosuppressive drugs such as prednisone and azathioprine may also be beneficial in LEMS, and paraneoplastic cases may respond to specific antitumor therapy.45,46 Therapeutic plasma exchange has been helpful in LEMS. Responses are usually less dramatic than those seen in myasthenia gravis, perhaps suggesting that a damaged nerve ending needs more time to heal than a motor end plate. IVIG has also been reported to be active in both the short and long term.45,46

Neuromyotonia and Limbic Encephalitis Neuromyotonia is characterized by muscle cramps, involuntary contractions, fibrillations, and fasciculations. There is evidence to suggest that some cases are caused by the presence of antibodies to VGKC in nerve fiberes. VGKC have a role in terminating action potentials, such that their blockage prolongs action potentials and enhances ACh release. Therapeutic plasma exchange has been followed by clinical improvement in individual cases.47 VGKC antibodies have also been found in some patients with limbic encephalitis characterized by memory impairment, often with preservation of general intelligence. Therapeutic plasma exchange has led to improvement in individual non-paraneoplastic cases, in concert with diminution in antibody levels.48

Stiff-Person Syndrome Stiff-person syndrome (SPS), originally known as stiff-man syndrome, is characterized by progressive rigidity and/or spasms of trunk and proximal limb muscles. About 60% of patients have high titers or circulating antibody to the 65-kD isoform of glutamic acid decarboxylase (GAD-65), which is the ratelimiting enzyme in the synthesis of γ-amino butyric acid (GABA), the neurotransmitter at many inhibitory central nervous system (CNS) synapses. When present, anti-GAD-65 is believed to have a pathogenic role, even though neuronal GAD is a cytoplasmic enzyme. CNS histology is normal in SPS, suggesting that anti-GAD causes functional rather than structural changes. Pathogenesis is more uncertain in patients who lack antibody to GAD. About 5% of cases are associated with cancer, usually breast cancer. Instead of anti-GAD, most such patients have antibody to amphiphysine, a 128-kD presynaptic vesicle protein. A few have antibody to a postsynaptic protein called gephyrin that is associated with receptors for GABA and for glycine, another inhibitory neurotransmitter. Symptoms often respond to diazepam or other agents that increase CNS GABA levels. Immunomodulatory therapies such as prednisone, TPE, and IVIG have also seemed beneficial in individual cases, but no controlled trials of TPE have been performed. In paraneoplastic cases improvement may follow removal of the malignancy.21(p156),49

Paraneoplastic Neurologic Syndromes In addition to LEMS, several other neurologic syndromes associated with malignant tumors are characterized by circulating antibody to structures in the nervous system. Paraneoplastic encephalomyelitis includes seizures, mental changes, and cerebellar and autonomic dysfunction. It is often associated with

636

anti-Hu (also called ANNA-1), an antibody to a 38- to 40-kD antigen in the nuclei of neurons and of small cell lung cancer cells. Paraneoplastic cerebellar degeneration is characterized by ataxia, dysarthria, and down-beating nystagmus. It may be associated with ovarian, breast, and small cell lung cancers, as well as with Hodgkin disease. About 40% of these patients have an antibody to 34- and 62-kD antigens in Purkinje cells (anti-Yo). Paraneoplastic opsoclonus-myoclonus syndrome produces both vertical and horizontal dysrhythmic conjugated eye movements. It may occur in children with neuroblastoma and in adults with lung, breast, or other tumors. Cases seen with breast or gynecologic cancers may have circulating anti-Ri (also called ANNA2), an antibody to 55- and 80-kD antigens in neuronal nuclei. Paraneoplastic SPS is characterized by stiffness and spasm in axial muscles. It is associated with antibody to amphiphysin, a 128-kD synaptic vesicle protein. Cancer-associated retinopathy produces photosensitivity and gradual vision loss. It is associated with anti-CAR, an antibody to antigens shared by retinal neurons and small cell lung cancer cells.50 Treatment of these syndromes is uniformly difficult. They seldom respond well to immunosuppressive drugs or to antitumor measures, even when these are otherwise effective. Therapeutic plasma exchange has been tried in these syndromes, usually with disappointing results.21(p142),50

Nonneoplastic Disorders with Central Nervous System Antibodies Rasmussen’s encephalitis is a rare, acquired disorder that begins in childhood, often following a viral infection. Seizures are a prominent feature; however, unlike patients with idiopathic epilepsy, those with Rasmussen’s encephalitis develop progressive, predominantly unilateral neurologic deficits including hemiparesis and mental retardation. The histopathology includes inflammation and atrophy of brain tissue, usually confined to one hemisphere.51 Recent studies have revealed circulating IgG antibody to the Glu R3 receptor for the CNS neurotransmitter glutamate, which may arise in response to a cross-reactive microbial antigen. Treatment with either TPE or IVIG has been followed by temporary improvement.20 Sydenham chorea is a movement disorder that may follow a group A streptococcal infection in children. Patients with Sydenham chorea may also exhibit obsessive-compulsive symptoms, while children who develop obsessive-compulsive behaviors, tics, and other neurologic symptoms in the absence of chorea may also have evidence of streptococcal infection (pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection; PANDAS).52 A cross-reactive antibody response to streptococcal antigens could mediate symptoms in some of these patients, and small controlled trials have indicated that both TPE and IVIG can be beneficial in either Sydenham chorea or PANDAS.20,21(p143),53

Multiple Sclerosis Multiple sclerosis (MS) is a disease characterized by localized neurologic dysfunction that is caused by demyelinated

Chapter 42: Therapeutic Plasma Exchange

“plaques” in the CNS. Two clinical patterns are observed. About 70% of patients will have acute attacks that resolve fully or partially (relapsing-remitting). The other 30% have slow, continual progression of disease (chronic progressive). The frequency of attacks in relapsing-remitting MS tends to decrease with disease duration, and some patients evolve to a chronic progressive pattern.54 Discrete areas or plaques of demyelination in white matter, which are easily visualized with magnetic resonance imaging (MRI), are the hallmarks of MS. Histopathologically these areas are initially inflammatory but progress to fibrosis.54 The mechanism of their appearance remains unexplained, but most authorities in the field suspect involvement of the immune system.55 Experimental allergic encephalomyelitis, an animal model of MS, can be induced by immunization with myelin basic protein (MBP) or other myelin proteins. Experimental allergic encephalomyelitis is mediated by T cells and can be passively transferred with T cells from immunized animals. Most evidence suggests a misdirected cellular immune response in MS as well, and it has been difficult to assign a primary pathogenic role to circulating antibody. Antibodies to MBP or myelin oligodendrocyte glycoprotein (MOG) can be found both in CNS lesions56 and in serum57 in some MS patients. Although they are predictive of progression to MS in patients with a clinically isolated demyelinating event,58 such antibodies do not produce MS in experimental animals.59 Also, anti-MOG does not correlate with histopathologic classification or apparent responsiveness to TPE.60 The study of treatments for MS is complicated by the natural history of the disease, particularly the tendencies for acute attacks to subside and for attack frequency to decline, but also by other spontaneous fluctuations in disease activity. Clinical measurement tools such as the Expanded Disability Status Scale are used to quantify improvement or progression of disability. Although these bring some degree of objectivity to clinical studies, they are subject to interrater variability.61,62 Immunosuppressive and immunomodulatory agents have been the mainstays of drug therapy in MS. Resolution of acute attacks is thought to be hastened by brief courses of either glucocorticoids or adrenocorticotrophic hormone, both of which may promote faster restoration of normal nerve conduction by decreasing edema and inflammation in and around new plaques. The relentless progression of disability is probably not halted by these measures, although aggressive treatment of optic neuritis with intravenous steroids may delay the onset of frank MS that often follows. Cyclosporine, total lymphoid irradiation, and cytotoxic immunosuppressants such as azathioprine and cyclophosphamide have only modest benefits that may not warrant the risks such drugs entail. Mycophenolate mofetil and low dose methotrexate have also been tried. Mitoxantrone and interferon-β have shown promise in recent studies, as has glatiramer acetate, a mixture of synthetic polypeptides that may modulate immune responses to MBP. These agents reduce the frequency of acute attacks and the appearance of new lesions seen with MRI. Immunomodulatory

monoclonal antibodies, such as alemtuzumab, daclizumab, natalizumab, and rituximab are also being investigated.62-64 Prophylactic administration of IVIG to patients with MS is reported to reduce the frequency of attacks and may slow clinical deterioration in relapsing-remitting patients. It has not as yet been shown to mediate improvement in chronic visual impairment or motor symptoms.64 The rationale for TPE in MS is uncertain, given the paucity of evidence that any circulating factor has a role in the etiology of acute attacks or chronic progression. Therapeutic plasma exchange has nevertheless been used, and encouraging results have been reported from uncontrolled studies. In controlled trials, however, it has been difficult to discern benefit, even with vigorous TPE regimens.14 The first randomized, double-blind, sham-controlled study in chronic progressive MS was reported to show significant benefit for patients receiving TPE in addition to cyclophosphamide and prednisone.65 The study was subsequently questioned because of anomalies in statistical analysis, and because dramatic recoveries in several of the TPE patients, not seen in subsequent trials, suggested that some relapsingremitting patients were misclassified and entered into the trial during attacks that would have improved spontaneously.61 Two later sham-controlled trials have not shown convincing benefit.66,67 A single trial suggested that TPE could be useful in a subset of patients with MS with prolonged severe demyelination68; however, that study combined results of MS patients with those of patients having neuromyelitis optica. The Therapeutics and Technology Assessment subcommittee of the American Academy of Neurology and the MS Council for Clinical Practice Guidelines have classified TPE as “of little or no value” in progressive MS and of uncertain value in acute severe demyelination in previously non-disabled individuals.69

Therapeutic Plasma Exchange in Hematologic and Oncologic Disorders Therapeutic plasma exchange has been tried in a variety of hematologic and oncologic conditions. These are listed in Table 42-4 along with their respective indications and intensity categories.

Monoclonal Proteins In addition to peripheral neuropathy (discussed above), four other syndromes associated with monoclonal immunoglobulins are regarded as clinical indications for TPE. Hyperviscosity, coagulopathy, and renal failure, which almost always occur in the setting of a malignant B-cell disorder, are discussed below; cryoglobulinemia is covered in a later section. Hyperviscosity was the first condition to be treated successfully with manual plasmapheresis, the precursor to TPE. The fullblown syndrome consists of neurologic symptoms, a bleeding diathesis, a peculiar retinopathy marked by alternating dilated and constricted segments in retinal veins, and hypervolemia caused by expansion of plasma volume. Symptoms are uncommon if the

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Table 42-4. Indication and Intensity of Treatment Categories for TPE in Hematologic and Oncologic Disorders Disorders

Antibody Specificities

ASFA Indication Category*

Intensity Category†

Hyperviscosity syndrome Hemolytic disease of the fetus and newborn ABO-incompatible marrow transplant Platelet alloimmunization and refractoriness Thrombotic thrombocytopenic purpura Hemolytic uremic syndrome Posttransfusion purpura Idiopathic thrombocytopenic purpura Autoimmune hemolytic anemia Aplastic anemia Pure red cell aplasia Coagulation factor inhibitors Catastrophic antiphospholipid antibody syndrome

Not applicable Rh(D) or other A or B HLA antigens vWF-cleaving enzyme Unknown HPA-1a or other Unknown Unknown Unknown Unknown Factor VIII or other Phospholipids

I II II NR I III III IV (II for IA) III III III III III

P, C R A R A A A R R R R A A, R

*I
standard first-line therapy; II
second-line therapy; III
controversial; IV
no efficacy. †

A
aggressive; R
routine; P
prolonged; C
chronic (see Table 42-2).

TPE
therapeutic plasma exchange; ASFA
American Society for Apheresis; vWF
von Willebrand factor; HPA
human platelet antigen; IA
protein A-silica immunoadsorption; NR
not rated.

relative serum viscosity is below 4 and become more likely when it exceeds 6. The hyperviscosity syndrome is most often seen in patients with Waldenström’s macroglobulinemia, who have IgM paraproteins, but it may also occur in multiple myeloma.70 At higher paraprotein levels a relatively large change in viscosity may follow a relatively small change in concentration. It is this nonlinear relationship that allowed the 2-unit manual plasmapheresis technique available in the 1950s to lower viscosity enough to relieve symptoms. Because most IgM paraproteins are roughly 80% intravascular, the same relationship also predicts that a one-plasma-volume automated exchange will provide a wide margin of safety and can therefore be repeated less frequently for hyperviscosity than is necessary for many other conditions. Viscosity measurements should guide therapy, of course, but treatment every 1 to 2 weeks may be adequate.70 Paraproteins may interfere in platelet and clotting factor interactions in the absence of hyperviscosity. Such coagulopathies are found in 60% of patients with macroglobulinemia, 40% of patients with IgA myeloma, and 15% of patients with IgG myeloma.71 In instances that are clinically significant, TPE therapy can help restore adequate hemostasis. Renal failure develops in 3% to 9% of patients with myeloma and confers a poor prognosis. In many cases, renal biopsy demonstrates accumulation of free light chains in renal tubules. Urinary excretion of light chains will greatly exceed the amount that could be removed by TPE if renal function is normal. The reverse may be true in renal failure; however, light chain levels return to baseline within hours after TPE.72 Three controlled studies have examined rates of recovery of renal function in myeloma patients who received TPE therapy. In one study, three of seven dialysis-dependent patients who received TPE recovered renal function, while none

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of five control patients did.73 In another, 13 of 15 patients in the treatment group recovered renal function compared with only two of 14 controls.74 A more recent controlled study enrolled 104 patients and assessed a composite outcome comprising death, dialysis, or a low glomerular filtration rate. Outcomes were not improved in the patients randomly assigned to receive TPE treatment.75 Thus, the value of TPE in this circumstance is questionable.

Alloantibodies to Blood Cells Alloantibodies to red cells and platelets may be problematic in a number of disease processes. Treatment by antibody removal has been tried in several of these. Hemolytic disease of the fetus and newborn was one of the first problems to be approached with automated apheresis instruments. In the hope of slowing fetal hemolysis, sensitized Dnegative mothers carrying D-positive fetuses underwent TPE to lower anti-D titers. Since the original reports, the number of sensitized mothers has declined as prophylaxis with Rh Immune Globulin has become more widespread. Furthermore, intrauterine transfusion with D-negative red cells has proven to be a better treatment for affected fetuses. For these reasons, TPE is seldom called for now, but it may still be useful in an occasional pregnancy when there is very early evidence of fetal involvement, because intrauterine transfusion is not feasible before about 18 weeks of gestation.76 Prompt, early institution of TPE was followed by successful delivery after multiple prior abortions in compelling case reports involving mothers with anti-M and anti-P.18 Therapeutic plasma exchange has also been used to remove isoagglutinins in the setting of hematopoietic stem cell transplantation. Allogeneic transplantation across a major ABO barrier (eg, group A donor to group O recipient) is feasible if a

Chapter 42: Therapeutic Plasma Exchange

hemolytic transfusion reaction to red cells in the transplant can be avoided. This was first accomplished in marrow transplants by exhaustive pretransplant TPE of the recipient; however, most centers have preferred to simply remove most of the red cells (eg, 20 mL remaining) from an incompatible marrow graft before transplantation.77 Peripheral blood stem cell collections include far fewer red cells (usually 10 mL). Such grafts can be infused safely without any manipulation. Red cell engraftment may be delayed in this situation. Therapeutic plasma exchange has been used, both before transplant to avoid delayed engraftment and after transplant to correct it, with uncertain results in both cases.78 Transplantation of a solid organ, such as a liver or kidney, across a major ABO barrier can result in hyperacute rejection and is usually avoided for this reason. When organ availability is limited, however, clinical circumstances have sometimes led to transplantation of an ABO-incompatible liver or kidney after extensive TPE of the recipient, often with a satisfactory outcome.19,79 Organ transplantation across a minor ABO barrier (eg, group O donor to group A recipient) does not carry an increased risk of rejection. Some patients, however, develop hemolytic anemia caused by isoagglutinins derived from B lymphocytes in the transplant. Red cell destruction mediated by these “passenger lymphocytes” is most often seen in heart-lung or liver transplantation, in which the volume of lymphoid tissue transplanted is relatively high, and is usually evident by 1 to 3 weeks after transplantation.80 Severe hemolysis may improve after TPE and compatible (eg, group O) red cell transfusion.81 Alloimmunization to HLA or platelet antigens can cause refractoriness to platelet transfusions. To restore responsiveness, antibody removal by means of TPE or a protein A-silica column has been attempted. Intravenous immune globulin has also been tried.18 However, the results have been inconclusive and the best option for alloimmunized patients is transfusion of compatible platelets.

A convincing account of the pathogenesis of TTP has been offered. It involves a plasma enzyme (a metalloproteinase called ADAMTS13 on the basis of structural features) that cleaves ultralarge von Willebrand factor (ULvWF) multimers secreted by endothelial cells, yielding the smaller vWF polymers found in normal plasma. Children with relapsing TTP have an inherited deficiency of ADAMTS13,83 while in idiopathic adult cases an “acquired deficiency” arises because of the formation of an autoantibody inhibitor to the enzyme.84,85 In either case, persistence of ULvWF in the circulation promotes inappropriate adherence of platelets to endothelial cells and to each other, leading to consumptive thrombocytopenia and to microvascular obstructions that cause mechanical trauma to red cells and varying degrees of end-organ ischemia. Periodic plasma infusion may abort or prevent attacks in congenitally deficient patients by supplying active enzyme.83 Idiopathic cases respond better to TPE (78% response rate vs 63% for plasma infusion in the Canadian study),86 presumably because exchanges remove inhibitory antibody as well as replacing the deficient enzyme. Exchanges are usually carried out daily until the platelet count and LDH (as a marker of hemolysis) have normalized. Various immunosuppressive maneuvers, including glucocorticoid therapy, vincristine, rituximab, and splenectomy have been advocated as adjunctive treatments. Discovery of a causative autoantibody provides support for their use in idiopathic, but not in congenital, cases.18,21(p159) A syndrome resembling TTP has been identified in some patients taking the antiplatelet drugs ticlopidine87 and clopidogrel.88 Antibody to ADAMTS13 has been detected in such cases,89 and TPE has been reported to improve outcome (76% survival vs 50% for unexchanged patients in a retrospective study of ticlopidine recipients).87 ADAMTS13 deficiency is generally not found in TTP-like syndromes associated with other drugs such as quinine or cyclosporine.90

Thrombotic Thrombocytopenic Purpura and Hemolytic Uremic Syndrome

Hemolytic Uremic Syndrome The hemolytic uremic syndrome (HUS) usually involves microangiopathic hemolysis, renal insufficiency, and mild-tomoderate thrombocytopenia. It occurs in children in a selflimited form that follows infection with a verotoxin-producing Escherichia coli (diarrhea positive); however, not all pediatric cases have this association (diarrhea negative). Both a familial and a nonfamilial form may be seen in adults. Some nonfamilial cases are associated with prior chemotherapy or stem cell transplantation. Because of the striking overlap in clinical manifestations, it was long supposed that the pathogenesis of HUS was the same as, or similar to, that of TTP. Largely on this basis, TPE and protein A-column therapy have been recommended for diarrheanegative childhood HUS and for adult HUS. Responses have generally been less favorable than in TTP, especially in HUS associated with chemotherapy or hematopoietic stem cell transplantation.82,91,92

Thrombotic Thrombocytopenic Purpura Thrombotic thrombocytopenic purpura is characterized by microangiopathic hemolytic anemia and thrombocytopenia, often severe. Central nervous system changes, fever, and renal abnormalities may be seen in advanced cases, although frank renal failure is unusual. A rare relapsing form begins in childhood, but the majority of cases in adults are sporadic, with women accounting for 70%. Thrombotic thrombocytopenic purpura is most often idiopathic but may be seen in association with other illnesses, such as systemic lupus erythematosus (SLE) and HIV infection, as well as with certain drugs. Idiopathic TTP was formerly assigned a mortality rate of 90%, but empiric studies in the 1970s and 1980s demonstrated much improved survival in patients receiving daily TPE with plasma replacement. Some patients, including children with the relapsing form of the disease, will respond to simple plasma infusion.82

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Studies of ADAMTS13 in adult patients with familial and nonfamilial forms of HUS have revealed neither severe deficiency nor inhibitory activity.84 Thus, the hypothesis that HUS has the same pathogenic mechanism as TTP would seem to be in error. Deficiency of complement factor H has been identified in some patients with familial HUS.93 This might provide some rationale for plasma replacement therapy; however, for most cases of HUS the role of TPE is unclear at this time. This is also true for the role of TPE in the HELLP syndrome (hemolysis, elevated liver enzymes, and low platelet count) in pregnant women,94 which shares clinical features with TTP, HUS, and preeclampsia.

Posttransfusion Purpura Posttransfusion purpura (PTP) is a rare syndrome in which the platelet count falls to dangerously low levels about 1 week after an allogeneic transfusion. Patients who develop the syndrome lack the common allele for one of the platelet-specific glycoprotein antigens, most often the HPA-1a antigen on glycoprotein IIIa. Most patients have had multiple prior transfusions or pregnancies, and have been immunized thereby to the platelet-specific antigen coded by the prevalent allele. The transfusion that precedes the illness appears to stimulate an anamnestic increase in the titer of IgG platelet-specific alloantibody, most often anti-HPA-1a. Posttransfusion purpura is self-limited and should resolve without treatment after a few weeks; however, bleeding complications, including fatal CNS hemorrhage, may occur in the interim.95 Although it is somehow linked to a platelet-specific alloantibody response, the mechanism of extensive destruction of antigen-negative autologous platelets in PTP remains uncertain. The following four possibilities have been proposed: 1) immune complexes consisting of platelet antigen and platelet antibody bind to autologous platelets and mediate their destruction, 2) soluble platelet-specific alloantigen derived from the transfusion is adsorbed by autologous platelets, 3) a simultaneous platelet autoantibody response occurs, or 4) a broad polyclonal alloimmune response produces some antibodies that cross-react with autologous platelets. Treatment of PTP is recommended, because many patients will have bleeding complications. High-dose glucocorticoids are usually given empirically and pulse methylprednisolone at 1 g/ day has been reported effective. Platelet transfusions, even those from antigen-negative donors, seldom raise the platelet count but are likely to cause severe reactions. Daily TPE usually promotes a rise in platelet count within several days and is thought to be an effective treatment for this reason, even though no controlled trials have been conducted.96 Exchanges usually include FFP replacement to avoid a superimposed humoral coagulopathy. Intravenous immune globulin produces a similarly rapid increase in platelet count and has become the favored treatment modality for this group of patients.97

Idiopathic Thrombocytopenic Purpura Idiopathic thrombocytopenic purpura (ITP) is an autoimmune illness affecting platelets. Most patients have an autoantibody of

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the IgG class directed against a platelet-membrane glycoprotein antigen. Idiopathic thrombocytopenic purpura is sometimes accompanied by warm autoimmune hemolytic anemia (WAIHA; Evans syndrome). In pediatric patients, ITP is acute and selflimited; recovery is the rule regardless of treatment. Adults with ITP, most of whom are women, seldom recover without treatment and their disease often progresses to a chronic form. An ITP syndrome may also be seen in patients with SLE or HIV.98,99 The goal of treatment in ITP is to prevent bleeding. Fortunately most of the circulating platelets in patients with ITP are relatively young and have above-average hemostatic activity, so that a normal platelet count is not needed to avoid hemorrhage. Glucocorticoids, splenectomy, and IVIG are the mainstays of therapy,98,99 Large intravenous doses of anti-D will often raise the platelet count in Rh(D) positive patients because excessive red cell destruction occupies phagocytic cells and inhibits platelet destruction. A few favorable anecdotal reports of TPE in ITP appeared in the 1970s, and one small trial suggested a lower rate of splenectomy in exchanged patients but a later study found no long-term benefit associated with such therapy.99 Given this experience, enthusiasm for this approach has waned.21(p134) Favorable responses to protein A-silica column treatment have been reported in ITP associated with HIV infection,100 as well as in patients with chronic ITP without HIV.101 The mechanism of action of protein A in this effect is unknown. A purely subtractive mechanism (ie, removal of platelet antibody) seems unlikely, because responses have been reported when as little as 250 mL plasma per week was treated. Bearing this uncertainty in mind, the protein A-silica column remains an option for chronic ITP refractory to more standard therapies,21(p134) and is discussed in greater detail in Chapter 41.

Autoimmune Hemolytic Anemia Autoimmune hemolytic anemia (AIHA) is caused by autoantibodies to red cells. Such antibodies are classified as either “cold” or “warm” agglutinins, depending on the temperature of maximal activity. Cold agglutinins are usually IgM antibodies directed against the I/i antigens; they bind most strongly at low temperatures and may produce a syndrome of complement-mediated intravascular hemolysis (cold agglutinin disease; CAD). Warm agglutinins are usually IgG and are often directed against an antigen that does not appear on Rhnull cells; they bind better at body temperature and produce a predominantly extravascular hemolytic syndrome, WAIHA. Autoimmune hemolytic anemia can be idiopathic but can also be associated with infections, lymphoproliferative disorders, or other autoimmune diseases.102 Most patients need treatment. Standard therapy is aimed at lowering antibody production and inhibiting destruction of sensitized cells. Glucocorticoids, IVIG, and splenectomy are often effective in WAIHA, and other immunosuppressive drugs may be tried if these measures fail. All of these approaches are less successful in CAD.102 Therapeutic plasma exchange to deplete circulating antibody has been tried in both WAIHA and CAD when conventional

Chapter 42: Therapeutic Plasma Exchange

treatments have failed. Because the IgM antibodies in CAD are predominantly intravascular and only loosely bound to cells, their removal by TPE should be relatively efficient. Such therapy, when added to conventional drug treatment, has been reported to lower antibody titers and transfusion requirements, albeit only temporarily. In WAIHA much of the circulating antibody is bound to red cells; TPE has been tried in this disorder also, but it is less likely to be helpful.21(p118) A nonrandomized study compared responses to red cell transfusions in five AIHA patients who underwent TPE before some transfusions and four other patients who did not. Responses were significantly better in the patients who did not receive TPE and were not improved by TPE in the patients who did, suggesting that TPE is not effective for this purpose.103

Pure Red Cell Aplasia and Aplastic Anemia Pure Red Cell Aplasia and Aplastic Anemia are marrow disorders. In the former there is reticulocytopenic anemia, while the latter leads to pancytopenia. At least some cases of both conditions likely have an immunologic basis. Allogeneic marrow transplantation is the preferred treatment for severe aplastic anemia if a suitable donor is available, but immunosuppressive therapies, such as glucocorticoids, cytotoxic drugs, cyclosporine, and antithymocyte globulin may be effective, especially in milder cases. In the serum of a minority of patients it is possible to demonstrate a factor, probably antibody, that inhibits the growth of marrow-derived precursor cells in culture.104 This provides a rationale for TPE, which has been reported in both disorders in a few cases. Results in aplastic anemia have been mixed; responses appear to be more likely in patients with serum inhibitory activity. All reported instances of TPE treatment for pure red cell aplasia have led to improvement, which is sometimes quite dramatic in patients with serum inhibitory activity. Thus, although TPE is not a primary therapy for either disorder, it can be offered to patients who have failed to improve after receiving conventional treatment, especially those found to have serum inhibitory factors.21(p117)

Coagulation Factor Inhibitors Coagulation factor inhibitors are IgG antibodies to components of the clotting cascade. They interfere with clotting by inactivating the targeted factor. Inhibitors may be autoantibodies that arise in individuals with no prior bleeding. Alternatively they may be alloantibodies that form in genetically deficient patients after exposure to “foreign protein” in the course of factor replacement therapy. Factor VIII is the clotting protein most often affected by antibodies of either type, and most of the following concerns Factor VIII inhibitors. The two goals of treatment for a patient with an inhibitor are control of individual bleeding episodes and suppression of inhibitor synthesis. Depending on inhibitor titer, the first goal can sometimes be achieved by infusion of high doses of human Factor VIII. Porcine Factor VIII, which cross-reacts

only partially with human Factor VIII antibodies, can be effective in the face of somewhat higher inhibitor titers, but its availability in recent years has been uncertain. For patients with the highest titers, Factor VIII-bypassing products such as recombinant Factor VIIa are needed. Therapeutic plasma exchange or selective immunoadsorption of IgG during a bleeding episode may reduce inhibitor titers enough to allow infused Factor VIII to bring about hemostasis. Suppression of inhibitor synthesis is approached with immunosuppressive measures, including high-dose glucocorticoids, cytotoxic agents, cyclosporine, IVIG, and rituximab.15,105,106 Tolerance-inducing protocols that include regular infusion of exogenous Factor VIII have been devised for patients with alloimmune inhibitors. In the so-called Malmö protocol, extensive TPE or IgG depletion with protein A-sepharose column procedures is used to reduce the inhibitor level at the onset of treatment so that infused “tolerizing” factor can circulate.107 Frequent, large (two to three plasma volume) exchanges with FFP replacement are recommended for patients with inhibitor. Central venous access is often required; placement of a catheter in a patient with a refractory bleeding diathesis is a challenge for all concerned and often mandates infusion of a Factor VIII-bypassing product for wound hemostasis.108 Therapeutic plasma exchange has also been reported for treatment of patients with antiphospholipid antibodies, which may interfere with in-vitro assays of coagulation, such as the partial thromboplastin time. In contrast to the inhibitory antibodies described above, however, they usually promote inappropriate coagulation in vivo and cause thrombotic events. Patients suffering thrombotic events in three or more organ systems are said to have catastrophic antiphospholipid antibody syndrome.109

Therapeutic Plasma Exchange in Rheumatic and Other Immunologic Disorders Therapeutic plasma exchange has been tried in a number of rheumatic diseases and other diseases that are considered to have an immune or autoimmune etiology. These are listed in Table 42-5 along with the author’s intensity guidelines and the indication categories assigned by ASFA.

Cryoglobulinemia Cryoglobulins are abnormal serum proteins that precipitate reversibly at 4ºC; some will precipitate at higher temperatures. Such precipitates always contain immunoglobulin, and immunoelectrophoretic or immunofixation analysis allows distinction of three types. Type I cryoglobulins consist of a single species of monoclonal immunoglobulin. These are usually found in B-cell lymphoproliferative disorders such as myeloma or Waldenström’s macroglobulinemia. Cryoglobulin levels are often quite high (500 mg/dL) and may cause Raynaud phenomenon or acral necrosis due to microvascular obstruction, as well as other symptoms. Type II cryoglobulins contain both monoclonal and polyclonal immunoglobulins. The former is usually an IgMκ

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Table 42-5. Indication and Intensity of Treatment Categories for TPE in Rheumatic and Other Immunologic Disorders Disorders

Antibody Specificities

ASFA Indication Category*

Intensity Category†

Cryoglobulinemia Rheumatoid arthritis Systemic lupus erythematosus Systemic vasculitis Polymyositis and dermatomyositis Goodpasture syndrome Rapidly progressive glomerulonephritis Renal transplantation Humoral rejection Presensitization Recurrent focal glomerulosclerosis Heart transplant rejection

IgG IgG dsDNA and others ANCAs Unknown Type IV collagen ANCAs

II NR (II for IA) III (IV for nephritis) NR IV I II

A, R, P, C R? R, P R? R? A R

HLA antigen HLA antigen Unknown HLA antigen

II II III III

A, R R R R

*I
standard first-line therapy; II
second-line therapy; III
controversial; IV
no efficacy; IA
protein A-silica immunoadsorption, NR
nor ranked. †

A
aggressive; R
routine; P
prolonged; C
chronic (see Table 42-2).

TPE
therapeutic plasma exchange; ASFA
American Society for Apheresis; IgG
immunoglobulin G; dsDNA
double-stranded DNA; ANCAs
antineutrophil cytoplasmic antibodies; NR
not rated.

with anti-IgG specificity, while the latter is polyclonal IgG bound to the IgMκ in an immune complex. Most cases occur in the context of chronic hepatitis C infection. They typically manifest a cutaneous vasculitis on the lower extremities and may have visceral manifestations of immune complex disease as well.110 Type III cryoglobulins are mixed polyclonal, often with IgM anti-IgG that binds IgG in immune complexes. These may arise in acute infections, such as hepatitis B, or in chronic inflammatory states such as severe rheumatoid arthritis. Clinical manifestations resemble serum sickness. If there is an underlying condition, cryoglobulin levels and related symptoms may decrease with treatment of this primary disorder, for example, chemotherapy for myeloma or interferon for hepatitis C virus infection. For idiopathic and secondary cases of mixed cryoglobulinemia, prednisone therapy often relieves symptoms, while alkylating agents may be useful in patients with severe symptoms resistant to prednisone. Therapeutic plasma exchange will reduce cryoglobulin levels and control symptoms, even in the absence of other treatments,111,112 but inconvenience and expense mitigate against such use. It should be started promptly for patients who seek treatment for severe acral ischemia or visceral manifestations of vasculitis, in whom it can help achieve control of symptoms until aggressive drug therapy takes hold.113 Patients with chronic vasculitic skin ulcers may also benefit.114 In all cases replacement fluids should be warmed to body temperature before infusion.

Rheumatoid Arthritis Rheumatoid arthritis (RA) is a disease of unknown cause that is more prevalent in women. It is the most common chronic inflammatory joint disease and a leading cause of disability. Most patients have rheumatoid factor, an IgM autoantibody to IgG; however, because this antibody is absent in many clinically typical

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cases, and because it is also found in patients who do not have arthritis, it is not likely to be directly involved in pathogenesis. Conservative treatment includes nonsteroidal antiinflammatory agents, oral glucocorticoids in low doses, and intraarticular steroids. More severely affected patients eventually receive slowacting “disease-modifying antirheumatic drugs” that are probably immunomodulatory, such as antimalarials, gold compounds, and methotrexate. Tumor necrosis factor and IL-1 inhibitors are also approved for treatment of RA. Therapeutic plasma exchange was tried for RA in the 1970s and 1980s, but controlled trials did not show benefit. There were subsequent reports of lymphapheresis, with or without accompanying TPE. Some controlled trials investigating this approach showed significant but short-lived benefit, while others did not. In practice, the prospect of only a modest chance of modest benefit from a costly, inconvenient therapy has discouraged treatment with therapeutic apheresis.17 A sham-controlled trial of 12 weekly protein A-silica column treatments produced improvement in 33% of 48 treated patients vs only 9% of 43 controls. Benefit persisted for about 8 months on average, and a subsequent course was again beneficial in seven of nine initial responders.115 Although its mechanism of action is unclear, this device has gained Food and Drug Administration approval for use in RA.

Systemic Lupus Erythematosus Systemic lupus erythematosus has long been regarded as the prototypic autoimmune disease. The most important diagnostic criterion, circulating antibodies to DNA, especially double-stranded DNA (anti-dsDNA), identifies patients who may have a variety of other autoantibodies and a disparate array of clinical syndromes in which skin disease, joint disease, cytopenias, or nephritis may be the sole or dominant problem.

Chapter 42: Therapeutic Plasma Exchange

Immunosuppressive measures are the cornerstone of therapy for SLE. Most patients are given prednisone in varying doses, and those with severe disease may also receive azathioprine or cyclophosphamide. The plethora of autoantibodies that seem relevant to clinical signs made SLE an obvious target for TPE. It was one of the first illnesses to be treated with automated TPE in the early 1970s, and early case reports and uncontrolled series suggested a favorable effect.17 Lupus nephritis is a particularly devastating manifestation in which glomerular deposition of immune complexes and antidsDNA is believed to have a prominent role in pathogenesis. Thus it seemed an attractive setting for randomized trials of TPE. A controlled trial with only eight patients suggested benefit.116 However, in a multicenter randomized controlled trial comparing oral cyclophosphamide plus TPE to oral cyclophosphamide alone, there was no advantage for the patients receiving TPE.117 A later international trial, which enrolled patients with a variety of severe manifestations, was structured to exploit enhanced sensitivity to a properly timed pulse dose of intravenous cyclophosphamide that was believed to follow pathogenic antibody removal by TPE.118 As mentioned above, subsequent work with knock-out mice deficient for the FcRn receptor suggests that enhanced susceptibility would not occur.4 In any case, this trial also failed to show any advantage for all patients treated with TPE119 or for a subgroup with nephritis.120 Thus large controlled studies have failed to confirm any worthwhile effect of TPE in SLE.

Systemic Vasculitis The term systemic vasculitis encompasses a group of disorders that cause inflammation in blood vessel walls and ischemic tissue damage. Vasculitis syndromes are conveniently classified on the basis of the size of vessels typically involved, but most are of unknown etiology. Immune complexes are found in patients with some syndromes, and autoantibodies such as antineutrophil cytoplasmic antibodies (ANCA) in Wegener’s granulomatosis (c-ANCA) and polyarteritis (p-ANCA), can be demonstrated in others. This has lent credence to the notion that humoral immune factors are somehow involved.121 Prednisone is the first-line therapy for most vasculitic syndromes and cyclophosphamide is often added in more severe cases. Randomized controlled trials in renal vasculitis,122 as well as in a group of patients with polyarteritis or Churg-Strauss angiitis,123 have shown little evidence that addition of TPE to drug therapy confers long-term benefit. Nevertheless, it may be requested for patients who are not responding to maximal drug therapy.

Polymyositis and Dermatomyositis Polymyositis and dermatomyositis are inflammatory diseases affecting skeletal muscle. A characteristic dermatitis involving the eyelids, knuckles, neck, and shoulders is also part of the latter condition. The usual clinical picture includes proximal weakness with biochemical evidence of muscle cell enzyme leakage; the diagnosis is confirmed by muscle biopsy. The natural

history is progressive fiber loss, eventually leading to profound, irreversible weakness. An autoimmune etiology is suspected, but circulating antibody specific for skeletal muscle has not been implicated. Initial treatment is high-dose prednisone, which can often be tapered to maintenance levels. Resistant disease is treated with azathioprine, methotrexate, an alkylating agent, or a combination. Controlled trials have also shown that IVIG infusion reduces muscle enzyme levels and improves strength temporarily.20 Several uncontrolled series were interpreted to show that TPE was beneficial, but they were unfortunately confounded by concurrent escalations in immunosuppressive drug therapy.20 A randomized controlled trial in which 12 patients received TPE, 12 received lymphapheresis, and 12 received sham apheresis, with no changes in drug therapy, showed no difference in the response rate among the three groups.124 Thus despite the successes with IVIG, there appears to be no role for TPE in the treatment of polymyositis.

Goodpasture Syndrome Goodpasture syndrome (GPS) is characterized clinically by pulmonary hemorrhage and rapidly progressive glomerulonephritis. Light microscopy of renal biopsies shows crescent formation in many glomeruli, while immunofluorescent and electron microscopy reveal linear subendothelial immune deposits that may also be evident in a lung biopsy. In 95% of cases there is a circulating antibody that binds to glomerular basement membrane (anti-GBM). Such antibodies are specific for a noncollagenous sequence near the carboxy terminus of the α3 chain of type IV collagen, which is found in appreciable quantities only in renal and pulmonary basement membranes. Untreated GPS progresses quickly and relentlessly, and most patients die of uremia or complications of lung hemorrhage.125 The preferred treatment for GPS is high-dose prednisone and cyclophosphamide, combined with aggressive TPE, to quickly reduce anti-GBM levels and minimize progression of tissue damage.19 Exchanges are usually carried out daily and may be continued for up to 2 weeks. It is prudent to give some FFP replacement in the latter part of each exchange to avoid a dilutional coagulopathy that might cause exacerbation of lung bleeding. A single controlled trial failed to show an advantage for GPS patients who received TPE; however, this study has been largely discounted because the TPE schedule (every 3 days) was not sufficiently aggressive and because the extent of renal damage at entry was worse in the TPE group than in controls.126 Early treatment is recommended because patients who are already dialysis-dependent at the onset of TPE are unlikely to recover renal function.127 It follows that the subset of patients whose renal biopsies show irreversible lesions are not likely to benefit from TPE unless they also have pulmonary hemorrhage.

Other Rapidly Progressive Glomerulonephritis In addition to GPS, there are two other categories of rapidly progressive glomerulonephritis (RPGN)—those with an immune

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complex disease, who have granular subendothelial immune deposits, and those with pauci-immune RPGN, who have scant immune deposits, if any. Light microscopic findings in both are similar to those found in GPS, with severe glomerular inflammation and crescent formation. Some cases also have associated lung hemorrhage. Patients in either group may have isolated renal disease or may have accompanying features that suggest a diagnosis of systemic vasculitis, mixed cryoglobulinemia, or Henoch-Schönlein purpura for granular-immune complex RPGN, or microscopic polyangiitis or Wegener’s granulomatosis for pauci-immune RPGN patients, most of whom test positive for ANCA.128 Therapies are similar for these two categories of RPGN, and some trials and series have included patients with both types. Virtually all patients receive prednisone and most receive either oral or intravenous cyclophosphamide. Therapeutic plasma exchange has been used extensively in patients with both types of disease. Two controlled trials, one published in 1988129 and the other in 1992,130 showed no advantage for patients who received TPE in addition to immunosuppressive drugs. However, a subgroup analysis in the second study suggested that patients who have dialysis-dependent renal failure are more likely to recover renal function if they receive TPE. No such trend was noticed in the more recent trial conducted by Guillevin and colleagues.123 A prospective, randomized trial by Stegmayr et al131 compared TPE with immunoadsorption. Among 38 patients with non-GPS RPGN, 87% of whom had ANCA, 70% avoided long-term dialysis. Therapeutic plasma exchange and immunoadsorption were equally effective. In another study of ANCA-positive patients with an elevated creatinine, 54% of dialysis dependent patients randomly assigned to receive TPE became dialysis independent compared to only 32% of those who received pulse methylprednisolone instead of TPE.132 Thus, while the role of TPE is not clearly defined in all types of RPGN, current evidence suggests efficacy in ANCA-positive patients who are dialysis dependent.

Solid Organ Transplantation In organ transplant recipients TPE has been used both to treat and to prevent rejection, as well as for recurrence of certain diseases in a transplanted organ. Photopheresis, which has also been tried for the same purposes, primarily in heart transplantation, is covered in Chapter 43.

Rejection Cellular immune mechanisms mediate most organ allograft rejection episodes; however, antibody-mediated rejection may occur rapidly in patients who have preexisting antibodies to ABO or HLA antigens expressed by the graft. Such “hyperacute” rejection is characterized histologically by neutrophil infiltration, fibrin deposition, and endothelial damage in small blood vessels; failure of the graft is mainly caused by ischemic damage. All treatments have been futile in hyperacute rejection, including TPE. So-called “vascular changes,” which may be seen microscopically in later rejection episodes, were formerly taken to indicate

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antibody-mediated rejection, even when immunofluorescence microscopy and tests for circulating antibody were negative.133 Standard posttransplant management for kidney and heart transplantation consists of prophylactic immunosuppression with glucocorticoids, a calcineurin inhibitor (cyclosporine or tacrolimus) and an antimetabolite such as azathioprine or mycophenolate mofetil. High-risk recipients and those who have rejection episodes in spite of these standard measures are treated with pulse steroids or T-cell antibody preparations or both.134,135 Case reports and uncontrolled series published in the late 1970s and early 1980s suggested TPE was beneficial in renal transplant rejection. Then five controlled trials19 were reported in the mid- and late 1980s. Four showed no significant benefit for patients receiving TPE in addition to standard drug therapy, even in the subgroups whose transplant biopsies showed “vascular” histologic changes. In the one study suggesting benefit, the mean treatment time was 10 to 11 months after transplant, when antibody-mediated rejection is less likely. The last and largest study concluded that TPE therapy for renal transplant rejection could no longer be recommended.136 Nevertheless, use of TPE for this purpose continued to be reported.19 In the more recent past, a new wave of enthusiasm for TPE has focused on patients with clinical and histologic evidence of acute rejection who also have circulating donor-specific antibody and/or deposition of complement component C4d in transplant biopsy tissue, suggesting a humoral mechanism. The antibody can be shown by a positive crossmatch with donor cells or by flow cytometric reactions with donor antigens. C4d deposition is demonstrated by immunofluorescence microscopy.133,137 Uncontrolled studies have been encouraging. Observations in the pretransplant period have shown that antibody titers can be reduced by TPE.138 Furthermore, in open trials, patients with refractory acute rejection who demonstrate donor-specific antibody and/or C4d have relatively good long-term graft survival ( 80%) after treatment regimens that include TPE and IVIG.139,140 A recent conference on antibody-mediated rejection recommended that demonstration of circulating antibody be considered an essential criterion for diagnosis of humoral rejection and that the effectiveness of TPE and IVIG in this entity be studied in “rigorous prospective, multicenter” trials.141 TPE has also been employed in cases of cardiac allograft rejection. Favorable outcomes have been reported in individual patients who were also receiving other therapies, but no controlled trials have been conducted. The criteria for a diagnosis of humoral rejection in this setting continue to evolve.137 “Vascular” histology is more difficult to detect or exclude in the endomyocardial biopsies performed to monitor cardiac allografts because few blood vessels are found in this part of the heart muscle. Immunofluorescence microscopy for IgG had been considered a reasonable criterion for humoral rejection; however, it has been suggested more recently that this diagnosis be made, and TPE employed, in patients with deteriorating cardiac function whose biopsies lack cellular infiltrates.142 In some such biopsies it has been possible to demonstrate capillary endothelial swelling, macrophage influx, and C4d

Chapter 42: Therapeutic Plasma Exchange

deposition.143 Controlled data to support the TPE recommendation are lacking, as are any published data correlating this clinical syndrome with circulating donor-specific antibody. Pretransplant TPE has been tried in organ transplant candidates who are presensitized to HLA antigens. Those whose sera react with lymphocytes from a large fraction of the population are less likely to have a compatible crossmatch with a cadaveric donor and hence have a lower likelihood of receiving a transplant. Prospective immunosuppression, combined with antibody removal by TPE or protein A-sepharose immunoadsorption, has been explored as a means to achieve a compatible crossmatch before transplantation and thereby prevent hyperacute rejection. Several centers have reported groups of patients who received kidney transplants after being prepared in this way and achieved quite respectable graft survival rates. Similar protocols have facilitated successful transplantation of ABO-incompatible kidneys and livers.19 In more recent studies, pretransplant TPE has facilitated elective living donor transplantation of HLA-crossmatch positive as well as ABO-incompatible organs.79,138,144 In summary, evidence from controlled trials has not shown global efficacy for TPE in reversing established rejection episodes in renal allografts. Anecdotal experience focusing on patients with circulating antibody and immunopathologic evidence of humoral rejection seems more promising, but controlled trials in such patients have yet to be published. Pretransplant antibody removal can make transplantation feasible for otherwise ineligible candidates. It can also facilitate transplantation of kidneys from living donors across ABO and donor-specific crossmatch barriers. Clarification of the role of TPE in cardiac transplantation awaits controlled studies targeting patients with documented humoral rejection and circulating donor-specific antibody.

Recurrence of Disease Focal glomerulosclerosis (FGS) is a disease that causes nephrosis and renal failure, predominantly in children. It recurs in about 30% of allograft recipients, which suggests that a humoral factor may have a role in its pathogenesis. A 50-kD plasma factor that binds to protein A has been implicated but has not been further characterized.145 Reduced proteinuria and improved renal function have been reported when recurrence in an allograft is treated with stepped-up immunosuppression and TPE.146,147 Goodpasture syndrome may occasionally recur in a transplanted kidney; however, this can usually be avoided by delaying transplantation until the anti-GBM response has subsided spontaneously. If the syndrome recurs in spite of this precaution, it should be treated promptly with TPE and cyclophosphamide.147 Allograft vasculopathy, a diffuse coronary artery disease that sometimes develops in transplanted hearts, is the leading cause of morbidity and mortality in heart transplant recipients who survive beyond 1 year. It may be related either to continuing hyperlipidemia or to chronic rejection. Selective depletion of LDLs, which is discussed in greater detail in the section on hypercholesterolemia, has been reported helpful in a few such patients with persistent lipoprotein abnormalities.148

Therapeutic Plasma Exchange in Toxic and Metabolic Disorders This section covers conditions in which removal of plasma constituents other than immunoglobulin is potentially beneficial. These are listed in Table 42-6 along with indication and intensity categories.

Hypercholesterolemia Familial hypercholesterolemia (FH) is a genetically determined deficiency of cell surface LDL receptors that interferes with cholesterol off-loading from LDL into cells and with the normal negative-feedback regulation of LDL synthesis, leading to highly elevated levels of circulating LDL, cholesterol (650 to 1000 mg/ dL), and lipoprotein(a) [Lp(a)]. Skin xanthomas and coronary atheromas develop in the first decade of life in homozygotes, and death from myocardial infarction before age 20 is common. Heterozygotes also have elevated LDL, cholesterol (350 to 500 mg/dL), and Lp(a) levels; they may develop xanthomas by age 20 and coronary atherosclerosis by age 30.149,150 Milder forms of hypercholesterolemia can be influenced by dietary modifications and are amenable to drug treatment with agents such as HMG-CoA reductase inhibitors, bile acid-binding resins, nicotinic acid, and ezetimibe, a new agent that inhibits gut absorption of both dietary and biliary cholesterol.150,151 However, FH homozygotes and some FH heterozygotes respond only modestly to these measures and remain at risk for premature death. Drastic surgical measures, such as ileal bypass, portacaval shunt, and liver transplantation may be recommended for such patients if they have evidence of coronary artery disease.149,150 Alternatively, removal of LDL and its associated cholesterol from the blood can be accomplished repeatedly by various modalities of therapeutic apheresis.150 A standard TPE will lower LDL and cholesterol levels by 50% or more, and long-term treatment every 1 to 2 weeks can lead to shrinkage of cutaneous xanthomas and regression of coronary artery deposits. Although TPE removes both LDL and Lp(a), it also depletes high-density lipoproteins (HDL), which

Table 42-6. Indication and Intensity of Treatment Categories for TPE in Metabolic Disorders Disorders

ASFA Indication Category* Intensity Category†

Homozygous familial hypercholesterolemia Refsum’s disease Overdose or poisoning Acute hepatic failure

II (I for LDL-P) II III III

C A, R, P A A

*I
standard first-line therapy; II
second-line therapy; III
controversial; IV
no efficacy; LDL-P
LDL-apheresis, ie, selective depletion of LDL. †

A
aggressive; R
routine; P
prolonged; C
chronic (see Table 42-2).

TPE
therapeutic plasma exchange; ASFA
American Society for Apheresis.

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are believed to have an antiatherogenic action. This disadvantage has stimulated efforts to deplete LDL semiselectively and on-line from patient plasma separated by an apheresis device, and then return the LDL-depleted plasma to the patient.150 Several systems designed to accomplish this goal are described in Chapter 43.

Refsum’s Disease Refsum’s disease results from deficiency of the peroxisomal enzyme phytanoyl-CoA hydroxylase, which participates in degradation of phytanic acid by α-oxidation. Accumulation of dietderived phytanic acid in plasma lipoproteins and in tissue lipid stores leads to symptoms, which may include peripheral neuropathy, cerebellar ataxia, retinitis pigmentosa, anosmia, deafness, ichthyosis, renal failure, and arrhythmias. Slow progression is the usual course, but rapid deterioration and even sudden death may follow a marked increase in plasma phytanic acid.152 Restriction of dietary intake of phytanic acid via dairy products, meats, and ruminant fats is the mainstay of treatment. It leads to gradual clearing of phytanate stores by slow ω- oxidation and gradual symptomatic improvement in most patients. Nutrition must be maintained, however, because mobilization of calories from endogenous fat can increase plasma phytanic acid levels acutely and cause clinical exacerbations. Therapeutic plasma exchange will remove large quantities of phytanic acid incorporated into plasma lipids.153 Selective lipoprotein depletion is also effective.154 Apheresis therapy is most appropriate for patients who have very high plasma phytanate levels and an associated exacerbation of symptoms. Skin disease, neuropathic symptoms, and ataxia usually improve as plasma levels drop. Cranial nerve defects usually do not.152

Drug Overdose and Poisoning Toxic effects may occur after exposure to excessive doses of pharmacologic agents or to harmful agents in the environment. Management techniques for both types of event are similar and may include removal of toxin still in the gastrointestinal tract, enhancement of renal elimination, and direct removal from blood by hemodialysis, hemoperfusion (eg, over charcoal columns), or TPE.155 Specific antidotes may also be given if available. Serious events are usually treated with multiple measures. Therapeutic plasma exchange has been reported to be beneficial, when combined with other therapies, in cases involving substances, such as methyl parathion, vincristine, and cisplatin, that bind tightly to plasma proteins. It has also been reported for severe hyperthyroidism, either endogenous or exogenous, in which its effectiveness may be limited by extensive binding of L-thyroxine to tissue proteins. Therapeutic plasma exchange has been reported in poisonings due to ingestion of the Amanita phalloides mushroom; however, diuresis clears more Amanita toxin.19 Unfortunately, the literature on this topic is older and entirely anecdotal. Furthermore, TPE has always been used in combination with other therapies that are presumably effective. This complicates formulation of firm, rational guidelines. Nevertheless it

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seems reasonable to offer TPE to a severely affected patient with an overdose or poisoning who has a high blood level of a toxic agent that binds to plasma proteins. It should also be noted that TPE has shown minimal or no beneficial effect in overdosage of drugs known to bind to tissue proteins and lipids, including barbiturates, chlordecone, aluminum, tricyclic antidepressants, benzodiazepines, quinine, phenytoin, digoxin, digitoxin, prednisone, prednisolone, tobramycin, and propranolol.19

Acute Hepatic Failure Acute hepatic failure may develop after a severe liver insult, such as overwhelming hepatitis B infection or acetaminophen overdosage. Cases may also be caused by drug reactions, Wilson’s disease, vascular anomalies, acute fatty liver of pregnancy, and a variety of toxins. Acute hepatic failure results in many metabolic imbalances and synthetic defects. Clinical symptoms include jaundice, coagulopathy, encephalopathy, and renal failure. The treatment of choice is liver transplantation, which leads to 60% to 80% long-term survival vs 60% mortality for untransplanted patients. Cerebral edema accounts for fatal outcomes.156 Conservative treatment is basically supportive. Fluid, electrolyte, and nutritional supplements are adjusted in response to metabolic abnormalities. Bowel sterilization with enteral antibiotics minimizes production of ammonia by intestinal bacteria. Pressors are infused if needed for hemodynamic support. Osmotic diuretics, sedatives, hyperventilation, and proper positioning are all employed to reduce intracranial pressure. Platelets and plasma products are infused to improve coagulation.156 Therapeutic plasma exchange with plasma replacement has seemed appealing as a means to restore metabolic homeostasis, remove toxic metabolites that may cause cerebral edema, and supply coagulation factors and other deficient plasma proteins in quantity without causing volume overload. Practical evaluations of this approach have produced mixed results.156 Some investigators have found TPE helpful in stabilizing and maintaining patients until an organ for transplant becomes available. Improvements in blood pressure, cerebral blood flow, and neurologic status were attributed to TPE in one study157; however, intracranial pressure, a key prognostic indicator, did not decrease. Hemoperfusion over activated charcoal, which will lower plasma ammonia levels, has also shown no advantage over intensive supportive care alone. Potential problems with extensive TPE arise from the diminished ability of patients with acute hepatic failure to metabolize the citrate in infused plasma. Accumulation of citrate leads to ionized hypocalcemia and to alterations in arterial ketone body ratios that may interfere with regeneration of hepatocytes.158 Thus, although TPE can partially reverse coagulopathy and other synthetic deficits in these patients, a favorable net impact on outcome has been difficult to demonstrate. Other methods for extracorporeal support of patients with liver failure have been investigated. Several devices place patient plasma in “metabolic contact” with hepatocyte suspensions of either human or porcine origin. One contains a suspension of

Chapter 42: Therapeutic Plasma Exchange

pig liver cells in the exterior space surrounding the hollow fibers in a filter cartridge. Patient plasma separated by an apheresis device and passed through a charcoal column to remove ammonia, flows through the hollow fibers before being returned to the patient. It is proposed that such devices could provide both detoxification and synthetic functions; however, evidence for the latter has been disappointing. Other devices aim to remove toxins via binding to various extracorporeal sorbents, such as activated charcoal or albumin molecules. None of these approaches has yet been shown to improve outcomes for acute liver failure patients in a large controlled trial, but research is ongoing.159

Conclusion Therapeutic plasma exchange is an effective therapy for a number of diseases, especially those mediated by paraproteins or autoreactive antibodies. Improvements in apheresis instruments that are outside the scope of this chapter have made it a very safe treatment as well. Thus, it should continue to have an important role in the management of selected diseases.

Disclaimer The author has disclosed no conflicts of interest.

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111. Berkman EM, Orlin JB. Use of plasmapheresis and partial plasma exchange in the management of patients with cryoglobulinemia. Transfusion 1980;20:171-8. 112. McLeod B, Sassetti R. Plasmapheresis with return of cryoglobulin depleted autologous plasma (cryoglobulinpheresis) in cryoglobulinemia. Blood 1980;55:866-70. 113. Bombardieri S, Maggiore Q, L’Abbate A, et al. Plasma exchange in essential mixed cryoglobulinemia. Plasma Ther Transfus Technol 1981;2:101-9. 114. McGovern TW, Enzenauer RJ, Fitzpatrick JE. Treatment of recalcitrant leg ulcers in cryoglobulinemia types I and II with plasmapheresis. Arch Dermatol 1996;132:498-500. 115. Felson DT, La Valley MP, Baldassare AR, et al. The Prosorba column for the treatment of refractory rheumatoid arthritis: a randomized, double-blind, sham controlled trial. Arthritis Rheum 1999;42:2153-9. 116. Huston DP, White MJ, Maltiolo C, et al. A controlled trial of plasmapheresis and cyclophosphamide therapy of lupus nephritis (abstract). Arthritis Rheum 1983;26(Suppl):S33. 117. Lewis EJ, Hunsicker LG, Lan SP, et al. A controlled trial of plasmapheresis therapy in severe lupus nephritis. N Engl J Med 1992;326:1371-9. 118. Euler HH, Schwab UM, Schroeder JO, et al. The lupus plasmapheresis study group: Rationale and updated interim report. Artif Organs 1996;20:356-9. 119. Schroeder JO, Schwab U, Zennet R, et al. Plasmapheresis and subsequent pulse cyclophosphamide in severe systemic lupus erythematosus. Preliminary results of the LPSG Trial (abstract). Arthritis Rheum 1997;40:S325. 120. Wallace DJ, Goldfinger D, Pepkowitz S, et al. Randomized control of pulse/synchronization cyclophosphamide/apheresis for proliferative lupus nephritis. J Clin Apher 1998;13:163-6. 121. Langford CA. Vasculitis. J Allergy Clin Immunol 2003;111:S602-12. 122. Pusey CD, Rees AJ, Evans DJ, et al. Plasma exchange in focal necrotizing glomerulonephritis without anti-GBM antibodies. Kidney Int 1991;40:757-63. 123. Guillevin L, Fain O, Lhote F, et al. Lack of superiority of steroids plus plasma exchange to steroids alone in the treatment of polyarteritis nodosa and Churg-Strauss syndrome. A prospective, randomized trial in 78 patients. Arthritis Rheum 1992;35:208-15. 124. Miller FW, Leitman SF, Cronin ME, et al. Controlled trial of plasma exchange and leukapheresis in polymyositis and dermatomyositis. N Engl J Med 1992;326:1380-4. 125. Pusey CD. Anti-glomerular basement membrane disease. Kidney Int 2003;64:1535-50. 126. Johnson JP, Moore J, Austin HA, et al. Therapy of anti-glomerular basement membrane antibody disease: analysis on the prognostic significance of clinical, pathologic, and treatment factors. Medicine (Baltimore) 1985;64:219-27. 127. Levy JB, Turner AN, Rees AJ, Pusey CD. Long-term outcome of anti-glomerlular basement membrane antibody disease treated with plasma exchange and immunosuppression. Ann Intern Med 2001;134:1033-42. 128. Little MA, Pusey CD. Rapidly progressive glomerulonephritis: Current and evolving treatment strategies. J Nephrol 2004;17:10-9. 129. Glöckner WM, Sieberth HG, Wichmann HE, et al. Plasma exchange and immunosuppression in rapidly progressive glomerulonephritis: A controlled, multi-center study. Clin Nephrol 1988;29:1-8.

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130. Cole E, Cattran D, Magil A, et al. A prospective randomized trial of plasma exchange as additive therapy in idiopathic crescentic glomerulonephritis. Am J Kidney Dis 1992;20:261-9. 131. Stegmayr BG, Almroth G, Berlin G, et al. Plasma exchange or immunoadsorption in patients with rapidly progressive glomerulonephritis: A Swedish multicenter study. Int J Artif Organs 1999;22.81-7. 132. de Lind van Wijngaarden RAF, Hauer HA, Wolterbeek R, et al. Clinical and histological determinants of renal outcome in ANCA-associated vasculitis: A prospective anlysis of 100 patients with severe renal involvement. J Am Soc Nephrol 2006;17:2264-74. 133. Colvin RB, Nickeleit V. Renal transplant pathology. In: Jennette JC, Olson JL, Schwartz MM, Silva FG. eds. Heptinstall’s pathology of the kidney. 6th ed. Philadelphia: Lippincott Williams & Wilkins, 2007:1347-490. 134. Zand MS. Immunosuppression and immune monitoring after renal transplantation. Semin Dialysis 2005;18:511-9. 135. Lindenfeld J, Miller GG, Shakar SF, et al. Drug therapy in the heart transplant recipient, Part I: Cardiac rejection and immunosuppressive drugs. Circulation 2004;110:3734-40. 136. Blake P, Sutton D, Cardella C. Plasma exchange in acute renal transplant rejection. Prog Clin Biol Res 1990;337:249-252. 137. Michaels PJ, Fishbein MC, Colvin RB. Humoral rejection of human organ transplants. Springer Semin Immunopath 2003;25:119-40. 138. Zachary AA, Montgomery RA, Ratner LE, et al. Specific and durable elimination of antibody to donor HLA antigens in renal-transplant patients. Transplantation 2003;76:1519-25. 139. Montgomery RA, Zachary AA, Racusen LC, et al. Plasmapheresis and intravenous immune globulin provides effective rescue therapy for refractory humoral rejection and allows kidneys to be successfully transplanted into cross-match-positive recipients. Transplantation 2000;70:887-95. 140. Crespo M, Pascual M, Tolkff-Rubin M, et al. Acute humoral rejection in renal allograft recipients: I. Incidence, serology and clinical characteristics. Transplantation 2001;71:652-8. 141. Takemoto SK, Zeevi A, Feng S, et al. National conference to assess antibody-mediated rejection in solid organ transplantation. Am J Transplantation 2004;4:1033-41. 142. Costanzo-Nordin MR, Heroux AL, Radvany R, et al. Role of humoral immunity in acute cardiac allograft rejection. J Heart Lung Transplant 1993;12:S143-6. 143. Fishbein MC, Koobashigawa J. Biopsy-negative cardiac transplant rejection: Etiology, diagnosis, and therapy. Curr Opin Cardiol 2004;19:166-9. 144. Montgomery RA, Zachary AA. Transplanting patients with a positive donor-specific crossmatch: A single center’s perspective. Pediatr Transplantation 2004;8:535-42. 145. Savin VJ, Sharma R, Sharma M, et al. Circulating factor associated with increased glomerular permeability to albumin in recurrent focal segment of glomerulosclerosis. N Engl J Med 1996;334:878-83. 146. Crosson JT. Focal segmental glomerulosclerosis and renal transplantation. Transplant Proc 2007;39:737-43. 147. Choy BY, Chan TM, Lai KN. Recurrent glomerulonephritis after kidney transplantation. Am J Transplant 2006;6:2535-42. 148. Thiery J, Meiser B, Wenke K, et al. Heparin-induced extracorporeal low-density-lipoprotein plasmapheresis (HELP) and its use in heart

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149.

150. 151.

152. 153.

transplant patients with severe hypercholesterolemia. Transplant Proc 1995;27:1950-3. Goldstein JL, Hobbs HH, Brown MS. Familial hypercholesterolemia. In: Scriver CR, Beuadet AL, Sly WS, et al, eds. The metabolic and molecular basis of inherited disease. New York: McGraw-Hill, 2001:1863-913. Marais AD, Firth JC, Blom DJ. Homozygous familial hypercholesterolemia and its management. Semin Vasc Med 2004;4:43-50. Rader DJ, Cohen J, Hobbs HH. Monogenic hypercholesterolemia: New insights in pathogenesis and treatment. J Clin Invest 2003;11:1795-803. Wills AJ, Manning NJ, Reilly MM. Refsum’s disease. Q J Med 2001;94:403-6. Gibberd FB. Plasma exchange for Refsum’s disease. Transfus Sci 1993;14:23-6.

154. Gutsche HU, Siegmund JB, Hoppmann I. Lipapheresis: An immunoglobulin-sparing treatment for Refsum’s disease. Acta Neurol Scand 1996;94:190-3. 155. Giorgi DF, Jagoda A. Poisoning and overdose. Mt Sinai J Med 1997;64:283-91. 156. Lee WM. Acute liver failure. N Engl J Med 1993;329:1862-72. 157. Larsen FS, Hansen BA, Ejlersen E, et al. Cerebral blood flow, oxygen metabolism and transcranial Doppler sonography during high-volume plasmapheresis in fulminant hepatic failure. Eur J Gastroenterol Hepatol 1995;8:261-5. 158. Saibara T, Maeda T, Onishi S, et al. Plasma exchange and the arterial blood ketone body ratio in patients with acute hepatic failure. J Hepatol 1994;20:617-22. 159. Rozga J. Liver support technology—an update. Xenotransplant 2006;13:380-9.

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Specialized Therapeutic Hemapheresis and Phlebotomy Robert Weinstein Professor of Medicine and Pathology, University of Massachusetts Medical School, and Chief, Division of Transfusion Medicine, UMass Memorial Medical Center, Worcester, Massachusetts, USA

A comprehensive, evidence-based analysis of apheresis therapies by the American Society for Apheresis (ASFA) recorded 40 disease states as Category I (apheresis as first-line therapy) or Category II (apheresis as adjunctive therapy) indications for therapeutic apheresis.1 Among these, 24 (60%) were indications for plasma exchange, consistent with prior surveys that found plasma exchange to be the predominant form of apheresis therapy in North America.2,3 At the same time, ASFA created a new category P (“pending”) to take into account potential treatment indications that are under investigation, in particular those that are in pivotal Phase III trials that may lead to approval by the US Food and Drug Administration (FDA).1,4 All of the potential indications in this category are specialized forms of apheresis that target specific fractions of the plasma or cellular compartments of the blood for processing or removal.1 Whereas specialized apheresis procedures have been in wide use for decades,5 some as a result of clinical trials and some on the basis of accumulated experience,6 the approval of new therapeutic indications, devices, and drugs is increasingly dependent on the presentation of carefully acquired supportive evidence.7-9 This applies to apheresis therapies as well.1,10,11 Accordingly, this chapter (which reviews therapeutic apheresis procedures that target, for processing, specific fractions of the plasma or cellular compartment of the blood) emphasizes those procedures that are supported by evidence.

Therapeutic Red Cell Apheresis John J. Abel of the Johns Hopkins University used the term “plasmapheresis” to describe a method for removing large quantities of plasma from experimental animals.12 The first reported series of cases in which the manual removal of plasma was employed for clinical purposes (treatment of Waldenström’s macroglobulinemia) was described as plasmapheresis.13 Thus, the

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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removal of red cells for therapeutic purposes can be referred to as erythrocytapheresis. This term particularly applies to the removal of red cells using automated blood processing instruments that are capable of selectively removing erythrocytes while returning the plasma, buffy coat cells, and additional isotonic saline to the patient.14-17 The use of automated blood processing equipment to exchange patient red cells for donor red cells is referred to as red cell exchange,18-21 although the term erythrocytapheresis is frequently used in reference to red cell exchange.1,22

Red Cell Exchange Therapeutic red cell exchange can be performed by manual exchange transfusion21,23(pp153-160) or with programmable automated blood processing (apheresis) instruments.18-20,22 This discussion focuses on automated red cell exchange. Basic features of automated blood processors that perform apheresis using centrifugation technology are detailed in Chapter 41. The machine operator enters the patient’s gender, height, and weight into the instrument’s computer and total blood volume is calculated.24,25 The programming functions of these instruments also accept input regarding the starting and desired ending hematocrit of the patient, the average hematocrit of red cell replacement units to be used, and the desired fluid balance (a default of 100% may be offered by the instrument). Finally, the operator enters the desired fraction of the patient’s own red cells (fraction cells remaining or FCR) that should remain in the patient’s circulation at the end of the procedure. The instrument calculates the volume of replacement fluid (eg, red cells of average hematocrit as programmed into the instrument’s computer) needed for the procedure.24 Calculation of the desired FCR is predicated on the targeted therapeutic endpoint of the red cell exchange.21,23,26-30 Thus (Formula 1): ⎛ starting endpoint ⎞⎟ ⎜⎜ parameter ⎟⎟⎟ ⎜⎜ hematocrit ⎟  FCR (as %)
100  ⎜ ⎜⎜ desired ending starting ⎟⎟⎟ ⎜⎜⎝ hematocrit parameter ⎟⎟⎠ where the specified parameter may be the % hemoglobin S, the % parasitized red cells seen on the peripheral blood film, etc.

Chapter 43: Specialized Therapeutic Hemapheresis and Phlebotomy

According to ASFA, red cell exchange is indicated (Category I or II) for treatment of severe manifestations of the protozoal infections Plasmodium falciparum and Babesia microti and for the management of sickle cell disease (SCD).1,6 Therefore, if a patient with sickle cell anemia whose hematocrit is 27% and whose starting hemoglobin S level is 100% is to undergo red cell exchange, and if the targeted endpoint hemoglobin S level is 30% and the desired ending hematocrit is 30%, then the FCR is calculated accordingly: ⎛ 27% 30% ⎞⎟ FCR (as %)
100  ⎜⎜ 
27% ⎜⎝ 30% 100% ⎟⎟⎠ At the end of the procedure, the patient’s own red cells will amount to 27% of the circulating red cell mass.

Sickle Cell Disease Sickled erythrocytes were first described in 1910 by Dr. JB Herrick, who noted the abnormally shaped red cells in the peripheral blood film of a dental student from the Caribbean island of Grenada,31 but the malady known now as sickle cell anemia was familiar to the people of central and west Africa.32,33 Caused by homozygosity for the mutant βS gene on chromosome 11,34-36 the disease likely resulted in a survival disadvantage in Africa.37 However, the gene frequency remained high because of the protection from P. falciparum malaria afforded to heterozygote children38 and because of social factors.39 The substitution of valine for glutamic acid as the sixth amino acid residue from the amino terminus of the hemoglobin β chain results in hydrophobic interactions between nearby hemoglobin S molecules such that hemoglobin S aggregates into large polymers when deoxygenated.40 The polymerization of hemoglobin S in vivo is the underlying basis for the hemolytic and vasoocclusive morbidity of SCD,26,40,41 and the exquisite dependence of polymerization on the concentration of hemoglobin S42,43 has provided a scientific basis, in concert with the strong clinical basis,44 for transfusion therapy in sickle cell anemia. For the most part, clinical studies related to transfusion management of SCD have focused on simple transfusion or manual exchange transfusion.23,26,27,44-50 The efficacy of manual vs automated red cell exchange in the treatment of SCD has not been directly studied in a clinical trial; however, red cell exchange can be completed more efficiently and quickly by automated than by manual methods.21 Automated red cell exchange has been shown to mitigate iron overload, while maintaining a low hemoglobin S level, in SCD patients receiving chronic treatment.51-53 Thus, it has entered into routine use in centers where therapeutic apheresis is available. Its role in the aspects of SCD for which it is indicated1,6 is discussed below (see Table 43-1). Manual exchange transfusion, or automated red cell exchange using discontinuous flow equipment, may be performed using isotonic saline, rather than red cells, as the replacement fluid in the early phases of the procedure in order to maximize the removal of hemoglobin-Scontaining red cells and avoid the gratuitous removal of normal red cells.23,51,53

Life- or Organ-Threatening Complications Red cell exchange is standard therapy for children with acute vaso-occlusive stroke (see below for discussion of stroke prevention) and should be performed shortly following documentation of thrombotic (rather than hemorrhagic) stroke by non-contrast computed tomography.21,23,44,54 The treatment goal should be a hemoglobin concentration between 9 and 10 g/dL and less than 30% hemoglobin S. Acute intervention, followed by chronic maintenance transfusion therapy, may limit early morbidity and mortality and prevent recurrence (see below).23,27,44,46 Acute multiorgan failure syndrome is a complication of severe pain episodes of sickle cell disease.21,54,55 It may present as an unusually severe pain episode in patients with sickle cell anemia or hemoglobin SC disease and is characterized by fever, accelerated hemolysis with a rapid decrease in hemoglobin and platelet count, nonfocal encephalopathy, and rhabdomyolysis.55 Beside the central nervous system, other organs, including liver and kidney, may be involved.55 Although standard red cell transfusion therapy may be effective if severe anemia is present, red cell exchange should be considered with higher hemoglobin levels.54 This syndrome may be less likely in patients who are on aggressive chronic transfusion regimens in whom the frequency of episodes of pain or acute chest syndrome has been observed to be reduced.50 In its 2000 report49 the National Acute Chest Syndrome Study Group (NACSSG) defined acute chest syndrome, the leading cause of death and hospitalization of patients with SCD,56,57 on the basis of presentation with a new alveolar infiltrate involving one or more complete lung segments (atelectasis excluded) and accompanied by chest pain, a fever 38.5ºC, tachypnea, wheezing, or cough. Although the NACSSG report did not describe a particular advantage of red cell exchange over simple transfusion, the trial was not randomized for the purpose of detecting such a difference.21,49 Many experienced practitioners recommend exchange transfusion for patients who present with diffuse pulmonary involvement or progressive respiratory decline despite simple transfusion, the goal being a hemoglobin no higher than 10 g/dL and less than 30% hemoglobin S.21,44,54 Although priapism has not been evaluated by ASFA as an indication for automated red cell exchange,1 it occurs in approximately 30% to 90% of males with SCD58-60 and thus may result in consultation requests to the apheresis service. Early claims of success in applying red cell exchange therapy to priapism after 12 to 24 hours of unresponsiveness to conventional treatments were unconvincing in that detumescence was rarely noted sooner than 24 to 48 hours after red cell exchange, Table 43-1. Red Cell Exchange in Sickle Cell Disease Acute or emergent

Acute vaso-occlusive stroke Multiorgan failure syndrome Acute chest syndrome refractory to standard management

Chronic

Prevention of stroke in high-risk children Secondary stroke prevention Prevention of iron overload in chronic transfusion recipient

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and often not until 2 to 7 days after treatment, and there were no controlled studies.44,61 A cautionary note regarding red cell exchange for the treatment of priapism was introduced with the description of acute, severe neurologic abnormalities in six boys with sickle cell anemia 1 to 11 days following partial exchange transfusion for priapism unresponsive to conservative therapy.62 The syndrome was characterized by severe headache at the onset, often associated with increased intracranial pressure, and further neurologic events ranging from seizure activity to obtundation requiring ventilatory support.62,63 The mean posttreatment hemoglobin of the affected children was 12.1 g/dL, and the syndrome, termed ASPEN (association of sickle cell disease, priapism, exchange transfusion and neurologic events), was attributed to cerebral ischemia after an acute rise in hemoglobin, concomitant decrease in hemoglobin S and release of vasoactive mediators during detumescence.62,63 A recent series64 described six adult men and one child with sickle cell anemia, six of whom received automated red cell exchange 28 to 48 hours after onset of an episode of priapism (one was treated 7 days after onset of priapism). All of them ultimately required surgical decompression procedures. Finally, a comprehensive review of 42 well-documented case reports of transfusion therapy in SCD-associated priapism evaluated the effectiveness of transfusion therapy vs conventional therapies in terms of time to detumescence.65 The mean time to detumescence with transfusion therapies was 10.8 days (26 cases) vs 8.0 days with conventional therapies (16 cases). Neurologic complications with transfusion therapy were described in nine cases, some with persistent long-term deficits. Primary and Secondary Prevention of Stroke Approximately 5% to 10% of untransfused children with SCD will have a cerebral infarction by age 20.45,66 Chronic transfusion therapy, given every 3 to 4 weeks, to maintain the level of hemoglobin S below 30% can improve the arteriographic appearance of affected cerebral vessels and reduce the risk of recurrent stroke from 66% to 90% to approximately 10%.45,46,54 Chronic automated red cell exchange can be substituted for simple transfusion.51-53 Some authors argue in favor of discontinuation of chronic transfusion therapy after 3 or more years67; however, reports of recurrent stroke rates of 50% or greater after discontinuation of transfusion therapy have led most sickle cell treatment programs to recommend indefinite prophylactic transfusion regimens.27,54,68,69 The utility of chronic transfusion therapy to decrease the risk of recurrent stroke in children with SCD27,45,46,54 and the demonstration that transcranial Doppler ultrasound was highly predictive of stroke risk in such children,70,71 led to the Stroke Prevention Trial in Sickle Cell Anemia (STOP trial), which sought to study the ability of transfusion therapy to prevent a first stroke in high-risk children with SCD.48 Time-averaged mean blood-flow velocity of at least 200 cm/sec in the internal carotid or middle cerebral artery70,71 and a stroke-free history were required for study entry.48 Over a period of approximately 2 to 3 years, transfusion therapy to maintain hemoglobin S below 30% without exceeding a hemoglobin concentration of 12 g/dL

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reduced the occurrence of stroke in the treatment group by 90% compared to the control group.48 A follow-up study (STOP 2)72 examined the effect of discontinuation of transfusion therapy after 30 months in children from the first STOP trial whose transcranial Doppler readings had reverted to normal. The study was ended prematurely after 79 of an intended 100 subjects had been enrolled. An interim analysis revealed that among 41 children who had been randomly assigned to discontinue transfusions, 14 had reverted to high-risk transcranial Doppler findings 4 to 9 months after stopping transfusions and two had had ischemic strokes. No neurologic events or high-risk Doppler findings were noted in the subjects randomly assigned to continue transfusion therapy. The National Heart, Lung, and Blood Institute recommends continuing transfusion therapy beyond 30 months.73 Transfusional Iron Overload There are no randomized, prospective comparisons of simple transfusion vs automated red cell exchange in the prevention of iron overload in children with SCD who require chronic transfusion therapy. However, four case series of 8 to 14 subjects51-53,74 have been reported in which children were either converted from a simple transfusion program to monthly red cell exchange or begun on red cell exchange early on. In addition, some in each series were on chelation therapy with desferrioxamine23 and some were not. In general, red cell exchange resulted in a 25% to 100% increase in red cell usage and a corresponding variable increase in donor exposures. However, serum ferritin tended to stabilize in those who were not on chelation therapy and significantly decrease in those who continued on chelation therapy. Some children who were begun on red cell exchange before development of iron overload did not accumulate iron as a result of their red cell exchange treatments.

Protozoan Disease Severe manifestations of malaria and babesiosis are ranked as Category II indications by ASFA, largely on the basis of anecdotal evidence.1,2 Malaria Severe malaria is caused by infection with P. falciparum.75 The infection results from injection of sporozoites into the bloodstream by the bite of a female Anopheles mosquito, the division of the malarial sporozoites into merozoites in the liver, and the development of the merozoites into ring-form trophozoites, and then schizonts, within invaded erythrocytes in the circulation.75 These asexual blood stages of P. falciparum are responsible for the symptoms of severe malaria that occur when infection is complicated by serious organ failure or metabolic dysfunction (see Table 43-2).75-79 The manifestations of severe P. falciparum malaria appear to derive from the adherence of infected erythrocytes to glycosylated molecules on endothelium and to platelet CD36 via P. falciparum erythrocyte membrane protein-1 (PfEMP-1), an adhesive protein encoded by the parasite genome and expressed on the surface of infected erythrocytes.80,81 The sequestration of parasitized

Chapter 43: Specialized Therapeutic Hemapheresis and Phlebotomy

Table 43-2. Severe Clinical Manifestations of Plasmodium falciparum Malaria General

Prostration or weakness

Cerebral malaria

Impaired consciousness Abnormal behavior Seizures (3 per 24 hours) Coma (persists at least 30 minutes after seizure)

Severe anemia

Hemolysis Hemoglobin 5 mg/dL or hematocrit 15% Bilirubin 2.5 mg/dL Macroscopic hemoglobinuria not due to G6PD deficiency

Fever Renal failure

Core body temperature 40ºC (104ºF) Creatinine 3.0 mg/dL Urine output 400 mL/24 hours in adults

Pulmonary abnormalities

Pulmonary edema Acute respiratory distress syndrome Hypoxemia

Hypoglycemia

Blood glucose 40 mg/dL

Shock

Systolic blood pressure 70 mm Hg

Hemostatic defects

Clinical and/or laboratory evidence of disseminated intravascular coagulation

Metabolic acidosis

Arterial pH 7.25 Plasma bicarbonate 15 mmol/L

Hyperparasitemia

5% of erythrocytes parasitized

erythrocytes in endothelium in the brain and other organs results in vascular obstruction and elaboration of proinflammatory cytokines such as tumor necrosis factor-α and interferon-γ that, in addition to other factors, may result in the severe manifestations of the disease.80,81 Thus, the use of exchange transfusion and automated red cell exchange in the treatment of severe malaria is based on an ability to rapidly reduce th e burden of parasitemia and the potential to thereby improve the rheologic properties of the blood and reduce the level of toxic mediators such as cytokines.29 Red cell exchange followed by plasma exchange, or automated whole blood exchange, has been described as an approach to the removal of both parasitized red cells and inflammatory mediators in the treatment of severe P. falciparum malaria. However, the evidence to support this approach is anecdotal at best.29 A meta-analysis of eight case-control studies did not find a survival advantage in using exchange transfusion as an adjunct to the treatment of severe P. falciparum malaria82; however, the authors speculated that automated red cell exchange might be a promising alternative (the studies analyzed employed manual whole blood exchange transfusion). Five case series (16 patients total) and one case report describe lowering of parasitemia by 80% to 90% using automated red cell exchange of 1.0 to 1.5 red cell volumes in approximately 2 hours, followed by rapid clinical recovery, in cases of severe P. falciparum malaria, including cerebral malaria.82-87 The United States Centers for Disease

Control and Prevention (CDC) recommends strong consideration of exchange transfusion or automated red cell exchange if parasite density is 10% or in case of complications such as cerebral malaria, acute respiratory distress syndrome (ARDS), or renal failure.88 Treatment may be repeated until parasitemia involves 1% of circulating red cells.29,88 Babesiosis Babesiosis in humans is a zoonotic disease and is spread to humans primarily through ticks of the genus Ixodes.89 In the Northeastern United States, where most cases of human babesiosis occur, the predominant organism is Babesia microti, the reservoir hosts are wild rodents, and the vector for transferring the protozoa from host to humans is the deer tick Ixodes dammini, the same tick that transmits Borrelia burgdorferi, causative agent of Lyme disease.89,90 The first two reported cases of human babesiosis, one from Europe91 and one from California,92 occurred in splenectomized individuals. The first case in a patient with an intact spleen was reported in Nantucket.93 When injected into the human bloodstream, the sporozoites of babesia are presumed to complex with C3 in the circulation and then enter erythrocytes by attaching to the C3b receptor.30 After asexual budding into four merozoites the parasite perforates the erythrocyte membrane, resulting in hemolysis. They are then free to infect other erythrocytes, and transform into dividing trophozoites (ring forms and tetrads visible on peripheral blood films).30 After an incubation period of 1 to 6 weeks following inoculation, infected patients develop a flu-like syndrome characterized by fever, fatigue, and malaise.30,90 Headache, chills, sweats, myalgia, and arthralgia are frequent complaints. Physical findings may include fever and splenomegaly, and jaundice and pallor may accompany marked extravascular hemolysis.94 Although most cases are subclinical or mild,94,95 severe manifestations, including disseminated intravascular coagulation, respiratory failure, and renal failure may occur.28 Immunocompromised or asplenic individuals are typically more severely affected.96 Although clinical trials are lacking, several case reports and case series suggest that, given the absence of an exoerythrocytic phase of infection, red cell exchange, whole blood exchange or red cell exchange followed by plasma exchange, combined with antibiotic therapy, can be beneficial in severe cases of babesiosis with 5% parasitemia.6,28,29,97 A single one- to two-volume red cell exchange can reduce the circulating population of parasitized erythrocytes by 85% to 90%.6

Erythrocytapheresis and Therapeutic Phlebotomy (Venesection) According to ASFA, symptomatic erythrocytosis and polycythemia vera, clinical entities that are characterized by an elevated circulating total red cell volume,98,99 are Category II indications for red cell volume reduction by erythrocytapheresis.1,6 In clinical practice, therapeutic phlebotomy, rather than automated erythrocytapheresis, is a mainstay of management.100,101 The International Council for Standardization in

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Table 43-3. Diagnostic Criteria for Polycythemia Vera Category

Polycythemia Vera Study Group (US)102

Pearson and Messinezy (British)114

World Health Organization115

A

1. Total RCV Male 36 mL/kg Female 32 mL/kg

1. Total RCV 25% above normal predicted mean

1. Total RCV 25% above normal predicted mean or hemoglobin 99th percentile of reference for age, gender, altitude

2. Arterial O2 sat 92%

2. Absent cause of secondary 2. Absent cause of secondary erythrocytosis erythrocytosis: Familial O2 sat 92% High-affinity hemoglobin EPO-producing tumor

3. Splenomegaly

3. Palpable splenomegaly

3. Splenomegaly

4. Abnormal marrow karyotype

4. Abnormal marrow karyotype (not bcr-abl)

B

Diagnosis

1. Platelets 400,000/µL

1. Platelets 400,000/µL

1. Platelets 400,000/µL

2. White cells 12,000/µL

2. Neutrophils 10,000/µL (12,500/µL in smokers)

2. WBC 12,000/µL

3. LAP 100

3. Splenomegaly (by imaging study)

3. Marrow biopsy reveals panmyelosis

4. Serum B12 900 pg/mL

4. EPO-independent BFU-E growth in vitro, or low serum EPO value

4. Low serum EPO levels

A1A2A3 or A1A2 any 2 B

A1A2A3 or A4 A1A2 any 2 B

A1A2 any other A or 2 B

RCV
red cell volume (“red cell mass”); LAP
leukocyte alkaline phosphatase; EPO
erythropoietin; BFU-E
burst-forming unit erythroid, a marrow-derived erythroid colony in culture.

Haematology recognizes absolute erythrocytosis in an individual whose measured total red cell volume (often referred to as “red cell mass”102) is more than 25% above the mean predicted value for a person of the same body surface area.98 This will be the case in men with a hematocrit of 60% or higher and women with a hematocrit of 56% or higher.98

Polycythemia Vera Polycythemia vera has been acknowledged as a distinct clinical disorder for more than a century.103,104 Classified as a bcr/ abl-negative myeloproliferative disorder, polycythemia vera is characterized by panmyelosis in the marrow and peripheral blood, splenomegaly, hyperviscosity of the blood, thrombosis and a tendency to evolve into either acute myeloid leukemia or myelofibrosis.99,101,105,106 Over 95% of cases are now known to be associated with an acquired point mutation, V617F, in exon 14 of the protein tyrosine kinase Janus kinase 2 (JAK2).107-111 The remaining 5% of patients appear to have mutations in exon 12 of JAK2, including some who may have a more benign phenotype (idiopathic erythrocytosis).112,113 Popular

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diagnostic systems for polycythemia vera are presented in Table 43-3.102,114,115 A revised diagnostic system that recognizes the predominance of JAK2 mutations in polycythemia vera has been proposed.116 Therapeutic Phlebotomy in Polycythemia Vera The increased whole blood viscosity that results from the expansion of total red cell volume in patients with polycythemia vera is the underlying basis of the life-threatening prothrombotic state and the headache, fatigue, dyspnea, cyanosis, and other signs and symptoms that characterize the disorder.101,105 Aggressive phlebotomy to a hematocrit below 45% in males of European ancestry and below 42% in females of European ancestory and in both genders of African ancestry is indicated for prevention of life-threatening thrombotic complications of polycythemia vera.101,105,117 High-risk patients (age 65 years or history of cardiovascular risk factors such as hypertension, diabetes, hypercholesterolemia, congestive heart failure, smoking, or thrombosis) should also be treated with cytoreductive chemotherapy (eg, hydroxyurea, busulfan, interferon-γ).101,117 Low-risk patients (age

Chapter 43: Specialized Therapeutic Hemapheresis and Phlebotomy

60 years, no cardiovascular risk, platelet count 1,500,000/µL) may initially be managed with phlebotomy alone.101,117 Low-dose aspirin (100 mg/day) is recommended for all patients without specific contraindications to its use.117,118 The pharmaceutical development of JAK2-selective kinase inhibiting agents may influence future management of polycythemia vera.119

Table 43-4. Secondary Erythrocytosis

Erythrocytapheresis in Polycythemia Vera A retrospective case series120 of 69 patients with polycythemia vera who underwent 206 isovolemic erythrocytapheresis procedures using 4% albumin as replacement fluid121 reported reduction of hematocrit from 56.8  5.6% to 41.9  6.6% after removal of 1410  418 mL of red cells with a hematocrit of 79.7  9.3%.120 A subset of 21 patients for whom close follow-up data were available, and whose hematocrit was reduced from 58  5.7% to 41.5  4.9% by a single erythrocytapheresis procedure maintained a hematocrit of 50% for a median of 6 months.120 The durability of response to a single procedure was associated with a median 70% inhibition of in-vitro erythropoietin-independent erythroid burst-forming unit growth that the authors attributed to iron removal during the apheresis procedure.122 This claim has not been confirmed. Automated erythrocytapheresis may be useful in polycythemia vera for the rapid induction of hematocrit lowering, followed by maintenance therapeutic phlebotomy, for emergent isovolemic hematocrit lowering in patients with acute thrombotic or microvascular complications, or to avoid perioperative thrombohemorrhagic complications in a patient with an uncontrolled hematocrit who requires urgent surgery.6,121 The volume of red cells to be removed (VR) during an erythrocytapheresis in order to achieve a desired hematocrit can be calculated as (Formula 2)121: ⎤ ⎡ (starting hematocrit desired hematocrit) ⎥ ⎢ VR
⎢ ⎥ 79 ⎥ ⎢  [ blood volu m e (mL/kg) ]  [ body weight (kg) ] ⎥⎦ ⎣⎢ Thus, for a 70-kg person with a blood volume of 70 mL/kg whose hematocrit is to be lowered from 68% to 55%, the volume of red cells to be removed is calculated as: VR
[(68 55) 79]  70  70
910 mL of red cells. Formula 2 can be used to estimate the volume of replacement fluid (crystalloid or colloid) needed to maintain fluid balance.121 An easy alternative is to program an automated apheresis instrument to perform a “red cell exchange” using replacement fluid with hematocrit of 0%, a target FCR of 100%, and a target hematocrit as desired (see Formula 1).

Secondary Erythrocytosis Secondary erythrocytosis refers to conditions that result in an elevated total red cell volume but are not clonal disorders of the marrow.98,99,101,102,123 Congenital and acquired causes have been

Type

Underlying Cause

Congenital

High oxygen-affinity hemoglobin124 Bisphosphoglycerate mutase deficiency125 Chuvash polycythemia126,127 Erythropoietin receptor mutation128

Acquired

Hypoxia-stimulated98,99,101,123 ● Cyanotic congenital heart disease ● Chronic lung disease ● High-altitude habitat ● Smoker’s erythrocytosis ● Carbon monoxide poisoning ● Chronic hypoventilation (sleep apnea) ● Renal artery stenosis Inappropriate erythropoietin production98,99,101,123 ● Renal cancer ● Hepatic cancer ● Cerebellar hemangioblastoma ● Endocrine tumors ● Uterine leiomyoma ● Polycystic kidney ● Meningioma Drug-mediated98,99,101,123 ● Androgen therapy ● “Blood doping” (surreptitious erythropoietin use) Multifactorial etiology ● Postrenal transplant erythrocytosis129

described, and predominantly involve the regulation or aberrant expression of erythropoietin or abnormalities of the erythropoietin receptor (see Table 43-4).98,99,101,102,123-129 A diagnostic investigation of a patient with suspected erythrocytosis is performed in order to 1) establish that a true state of erythrocytosis exists (eg, an elevated total red cell volume), 2) distinguish secondary erythrocytosis from polycythemia vera, and 3) determine the cause of secondary erythrocytosis, thereby guiding the approach to clinical management.101,102,116,123,130,131 Therapeutic Phlebotomy in Secondary Erythrocytosis The role of phlebotomy is less certain in secondary erythrocytosis than in polycythemia vera.123 As suggested by Table 43-4, secondary erythrocytosis is generally an adaptation to the disordered regulation of erythropoietin or to hypoxemia. In some cases the underlying cause can be treated medically or surgically, and in others the erythrocytosis represents a physiologic adaptation to a chronic condition. For example, adults with cyanotic congenital heart disease are not considered to be at heightened risk for thrombotic stroke despite mean hematocrits of 57.5%  7.2%132 and do not exhibit symptoms of hyperviscosity at hematocrits below 65% in the absence of dehydration or iron deficiency.133 In fact, microcytosis or a history of phlebotomy are significant risk factors for stroke in this population, as are atrial fibrillation and hypertension.134

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Isovolemic phlebotomy, with saline replacement, should be reserved for patients with moderate symptoms of hyperviscosity (ie, headache, slow mentation, visual disturbance, tinnitus, dizziness, etc), and who are neither dehydrated nor iron deficient.133 A program of therapeutic phlebotomy should not be undertaken purely for the sake of achieving a target hematocrit in an asymptomatic individual. Withdrawal of up to a unit of whole blood, replaced by 750 to 1000 mL of isotonic saline, has been recommended for relief of symptoms.133 Preoperative autologous blood donation if the hematocrit is above 65% may also be considered.133 Similar recommendations may refer to patients with high oxygen-affinity hemoglobin levels who have symptoms such as dizziness, dyspnea, or angina, which are believed to result, in part, from an expanded total red cell volume.101,123 There is no formal evidence that phlebotomy is beneficial, and a modest target (ie, a hematocrit 60% achieved by partial exchange transfusion) has been recommended.101,123 Patients with chronic hypoxic lung disease and erythrocytosis or with smoker’s erythrocytosis are best managed using medical therapy to deal with their underlying pulmonary disorder. Non-controlled studies suggest that phlebotomy to a hematocrit of 50% to 52% may improve exercise tolerance, alleviate headache and confusion, and otherwise ameliorate symptoms of hyperviscosity.101,123 Postrenal transplant erythrocytosis, defined as a hematocrit above 51% to 55%, occurs spontaneously in 15% to 20% of kidney transplant recipients in the first 8 to 24 months after engraftment.129,135-137 One fourth of cases remit spontaneously within 2 years of onset, the balance persisting for up to several years until chronic graft rejection supervenes.123 Male gender, retention of native kidneys, smoking, a rejection-free course with a well-functioning graft, and limited or no need for erythropoietin during the period of pretransplant hemodialysis have been listed as risk factors for postrenal transplant erythrocytosis.129,135 Hyperviscosity symptoms such as malaise, headache, plethora, lethargy, and dizziness are described as common among patients with this condition, and 10% to 30% develop significant thromboembolic complications.129,135 The pathogenesis appears to be multifactorial, and likely involves an interplay between endogenous erythropoietin production by the retained native kidney, the renin-angiotensin system, androgen secretion, insulin-like growth factors, and cytokines.129,136,137 One retrospective series reported 11 thromboembolic events including transient ischemic attacks and strokes, and venous thromboembolism in 10 of 53 (19%) patients with postrenal transplant erythrocytosis but in none of 49 control cases (p  0.001).135 This sort of experience has led to an appreciation of the need to control the red cell volume in these patients.129,135-137 The mainstay of treatment is angiotensin-converting enzyme inhibition or angiotensin-converting enzyme receptor blockade, sometimes in combination with theophylline, which lowers hemoglobin and hematocrit within 8 weeks with peak effect seen after up to 12 months.138-142 Cautious phlebotomy may be used initially to lower the hematocrit to 45% to 50%; however, using

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phlebotomy to maintain a target hematocrit over time results in iron deficiency.129,142 Erythrocytapheresis in Secondary Erythrocytosis Although automated erythrocytapheresis is seldom recommended for management of secondary erythrocytosis,121 it may be useful in circumstances where isovolemic procedures are called for, such as in cyanotic heart disease.133 One retrospective series reported 208 erythrocytapheresis procedures involving 61 patients, most of whom appeared to have chronic pulmonary insufficiency or cyanotic congenital heart disease (details were not provided).143 The volume of whole blood processed was 1322  446 mL in two to four cycles (discontinuous flow equipment) at a whole blood flow rate of 60 mL/minute, a mean of 576 mL (range 426-800 mL) of red cells was removed per procedure, mean hematocrit was reduced from 58.4%  4.5% to 42.7%  5.3%, and patients were maintained with a hematocrit below 50% for an average of 6.5 months after every procedure.143 Another descriptive case series claimed that hemoglobin, hematocrit, and blood viscosity could be lowered “drastically” for as long as 11 months by a single erythrocytapheresis procedure.14 Apheresis has not been reported in the management of posttransplant erythrocytosis.

Hereditary Hemochromatosis Although not covered in the 2007 ASFA review of indications for therapeutic apheresis,1,6 hereditary hemochromatosis is an important indication for therapeutic phlebotomy144 and erythrocyatapheresis has been described as an alternative to phlebotomy in its management.145 This is an inherited disorder that, untreated, results in iron deposition in, and damage to, the liver, heart, pancreas, and other organs.144,146,147 Its prevalence is approximately 1:200 among those of European ancestory.148-150 The most common genetic mutation, accounting for 90% of cases (and almost all cases in persons of Northern European ancestry) is homozygosity for a single missense mutation (G to A) at nucleotide 845 in the HFE gene on chromosome 6p21.149-153 This mutation results in substitution of cysteine with tyrosine at amino acid 282 and is referred to as the C282Y mutation.154 Abnormalities of the HFE gene may result in a defect in iron sensing in the deep crypt cells of gut epithelium and thus inappropriate iron uptake despite abundant iron stores in the body.149 The accumulation of iron in the liver and other organs slowly results in liver failure (cirrhosis, hepatocellular carcinoma), diabetes, hypogonadism, hypopituitarism, arthropathy, cardiomyopathy and heart failure, and skin pigmentation.149,152 A presenting syndrome of asthenia, arthralgia, and abnormal liver function has been described as classic for the clinical disease.147 Because of the central importance of iron loading in the pathogenesis of hereditary hemochromatosis, iron removal by therapeutic phlebotomy is the mainstay of treatment.155 Additional genetic associations with hereditary hemochromatosis, and diagnostic elements of the condition, are provided in Tables 43-5 and 43-6.

Chapter 43: Specialized Therapeutic Hemapheresis and Phlebotomy

Therapeutic Phlebotomy in Hereditary Hemochromatosis Therapeutic phlebotomy has been the primary mode of iron reduction in hereditary hemochromatosis for over a half century.144,155,160 Phlebotomy therapy should be started in all patients whose serum ferritin level is elevated (see Table 43-6) and should not be withheld from the elderly on the basis of age or from iron-loaded patients who have not developed clinical symptoms.144,155 A common treatment approach is to perform one phlebotomy per week (1 unit or 7 mL/kg of whole blood not to exceed 550 mL per phlebotomy) until the serum ferritin is 50 ng/mL.144,155 Thereafter, it is usually necessary to annually remove 3 to 4 units of blood to maintain the ferritin at that level. Malaise, weakness, fatigability, and liver transaminase elevations often improve during the first several weeks of treatment, but joint symptoms may initially worsen before eventually improving (if at all).144,155 Cardiomyopathy and cardiac arrhythmias may resolve with phlebotomy, but insulin-dependent diabetes generally will not. The risk of hepatocellular carcinoma will persist if cirrhosis was present before the onset of phlebotomy therapy.144,155 Hypogonadotrophic hypogonadism and thyroid dysfunction do not usually improve with phlebotomy; however, pituitary and gonadal function may improve if they were of recent onset at the time phlebotomy therapy was started.144

Table 43-5. Genetic Mutations Resulting in Hereditary Hemochromatosis Syndromes156,157 Type

Gene

Chromosome

Mutation

I* (classical)

HFE

6p21

Cys282→Tyr (C282Y) His63→Asp (H63D) Ser65→Cys (S65C)

IIa* (juvenile) IIb* (juvenile)

HJV (hemojuvelin) HAMP (hepcidin)

1q21

Gly320→Val (G320V)

19q13

93delG Arg56→Ter (R56X) Gly→71Asp (G71D) G-A, 14 5-prime UTR

III*

IV†

TFR2 (transferrin receptor)

7q22

Erythrocytapheresis in Hereditary Hemochromatosis Early reports from Europe14,15,17,161 suggested that automated erythrocytapheresis could efficiently remove red cells and deplete iron in patients with hemochromatosis. A German group from Munich reported successful lowering of iron in 14 patients with hemochromatosis, with intervals of 2 to 11 months between procedures.14 An Italian group15 treated 14 patients with a mean of 93 (range 21-203) erythrocytapheresis procedures over a mean (median) of 9.6 (24) months. A mean of 19 (range 4.2-40.6) grams of iron were removed, transferrin saturation was reduced from 90  8.7% to 17  10.6%, serum ferritin decreased from 3164  1488 ng/mL to 60.5  77.5 ng/mL, and hepatic iron was said to have normalized in all cases. A follow-up study from Munich17 described prospective observations on eight patients with hereditary hemochromatosis who were treated with isovolemic erythrocytapheresis (1000 mL removed) every 4 weeks until serum ferritin fell below 300 ng/mL. Iron depletion, thus defined, was achieved after a mean of 8.5 months during which a mean of 9.4 liters of red cells were removed in a mean of 8.9 procedures. Serum ferritin levels decreased from 2596  399 ng/ mL to 168  83 ng/mL and transferrin saturation fell from 91  6% to 19  10%. Maintenance erythrocytapheresis every 5 to 6 months prevented iron reaccumulation during 18 to 36 months of follow-up.17 A group from Zaragoza, Spain162 compared data from nine patients with hemochromatosis who were treated with erythrocytapheresis over a period of 2 years to nine similar patients who were treated with conventional phlebotomy therapy. The erythrocytapheresis procedures were performed using a discontinuous-flow blood processor via single-needle access. A mean of 275 mL of red cells were removed with each procedure. The median serum ferritin reduction with each erythrocytapheresis was 55 ng/mL compared to 17 ng/mL

Table 43-6. Some Diagnostic Considerations in Hereditary Hemochromatosis (absent a cause of secondary iron overload)147,148,155,158,159 Diagnostic Tool

Factors to Consider

Clinical clues

●

Tyr250→Ter (Y250X) Glu60→Ter (E60X) Met172→Lys (M172K) Gln690→Pro (Q690P)

● ●

● ●

SLC40A1 (ferroportin)

2q32

Asn144→His Ala77→Asp (A77D) Val162 del Asp157→Gly (D157G) Gln182→His (Q182H) Gly323→Val (G323V) Asp181→Val (D181V) Gly80→Val (G80V) Gly267→Asp (G267D)

● ● ●

Screening criteria

● ●

Diagnostic tests

● ● ●

*autosomal recessive † autosomal dominant

●

Celtic ethnicity Chronic asthenia Arthropathy (fourth and fifth metacarpophalangeal joints: “hemochromatosis handshake” Impotence Hyperpigmentation Liver abnormalities (transaminase elevation, hepatomegaly) Diabetes Cardiomyopathy Transferrin saturation 50% in men; 45% in women Serum ferritin: 200 ng/mL in premenopausal women; 300 ng/mL in men Hepatic iron index 1.9 Hepatic iron concentration 80 µmol/g dry weight Grade 3-4 hepatic iron deposition HFE gene analysis

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with phlebotomy. Patients treated with erythrocytapheresis achieved their iron depletion goal in one third the time required for those treated with phlebotomy.162 A prospective evaluation of erythrocytapheresis in 13 patients with hereditary hemochromatosis was reported from Palma de Mallorca, Spain.145 The volume of red cells to remove was calculated according to the patient’s blood volume and preprocedure hematocrit (as noted in Formula 2 above), and the goal of each procedure was to remove a maximum of 800 mL of red cells and reduce the patient’s hematocrit to 30%. Procedures were scheduled every 2 weeks, but the treatment interval would be extended for another 2 weeks if the starting hematocrit was found to be 36%. Starting serum ferritin was (mean  SD) 1517  1329 ng/mL, and was reduced to 20  6.5 ng/mL after 6.7  2.9 months and 13.5  7.2 apheresis sessions. A mean of 565.5  152 mL of red cells was removed with each procedure; this translated into removal of 878  315 mg of iron per month. Four of the 13 patients underwent a maintenance erythrocytapheresis procedure after 6, 14, 16, and 16.5 months.145 A report from Buenos Aires163 described a curious regimen of isovolemic erythrocytapheresis in combination with recombinant human erythropoietin, 4000 units subcutaneously three times per week (approximately 150 µg/kg/week) and folic acid 10 mg daily by mouth, in nonanemic patients with hereditary hemochromatosis. Ten men with hereditary hemochromatosis were subjected to erythrocytapheresis when their hematocrit reached 30% (or hemoglobin 10 g/dL), and the volume of red cells collected was calculated using Formula 2 as described above but in no case was greater than 30% of the circulating red cell mass. Procedures were performed with both continous- and discontinuous-flow equipment using a three-phase apheresis protocol: collection of the red cell product, infusion of isotonic saline to replace the removed red cell volume, return of autologous plasma and buffy coat cells. A median of 3.1 erythrocytapheresis procedures (range 2.4-4.4) were performed each month during which a median of 2098 mL (1648-3677) of red cells were collected. It took a median of 9 (3-14) days for the hematocrit to reach 34% (or hemoglobin to rise to 10 g/dL) between procedures. Serum ferritin was reduced from a mean (range) of 2960 (1057-7200) ng/mL to 270 (184-398) ng/mL in a median of 3.25 (1.0-7.5) months.163 More recently, a pilot study from the Netherlands164 reported results from six patients with hereditary hemochromatosis who received a mean (range) of 9.8 (5-18) erythrocytapheresis procedures over 4.8 (2-9) months using discontinuous-flow equipment. An estimated mean (range) of 465.2 (360-579) mg of iron were removed per procedure and a mean total of 4.38 (2.69-7.97) grams of iron were removed. Initial serum ferritin was 1419 (798-2541) ng/mL and final ferritin was 54 (39-90) ng/mL.163a In summary, erythrocytapheresis in the treatment of hereditary hemochromatosis is supported by Type II-3 evidence1 and may be an acceptable alternative to conventional schedules of

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whole blood phlebotomy in selected patients. This indication has not yet been reviewed by ASFA and thus has not been assigned an indication category as of this writing.1

Therapeutic Platelet Apheresis Thrombocytapheresis is a term that describes the selective removal of platelets from a patient for therapeutic purposes, using a blood processing (apheresis) device.1 The 2007 ASFA review of indications for apheresis therapy lists symptomatic thrombocytosis as a Category II indication for thrombocytapheresis.1,6 Athough not made clear without a careful reading of the text,6 this designation refers to primary thrombocytosis, as results from a clonal (myeloproliferative) disorder of the marrow.164 Thrombocytapheresis for prophylaxis in asymptomatic patients or to lower the platelet count in cases of secondary or reactive thrombocytosis164 is listed as a Category III (ie, unproven) indication because data either conflict or are insufficient to demonstrate benefit in these circumstances.1,6 Some prominent causes of primary and secondary thrombocytosis are listed in Table 43-7.164-166 Secondary thrombocytosis per se does not convey a risk of thromboembolic morbidity absent confounding factors such as malignancy or major surgery.165,167 Even then, antiplatelet agents should be the first option for treatment.166 In any case, treatment of the underlying cause is the prime factor in the resolution of secondary thrombocytosis.164 In fact, given the absence of risk posed by the platelet count in secondary thrombocytosis, the platelet count may be considered a laboratory sign of an underlying condition that should be investigated.164

Table 43-7. Some Causes of Primary (Clonal) and Secondary (Reactive) Thrombocytosis164-166 Type

Possible Causes

Primary thrombocytosis

Essential thrombocytosis Polycythemia vera Chronic myelogenous leukemia Myelofibrosis Myelodysplastic syndrome (5q-)

Secondary thrombocytosis

Acute hemorrhage Chronic blood loss with iron deficiency Tissue damage or trauma Malignancy Acute or chronic inflammation Acute or chronic infection Physical exercise Rebound from chemotherapy or immune thrombocytopenic purpura Medication

Chapter 43: Specialized Therapeutic Hemapheresis and Phlebotomy

Consequences of Thrombocytosis in Primary Thrombocytosis (Essential Thrombocythemia) Thrombocytosis with thromboembolic complications is a feature common to all of the chronic myeloproliferative disorders,168,169 but essential thrombocythemia is the one most likely to be diagnosed in a patient who presents with thrombocytosis.165 Thus, the remainder of this discussion will focus on essential thrombocythemia. Both thrombotic and hemorrhagic complications result in patients with essential thrombocythemia.164,170 Factors that increase risk of thrombosis by threefold or more include age greater than 60 years and cardiovascular risk factors such as hypertension, hypercholesterolemia, and a prior thrombotic event.171-173 There is no evidence in favor of prophylactic treatment of low-risk, asymptomatic patients, regardless of platelet count, but high-risk and symptomatic patients are treated to lower their platelet count to below 400,000/µL. In a cohort of 126 subjects with a median age of 31 years (range 5-40) smoking appeared to neutralize the protective effect of age less than 60 years.174 The United Kingdom Medical Research Council Primary Thrombocythemia 1 Study has established hydroxyurea plus lowdose aspirin as the treatment of choice.175 Whereas the actuarial rate of first thrombosis in an untreated high-risk population is approximately 26% at 2 years, the rate decreases to about 4% at 2 years with hydroxyurea and aspirin.175,176 As the platelet count rises above 1.0 to 1.5 million/µL, there is increasing evidence of platelet dysfunction that may result in a risk of mucocutaneous hemorrhage in 35% or more of patients with essential thrombocythemia. This platelet count is an indication for cytoreductive therapy.164,170,177,178 While several in-vitro platelet function abnormalities can be demonstrated,170 the most significant functional consequence of hyperthrombocytosis is an acquired von Willebrand syndrome that results from the accelerated clearance of hemostatically competent large multimers of von Willebrand factor from the circulation.179-184 Hemostatic function appears to improve with reduction of the platelet count.179,181,183,184

Therapeutic Thrombocytapheresis in Primary Thrombocytosis Rapid lowering of an elevated platelet count, using apheresis and/or chemotherapy, is indicated for patients with myeloproliferative disorders who present with clinical syndromes of microvascular thrombosis such as digital or cerebral ischemia.170 Several case series and case reports have reported successful, rapid lowering of the platelet count in symptomatic patients in whom chemotherapy was either not an immediate option or was judged to have an insufficiently rapid effect.168,185-189 Procedures in which 1.5 to 2.0 blood volumes are processed, and crystalloid replacement fluids are used to manage fluid balance, can lower the platelet count by 30% to 60%.6,185,189,190 However, thrombocytapheresis without concomitant chemotherapy is not a practical means for controlling the platelet count beyond the acute setting.185,187,189 Thus, symptomatic thrombocytosis is a Category II indication for thrombocytapheresis.1 Weekly thrombocytapheresis, beginning in the 5th gestational week, has

been used in the management of a high-risk pregnant patient with essential thrombocythemia.190

Therapeutic White Cell Apheresis Hyperleukocytosis (white cell count 100,000/µL) with leukostasis is a Category I indication for therapeutic leukocytapheresis performed by centrifugation using discontinuous- or continuous-flow equipment.1,6,191-195 Leukocytapheresis by selective adsorption techniques is currently under study in the United States and elsewhere for the treatment of inflammatory bowel disease, which ASFA has designated a Category P (pending) indication.1,6,196

Leukocytapheresis for Hyperleukocytosis Hyperleukocytosis is a risk factor for early mortality, often from pulmonary and/or central nervous system hemorrhage, in adults and children with acute myeloblastic leukemia.191,197 It occurs in 5% to 13% of newly presenting cases of adult acute myelogenous leukemia and 12% to 25% of pediatric acute myelogenous leukemia.192,198 The reported incidence in acute lymphoblastic leukemia ranges from 10% to 30%.198 Mortality rates of 20% to 40% have been reported.198 Children with acute lymphoblastic lymphoma are less susceptible to intracerebral hemorrhage if the white cell count is below 400,000/µL.191,199 Clinical features of hyperleukocytosis with leukostasis include respiratory distress, hypoxemia, diffuse interstitial or alveolar infiltrates on chest x- ray, confusion, somnolence, stupor or coma, headache, dizziness, tinnitus, gait instability, or visual disturbances.198 Physical examination may demonstrate papilledema, dilated retinal veins and/or retinal hemorrhages, cranial nerve defects, or meningeal signs.198 Metabolic derangements caused by tumor lysis may include hyperkalemia, hyperuricemia, hypocalcemia, and hyperphosphatemia and may result in renal failure and early death.191,192,197 Pulmonary, central nervous system, cardiac, and other organ morbidity results from obstruction of small vessels by aggregates of leukemic blasts with resulting infarction and hemorrhage.200 Coagulopathy results from release of lysozomal enzymes from myeloid blasts, disseminated intravascular coagulation, and thrombocytopenia resulting from marrow failure.198-200 A standard treatment approach to hyperleukocytosis includes intravenous hydration, lowering of plasma uric acid using allopurinol or urate oxidase and, if urate oxidase is not used, administering intravenous sodium bicarbonate to alkalinize the urine.199 Hydroxyurea may be prescribed to rapidly lower the total circulating nucleated cell count without precipitating a tumor lysis syndrome.198 Induction chemotherapy may be used for this purpose but may precipitate tumor lysis syndrome and hemorrhage.200 The processing of 1.5 to 2.0 blood volumes, using crystalloid or colloid fluids to maintain fluid balance, with or without a sedimenting agent such as 6% hydroxyethyl starch to enhance the separation of white cells from red cells, can reduce the circulating white cell count by up to 60%.6,193,194,201 Leukocytapheresis before

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initiation of definitive induction chemotherapy has been reported to lower the leukemic blast count to 100,000/µL in approximately 60% of patients, and to lower the incidence of early death (Day 21 after admission) by half, but does not affect long-term survival and may not prevent death from leukostasis in patients who present with severe symptoms of pulmonary or cerebral leukostasis before initiation of apheresis.191,193,195,201 In any case, leukocytapheresis is not initiated without the above-mentioned measures (eg, hydration, uric acid lowering, and hydroxyurea) and should not be considered primary therapy in patients who present with high blast counts but without symptoms of leukostasis. Thus, leukostasis is appropriately classified as a Category II indication for leukocytapheresis.1,194,199,201

Leukocytapheresis for Inflammatory Bowel Disease Idiopathic inflammatory bowel disease principally refers to ulcerative colitis and Crohn’s disease, similar but distinct disorders of intestinal inflammation.202-204 Both may present with fever, diarrhea, rectal bleeding, weight loss, and abdominal pain.203 Whereas perianal disease and anal strictures and fistulas are common in Crohn’s disease, they are not features of ulcerative colitis. Also, ulcerative colitis is exclusive to the colon, while Crohn’s disease involves the ileum in two-thirds of patients.203 The majority of patients with ulcerative colitis exhibit positive serology for perinuclear-staining antineutrophil cytoplasmic antibodies but few are positive for anti-Saccharomyces cerevisiae.203-204 Medical therapy for both disorders includes oral aminosalicylates, oral or parenteral steroids, immunomodulators (including azathioprine or mercaptopurine), and biological agents such as the antitumor necrosis factor agent infliximab.205-208 Interest in leukocytapheresis in the treatment of severe inflammatory bowel disease derives from the observed infiltration of the intestinal mucosa in these disorders by granulocytes and macrophages that produce marked mucosal tissue injury through the release of reactive oxide compounds, matrix metalloproteinases, and proinflammatory cytokines.203,208,209 Two leukocyte adsorption columns are currently in use in Europe and Japan: a column of cylindrical nonwoven polyester fibers that preferentially binds granulocytes and lymphocytes (Celsorba, Asahi Kasei Kuraray, Tokyo, Japan) and a column of cellulose acetate beads that binds primarily granulocytes and monocytes (Adacolumn, JIMRO, Takasaki, Japan).196 Reports of case series and small randomized (but not blinded) trials have claimed clinical benefit for both devices.1,196 The Adacolumn is currently in Phase III pivotal trials in the United States; thus, inflammatory bowel disease is listed as a Category P (pending) indication for adsorption leukocytapheresis (designated “adoptive cytapheresis” by ASFA1,6) until the outcome of the trial is known.210

Extracorporeal Photochemotherapy Extracorporeal photochemotherapy (ECP, also referred to as “photopheresis”) describes a procedure in which circulating

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mononuclear cells are collected by centrifugal apheresis, exposed to 8-methyoxypsoralen (8-MOP, a photoactivating agent), and then exposed to ultraviolet A (UVA) light. The treated cells are then reinfused into the patient. The only commercially available closed system is UVAR-XTS (Therakos, Exton, PA). A typical procedure, using single-needle access, consists of six cycles of processing 125 mL of blood or three cycles of processing 225 mL of blood to collect a buffy coat volume of approximately 240 mL. The instrument mixes the collected buffy coat cells with saline and 8-MOP (17 µL per ml of buffy coat) and then pumps the cells through a 1-mm plastic film between two banks of fluorescent UVA lamps (352 nm, 1-2 J/cm2) to affect photoactivation. Whereas red cells and plasma are returned to the patient at the end of each collection cycle, the treated buffy coat product is returned following the last cycle. A full procedure is completed in approximately 3 hours.211,212 The precise mechanism of action of ECP is unknown,211,213 but available evidence suggests that it works through a process of immunomodulation.212-214 Photoactivated 8-MOP intercalates within nuclear DNA of normal and malignant T lymphocytes and causes apoptosis of treated T cells, but not treated monocytes, within 24 hours after ECP.215,216 The two main immunologic consequences of phagocytosis of apoptotic cells appear to be induction of major histocompatibility Class I-restricted CD8 cytotoxic T lymphocytes through the antigen-presenting activity of human dendritic cells and the elaboration by monocytes and macrophages of immunosuppressive cytokines including interleukin (IL)-10 and IL-1 receptor antagonist.213 Further, ECP results in an increase in CD83, CD86 plasmacytoid (DC2) dendritic cells with a concordant diminution in CD80, CD123 monocytoid (DC1) dendritic cells. The DC2 cells stimulate Th2 T-helper cells to secrete inhibitory cytokines (eg, IL-4, IL-10, IL-13) while inhibiting stimulation of Th1 T-helper cells that secrete proinflammatory cytokines (eg, IL-2, interferon-γ) and thus inhibiting Th1-mediated alloreactivity.214 ECP also appears to result in an increase in a population of circulating CD4, CD25, CD69 CTLA-4 regulatory T cells that are immunosuppressive and are involved in transplant tolerance.217 Such induced immunologic responses to ECP are presumed to explain observed clinical benefit in diverse conditions such as cutaneous T-cell lymphoma, chronic graft-vs-host disease (GVHD), and cardiac transplant rejection.211-216,218 In 1998 the United States Centers for Medicare and Medicaid Services (CMS) approved Medicare reimbursement for ECP in the palliative treatment of skin manifestations of cutaneous Tcell lymphoma (CTCL) not responsive to other therapy. In 2006 CMS further determined that ECP is “reasonable and necessary” for treatment of acute cardiac allograft rejection and chronic GVHD refractory to standard immunosuppressive drug treatment.219 ASFA has listed treatment of heart transplant rejection and cutaneous manifestations of GVHD as Category II indications for ECP, and erythrodermic CTCL and prophylaxis of heart transplant rejection as Category I indications for ECP.1,6 Some

Chapter 43: Specialized Therapeutic Hemapheresis and Phlebotomy

ECP regimens used in the treatment of the Category I and II indications1,6 are described in Table 43-8.212,214,221-229

Cutaneous T-Cell Lymphoma ASFA has listed the treatment of erythrodermic CTCL as a Category I indication for ECP.1,6

Background The CTCLs are a heterogeneous group of extranodal nonHodgkin lymphomas of T-cell origin that target the skin.229,230 Mycosis fungoides, the most common form of CTCL, accounts for almost half of all primary cutaneous lymphomas.230 It is largely a disease of adults (median age 55-60 years at diagnosis) and typically presents as an indolent disorder that progresses slowly over years from patchy skin involvement to infiltrated plaques, tumors, and widespread disease.230 Whereas localized (eg, nonerythrodermic) mycosis fungoides is adequately managed with topical therapies, the application of ECP in erythrodermic mycosis fungoides is recommended by ASFA and by the British Photodermatology Group and UK Skin Lymphoma Group.1,211 Whereas limited-stage mycosis fungoides does not shorten life expectancy, advanced-stage disease may be associated with a 10-year disease-specific survival of 20%.230 Sézary syndrome is defined as a triad of erythroderma, generalized lymphadenopathy, and the presence of neoplastic T cells (Sézary cells) in the Table 43-8. Regimens of Extracorporeal Photochemotherapy (ECP) Indication

Treatment

Cutaneous T-cell lymphoma

ECP on 2 consecutive days once per month; continue for 6 months before declaring treatment failure212,229

Cardiac allograft rejection

Prophylaxis220,221 Month 1 after transplant: ECP day 1and 2, 5 and 6, 10 and 11, 17 and18, 27 and 28 ● Month 2 and 3 after transplant: ECP on 2 successive days every 2 weeks ● Month 4-6 after transplant: ECP on 2 successive days every 4 weeks ●

Acute (moderate) rejection222,223 ECP procedures on 2 consecutive days at weekly episode intervals as needed to resolve rejection as indicated by endomyocardial biopsy

●

Recurrent/refractory rejection224,225 ECP on 2 consecutive days weekly for 1 month, then every 2 weeks for 2 months, then monthly for 3 months (total of 22 procedures)

●

Chronic rejection226 Beginning within 1 month of transplantation: ECP on 2 successive days every 4 weeks for 12 months, every 6 weeks for next 6 months, every 8 weeks during next 6 months

●

Chronic graft-vs-host disease

ECP on 2 consecutive days every 1-2 weeks, then consider monthly interval; treat at least 6 months before declaring treatment failure214,227,228

skin, lymph nodes, and peripheral blood.230 Diagnostic criteria were recommended by the International Society for Cutaneous Lymphomas for the purpose of identifying patients with a worse prognosis compared to other erythrodermic forms of CTCL (notably erythrodermic mycosis fungoides).231 These include 1 in 1000 circulating Sézary cells per µL of blood; a CD4/CD8 ratio 10 caused by expansion of circulating CD4 T cells and/ or aberrant loss or expression of pan-T-cell antigens (CD2, CD3, CD4, CD5) by flow cytometry; demonstration of a circulating T-cell clone by molecular or cytogenetic methods; or a cytogenetically abnormal T-cell clone.231 The prognosis is poor, with median survival of 2 to 4 years and 5-year survival of approximately 24%.229 A retrospective cohort study from Stanford of 106 patients with erythrodermic mycosis fungoides and Sézary syndrome identified age 65 years, clinical Stage IV and circulating Sézary cells 5% of total lymphocytes as independent negative prognostic factors for survival.232 Median survivals of patients with none, one, or more than one of these adverse prognostic factors were 122 months (n
36), 44 months (n
39), and 18 months (n
31), respectively (p 0.005).232

Extracorporeal Photochemotherapy in the Treatment of Cutaneous T-Cell Lymphoma The 1987 report by Edelson et al233 of a successful pilot trial of ECP, in which 27 of 37 treatment-resistant patients with CTCL experienced an average 64% decrease in cutaneous involvement after 22  10 weeks, led to FDA approval of ECP for the treatment of advanced CTCL later that year.234 Long-term follow-up of the original 29 patients with erythrodermic CTCL from the pilot study of Edelson et al233 reported that median survival of the treated patients was 60.33 months from diagnosis and 47.9 months from the start of ECP. Four of the six patients who achieved complete responses in the original study remained in complete remission.235 A retrospective study from London compared survival of patients with Sézary syndrome who had not received ECP to survival of patients who had.236 Median survival was 39 months (range 1-138) in the ECP group (n
29) and 26.5 months (12-67) in the non-ECP group (n
15), a nonsignificant difference (p
0.12).236 All patients had been demonstrated to have a circulating T-cell clone by testing based on polymerase chain reaction, whereas this was not the case in Edelson’s study.233 Between 1987 and 2001, 21 studies were reporeed in which a total of 485 patients were treated using ECP.211,212,237 Although most patients in these studies had erythrodermic CTCL, most studies did not report the response rates separately for the erythrodermic subjects.237 In addition, responses were defined as 25% skin clearing in some studies and 50% skin clearing in others, and complete responses were defined as either 75% to 100% skin clearing, 90% skin clearing, or 100% skin clearing.212 Nonetheless, overall response rates reported in these studies ranged from 31% to 87.5%, and complete response rates ranged from 0% to 54% (20%-30% in most studies).211,212,237 The variability in response rates has been attributed to

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differences in entry criteria, prior or concurrent therapy, duration between diagnosis and application of ECP, the ECP protocol followed, and definition of response.211 In one analysis of 23 patients with Sézary syndrome treated with ECP as the sole therapy for up to 1 year,237 improvement in skin score was positively associated with a reduction in circulating Sézary cells as a percentage of total white cell count (p
0.03). Neither the absolute change in total white cell count, lymphocyte count, CD34 count, CD8 count, nor the Sézary count distinguished the 13 patients who responded to treatment from the nonresponders. Nor were age, gender or prior treatment with chemotherapy associated with responder status. Baseline lymphocyte count was associated with change in skin score at 6 months (p
0.046) but not at 12 months. Sézary cell count as a percentage of total white cell count was the only baseline variable that was a significant predictor of responder status at 6 months: a 1% increase in % Sézary cells at baseline was associated with an odds ratio of 1.07 of becoming a responder (p
0.021) at 6 months but not at 12 months.238

ASFA has listed prophylaxis of heart transplant rejection as a Category I indication for ECP and treatment of heart transplant rejection as a Category II indication for ECP.1,6 The Category I designation for heart transplant rejection prophylaxis is apparently determined on the basis of two small randomized pilot studies.220,221

coronary disease and 45% for noncoronary cardiomyopathy, with an average recipient age of (mean  SD) 50.7  12.5 years (range 18-77).239 Treatment for an episode of allograft rejection was associated with a 26% increase in mortality risk before hospital discharge and a 48% increase in mortality risk after discharge but within the first postoperative year.239 From January 1992 to June 2006, acute allograft rejection accounted for 12% of deaths, and after 5 years allograft vasculopathy and late graft failure (presumed to be the result of chronic rejection and consequent allograft vasculopathy) combined to account for 30% of deaths.239 Immunosuppressive therapy to prevent allograft rejection has included perioperative lymphocyte antibody (polyclonal antilymphocyte or antithymocyte globulin, OKT3, or IL-2 receptor antibodies), and chronic postoperative calcineurin inhibitors (eg, tacrolimus, cyclosporine), mycophenolate mofetil, sirolimus, and others.239 Cardiac allograft rejection is defined and classified on the basis of histologic findings obtained through surveillance endomyocardial biopsy following transplantation, and may not present as symptoms of allograft dysfunction.240 Because of the association between rejection, graft failure, and death, biopsydetected evidence of rejection should result in adjustment in the immunosuppressive regimen in order to successfully resolve the episode of rejection.222,225,239,240 Thus CMS has only approved reimbursement for severe, drug-refractory rejection.219 The histologic criteria for acute cellular rejection are summarized in Table 43-9.

Background Between 1990 and 2006, approximately 3000 to 4500 heart transplant procedures were reported each year to the registry of the International Society for Heart and Lung Transplantation.239 Between 1 January 2004 and 30 June 2006, 41% of heart transplant procedures were performed for heart failure caused by

Extracorporeal Photochemotherapy in the Treatment of Cardiac Allograft Rejection Studies in support of ECP for treatment of cardiac allograft rejection focus on the effects of ECP on endomyocardial biopsy findings, rather than on survival or graft function.211,219 Two pilot studies have shown evidence that the risk of acute cellular

Cardiac Allograft Rejection

Table 43-9. Initial and Modified International Society for Heart Transplantation Criteria for Cellular Allograft Rejection 1990241

Severity

2005242

No rejection

Grade 0

No rejection

Grade 0 R

No rejection

Mild rejection

Grade 1

A – Focal: Focal perivascular and/or interstitial infiltrate without myocyte damage B – Diffuse: Diffuse infiltrate without myocyte damage

Grade 1 R

Interstitial and/or perivascular infiltrate up to one focus of myocyte damage

Moderate rejection

Grade 2

Focal: One focus of infiltrate with associated myocyte damage A – Focal: Multifocal infiltrate with myocyte damage B – Diffuse: Diffuse infiltrate with myocyte damage Diffuse, polymorphous infiltrate with extensive myocyte damage with vasculitis and with or without edema and/or hemorrhage

Grade 2 R

Two or more foci of infiltrate with associated myocyte damage

Grade 3 R

Diffuse infiltrate with multifocal myocyte damage with or without edema, hemorrhage, vasculitis

Grade 3

Severe rejection Grade 4

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rejection episodes can be decreased by incorporating ECP into the prophylactic immunosuppressive regimen of cardiac allograft recipients without increasing the risk of infection caused by immunosuppression.220,221 Another prospective pilot trial has reported that development of panel-reactive antibodies (responsible for chronic rejection) and coronary artery intimal hyperplasia (a pathogenetic mechanism of graft failure in chronic rejection) are mitigated by the addition of ECP to the posttransplant immunosuppressive regimen for the first 2 years after transplant surgery.226 Dall’Amico and colleagues in Padua, Italy reported a prospective pilot trial in eight patients, with recurrent acute rejection episodes despite immunosuppression, who were treated with 2 consecutive days of ECP every 4 weeks for 6 months.243 Seven benefited with a reduction in the number and severity of rejection episodes; reduction in daily prednisone, cyclosporine, or azathioprine doses; and improvement on endomyocardial biopsy specimens. The investigators conducted two follow-up studies—one a prospective uncontrolled study in 22 patients with two or more acute rejection episodes224 and the other a prospective uncontrolled study of 11 patients with recurrent severe acute rejection episodes.244 They reported that an intensive regimen of ECP (see Table 43-9) is highly effective in resolving and preventing recurrent acute rejection refractory to conventional immunosuppression, but cautioned that most acute rejection episodes, particularly nonsevere episodes, could be effectively managed using medical regimens without ECP.219 Other small case series and case reports have presented corroborating data.222,223,225,245,246 Evidence to support ECP in the management of renal or lung transplant rejection are insufficient to recommend its use in these settings.1,211

Graft-vs-Host Disease The ASFA 2007 evidence-based review of apheresis therapies lists skin manifestations of GVHD as a Category II indication for ECP.1,6 CMS considers ECP reasonable and necessary for the treatment of chronic GVHD that is refractory to standard immunosuppressive drug therapy.219

Background GVHD in hematopoietic progenitor cell transplant recipients occurs when T cells of donor origin (either transplanted with, or that develop from, the graft) interact with tissue in the HLA-matched but genetically nonidentical host.247 Classical acute GVHD develops within 100 days of transplantation with skin manifestations that vary from an erythematous morbilliform rash to epidermal necrolysis, mucosal inflammation causing diarrhea and abdominal cramping, and abnormalities of liver function tests.247,248 GVHD that develops beyond 100 days of transplantation, or persists more than 3 months, is traditionally referred to as chronic GVHD, and is characterized by an oral, ocular, and mucous membrane sicca syndrome; skin involvement; scleroderma; bronchiolitis obliterans; joint contractures; myofasciitis; esophageal stricture; or other fibrotic

complications in various organ systems.247,249 The cumulative incidence of acute GVHD is approximately 12% to 75%, and the cumulative incidence of chronic GVHD is approximately 15% to 70% after hematopoietic cell transplantation, depending on whether the donor-recipient pair are related or unrelated and on whether a myeloablative or nonmyeloablative conditioning regimen was used.249-251 The onset of acute GVHD may be delayed beyond 100 days in some patients such as those who receive nonmyeloablative conditioning regimens, and chronic GVHD may present within the first 2 months after transplantation in certain circumstances. 250,251 A consensus conference of the National Institutes of Health has proposed a categorization of GVHD based on these observations, taking into account the clinical overlap syndromes between the two (see Table 43-10).252 Accurate diagnosis and staging is important in that recurrent or late onset acute GVHD may not require prolonged therapy as is required with chronic GVHD, while overlap syndromes may require shorter courses of typical treatments for chronic GVHD.253

Extracorporeal Photochemotherapy in the Treatment of GVHD The application of ECP to the treatment of GVHD has been extensively reviewed.211,219,254-256 There are no controlled, blinded, randomized trials, and most reports have been case reports, case series, or retrospective analyses, although some prospective analyses are available.219,254 Among the larger prospective reports is one from a London group that treated 28 patients who had developed steroid-refractory chronic GVHD following HLA-matched allogeneic marrow or peripheral blood progenitor cell transplant.227 Among the patients, 27 were classified as having extensive chronic GVHD257 and 20 had involvement of more than 50% of their skin surface. Patients were given ECP on 2 consecutive days every 2 weeks for the first 4 months, and monthly thereafter. ECP was initiated a median of 34 months (range 10-167) after transplantation and 23 months (range 2164) from the onset of chronic GVHD. Of the 21 patients with cutaneous involvement who were evaluable, a 25% reduction in skin involvement was noted in eight (38%) after 3 months and in 10 (48%) after 6 months, and a statistically nonsignificant improvement in liver function tests was noted.227 The authors contrasted their results to those of a retrospective report of 15 patients with extensive chronic GVHD who received ECP on a similar schedule, beginning a median of 12 months (range 344) after transplantation. Among those 15 patients, 12 (80%) achieved complete resolution of cutaneous manifestations of GVHD, seven of 10 (70%) patients with liver involvement had complete responses, and all 11 patients with oral mucosal disease had complete responses.258 A group from Boston enrolled 25 patients with extensive, steroid-refractory chronic GVHD in a prospective uncontrolled trial to evaluate ECP for skin and visceral involvement.259 ECP was performed in the outpatient setting for 2 consecutive days every 2 weeks on 17 patients and weekly on eight patients who

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lived a long distance from the treatment site. The median time from transplantation to initiation of ECP was 790 days (range 242-2928), and ECP continued, until best response or disease progression, for a median of 9 months (range 3-24). Sixteen patients (64%) responded (50% improvement in skin rash; 50% improvement in bilirubin) in at least one site of disease. Twenty improved with respect to cutaneous involvement, including softening of sclerodermatous changes in six. Six of 13 with oral mucosal ulcers experienced healing of their ulcers during

ECP. Three of six patients with joint involvement had increased range of motion.259 An Italian group evaluated ECP in a prospective, uncontrolled study of 32 patients with chronic GVHD who began treatment a median of 5 months (range 1-56) after hematopoietic cell transplantation.260 All but five had cutaneous involvement. The treatment schedule was the same as that used by the London group.227 The investigators reported that 16 patients (50%) achieved a complete response and were able to have their

Table 43-10. Syndromes of Acute and Chronic Graft-vs-Host Disease (GVHD)252 Clinical Onset After Transplantation

Features of Acute GVHD

Features of Chronic GVHD*

Classical acute GVHD Persistent, recurrent, late onset

100 days 100 days

Skin Erythema ● Maculopapular rash Mouth ● Gingivitis ● Mucositis ● Erythema ● Pain GI tract ● Anorexia ● Nausea ● Vomiting ● Diarrhea ● Ileus ● Cholestatic hepatitis

None

Classical chronic GVHD

No time limit

None

Skin Poikiloderma ● Lichen planus ● Scleroderma Mouth ● Lichen-type features ● Hyperkeratotic plaques ● Restricted opening Genitalia ● Lichen-planus ● Vaginal stenosis GI tract ● Esophageal web ● Esophageal strictures Lung ● BOOP (biopsy) Muscle ● Fasciitis Skeletal ● Joint stiffness ● Contractures

●

●

Overlap syndrome

No time limit

Yes; same as above

*Any of these features are considered sufficient to establish a diagnosis of chronic GVHD GI
gastrointestinal; BOOP
bronchitis obliterans with organizing pneumonia.

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Yes; same as above

Chapter 43: Specialized Therapeutic Hemapheresis and Phlebotomy

immunosuppressive drug doses decreased by at least half (the “determinant” group). In seven patients (22%) ECP was declared ineffective in that either progression was observed in at least one involved organ system, it was necessary to increase the dose of immunosuppressants, or immunosuppressants were not reduced by 50% but no clinical complete responses were obtained. The remaining nine patients (28%) were considered responders if their outcomes were better than the “ineffective” group but not as good as the “determinant” group. Thus, with an overall response rate of 78%, the authors concluded that ECP is beneficial for steroid-refractory chronic GVHD.260 A pilot study from Vienna is the principal source of data regarding the use of ECP to treat steroid-refractory acute GVHD.261 In this prospective, unblinded, nonrandomized study, 21 patients who underwent marrow (n
19) or peripheral blood progenitor cell (n
2) transplantation and who experienced acute GVHD unresponsive to corticosteroids and cyclosporin A, were treated with ECP on 2 consecutive days (one cycle) at 1- to 2-week intervals until improvement and then every 2 to 4 weeks until maximal response was reached. ECP was tapered thereafter on an individual basis. The onset of acute GVHD occurred a median of 19 days (range 13-33) after transplantation and drug therapy was given for a median of 21 days (range 9-49) before the start of ECP. The maximal response to ECP was achieved after a median of four cycles (range 1-13) or 2 months (range 0.5-6) of treatment. Complete resolution of acute GVHD was achieved in 60% of patients after 3 months of ECP. Three months after initiation of ECP, complete responses were obtained in 60% of patients with cutaneous manifestations, 67% of patients with liver manifestations, and no patients with gut manifestations of acute GVHD.261 Overall transplant-related mortality was similar to that achieved using conventional second-line immunosuppressive therapy for steroid-resistant acute GVHD.253 Corroborating data in support of the benefit of ECP in mucocutaneous chronic GVHD are provided by smaller prospective and several retrospective studies.262-267 At present, the report of the combined workshop of the British Photodermatology Group and the UK Skin Lymphoma Group on evidence-based practice of photopheresis considers the available evidence sufficient to recommend ECP for the treatment of steroid-refractory chronic GVHD but not for acute GVHD.211 CMS also supports the use of ECP as reasonable and necessary for patients with chronic GVHD whose disease is refractory to standard immunosuppressive drug treatment but does not consider acute GVHD in its analysis.219 ASFA considers skin manifestations to be a Category II indication for ECP but does not distinguish chronic from acute in its recommendation.1,6

addressed in Chapter 42. The discussion herein focuses on therapeutic procedures that, rather than removing whole plasma, are designed to remove a specified fraction of the plasma and return the processed plasma to the patient. A pathogenic plasma substance (for example, a specific autoantibody) is usually present at relatively low levels in the circulation: a typical plasma exchange may remove 150 g of healthy plasma protein in order to eliminate 1 to 2 g of offending substance.268 To minimize the sacrifice of healthy plasma proteins, selective extraction of pathologic plasma constituents has been proposed for the treatment of patients with disorders that might otherwise be treated using TPE.269

Immunoadsorption Apheresis

Specialized Therapeutic Plasma Processing

Immunoadsorption systems employ the principle of affinity chromatography and make use of immobilized sorbents or ligands that have enhanced or specific binding affinity for a specific antigen, antibody, immune complex, or other substance in the patient’s circulation.269,270 Examples include 1) staphylococcal protein A or sheep antihuman immunoglobulin (IgG) antibody for extraction of IgG and immune complexes from the circulation, 2) sheep antihuman low-density lipoprotein (LDL) or apolipoprotein B antibody for extraction of LDL, 3) synthetic blood group substances for removal of ABO isoagglutinins, and 4) DNA for removal of DNA antibody. Two immunoadsorption systems that have received approval from the US FDA are the staphylococcal protein A-agarose column (Immunosorba, Fresenius HemoCare, Redmond, WA),271 and the staphylococcal protein Asilica column (Prosorba, Fresenius HemoCare).272 Protein A is a cell-wall constituent of the Cowan I strain of Staphylococcus aureus. Mammalian IgG binds to five homologous regions at its amino terminus, but interaction of protein A with other plasma proteins is insignificant. Protein A interacts strongly with IgG1, IgG2, and IgG4, but only to a variable extent with IgG3, IgM, and IgA. The affinity of protein A is higher for complexed IgG than for free IgG. Processing of 2.5 plasma volumes using a protein A-agarose column resulted in a 97% reduction in IgG1, a 98% reduction in IgG2, a 40% reduction in IgG3, a 77% reduction in IgG4, a 56% reduction in IgM, and a 55% reduction in IgA, while plasma levels of albumin, fibrinogen, and antithrombin were reduced by less than 20%.270 Thus, in principle, plasma adsorption with protein A affinity columns permits the processing of more plasma than does TPE without unacceptable loss of other essential plasma constituents.268 The commercial distribution of both protein A immunoadsorption systems in the United States was discontinued in 2006.6 The use of these devices in a variety of clinical conditions has been extensively reviewed.268 A brief discussion of current Category II or P indications1,6 for therapeutic immunoadsorption follows.

In its 2007 evidence-based review of apheresis therapies, ASFA lists 30 Category I, II, or P indications for therapeutic plasma processing, 24 of which are indications for therapeutic plasma exchange (TPE).1 The physiology of TPE and its indications are

Immune Thrombocytopenic Purpura Immune thrombocytopenic purpura (ITP, also known as idiopathic thrombocytopenic purpura) presents with thrombocytopenia and mucocutaneous bleeding and is caused by accelerated

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clearance of IgG-coated platelets that interact with Fcγ receptors on tissue macrophages, primarily in the spleen and liver.273 Antibodies against platelet surface glycoproteins, typically glycoprotein IIb/IIIa or Ib/IX, may be naturally occuring, a result of ingestion of medication, or a manifestation of autoimmune or lymphoproliferative disorders.273 Treatment focuses on immunosuppressants, with corticosteroids serving as traditional firstline therapy, and splenectomy often used in the case of steroid failure.273,274 Other effective or promising treatments, including intravenous IgG, anti-D immunoglobulin, and rituximab have proven more popular among practitioners than immunoadsorption for the treatment of otherwise refractory cases,273-276 and a new generation of therapies for ITP may yet supercede current treatment approaches.277 Immunoadsorption with the Prosorba column was reported effective in a group of 72 patients with ITP who had failed to improve on at least two other therapies.276 An increased platelet count was achieved in 33 (46%) of refractory ITP patients who had failed at least two other treatments (including steroids and splenectomy), but responses in seven of the 33 responders lasted less than a month.276 The remaining (79%) responding patients did not experience a relapse of thrombocytopenia over a mean follow-up period of 8 months.276 In another report, six of 10 platelet-refractory, alloimmunized patients achieved at least a doubling of their platelet count after Prosorba treatment.278 In both reports, many of the patients continued to receive other therapies, and there was no control group. Furthermore, little or no corroborating data have been published. Nonetheless, ASFA considers refractory ITP a Category II indication for immunoadsorption.1,6

Rheumatoid Arthritis The Prosorba column was approved by the FDA “… for use in the therapeutic reduction of the signs and symptoms of moderate to severe rheumatoid arthritis (ra) in adult patients with long-standing disease who have failed or are intolerant to diseasemodifying anti-rheumatic drugs (dmards).”279 The serendipitous observation of apparent improvement in symptoms of patients with rheumatoid arthritis who were receiving Prosorba treatment for ITP resulted in a pilot study in which nine of 11 patients with refractory rheumatoid arthritis showed apparent benefit.280 A properly conducted pivotal randomized trial assessed Prosorba vs placebo in 91 patients who had failed to respond to methotrexate or at least two other second-line drugs.281 Efficacy was assessed 7 to 8 weeks after completion of 12 weekly true or sham treatments. An independent data safety monitoring board stopped the trial after a planned interim analysis because 31.9% of the 47 patients in the Prosorba arm reached the defined therapeutic endpoint while only 11.4% of the 44 sham-treated patients did (p
0.019). Subsequent reported experience was consistent with the study findings.282 As it happened, Prosorba was introduced almost simultaneously with a new generation of anti-rheumatic drugs (including leflunomide, enteracept, and infliximab) and these quickly overshadowed Prosorba in the management of severe rheumatoid arthritis.283,284 Although it has been suggested

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that Prosorba may be preferable for patients in whom chronic infection renders potent immunosuppressive agents less desirable,285 its market withdrawal renders the issue moot, at least in the United States.

Coagulation Factor Inhibitors Alloantibody inhibitors to coagulation factors may arise in approximately 20% of patients with severe hemophilia A (Factor VIII deficiency) and 12% of patients with severe hemophilia B (Factor IX deficiency).286 The risk may be higher in patients treated solely with recombinant, as opposed to plasma-derived, clotting factor replacement.287 Autoantibody inhibitors, most typically directed against Factor VIII (“acquired hemophilia”) occur at a rate of approximately one person per million per year, and 40% to 50% of cases are seen in association with other conditions including pregnancy, autoimmune disorders, hematologic or other malignancies, and use of drugs such as antibiotics or anticonvulsants.288 Nilsson289 first reported the use of protein A-Sepharose (Sigma-Aldrich Co., St. Louis, MO) adsorption, in combination with immunosuppression, in the management of a patient with hemophilia B and a high-titer Factor IX inhibitor. A number of case reports and small case series have since reported successful lowering of inhibitor titer using the Prosorba, Immunosorba, or TheraSorb-Ig [sheep antihuman immunoglobulin coupled to Sepharose (Miltenyi Biotech GmbH, Bergisch Gladback, Germany)] columns in patients with congenital and acquired hemophilia.290-294 In these procedures, approximately 2.5 to 3.0 plasma volumes are processed at up to 50 mL/minute (plasma flow rate), and a series of one to six procedures is performed at 1- to 2-day intervals, thus lowering inhibitor titers by as much as 90% to 100%.290,294 Patients with high-titer inhibitors must receive concurrent immunosuppressive treatment in order to prevent rebound of the inhibitor after several days or in order to induce tolerance to Factor VIII or IX.288,290 Because of the use of concurrent medication (eg, Factor VIIa, activated prothrombin complex concentrates, large doses of human Factor VIII concentrates, porcine Factor VIII) it is difficult to assess the specific contribution of the apheresis procedure to the achievement of hemostatic control in reported cases.288 Because of this, and in the absence of clinical trial data, ASFA has designated management of coagulation factor inhibitors a Category III indication for immunoadsorption apheresis (paucity of data).1,6 Immunoadsorption may be useful as an adjunct to early management of patients with inhibitors who are hemorrhaging or who need surgery too emergently to await the outcome of immunosuppressive therapy and who do not respond adequately to factor replacement therapy.290,292-294 And as stated earlier, the removal of the relevant devices from the US market renders the issue moot in the United States. Dilated Cardiomyopathy Dilated cardiomyopathy (DCM) describes a cardiac disorder in which the left ventricle is dilated and exhibits impaired contraction.

Chapter 43: Specialized Therapeutic Hemapheresis and Phlebotomy

It may be idiopathic, familial, post-viral, alcohol- or drug-induced, or related to other cardiac disease. Both ventricles may be affected. It presents with heart failure, often progressive, and may be complicated by arrhythmias, thromboembolic events and sudden death.295 It is the most frequent antecedent cause of heart transplantation throughout the world.239 Approximately 60% to 80% of patients with DCM harbor autoantibodies directed against cardiac myosin heavy chain, myocardial β1-adrenergic receptors, or other cardiac tissue with a predominance of antibodies of the IgG 3 subclass.296-301 Antibodies directed against cardiac tissue targets, similar to those involved in DCM, induce structural and functional changes consistent with DCM in animal models.302,303 The potential relevance of heart antibodies to clinical DCM was explored in a pilot study of eight patients with DCM and advanced congestive heart failure who were subjected to a series of four or five immunoadsorption procedures per week over 2 weeks using the Therasorb-Ig system.304 β1-adrenoreceptor autoantibody levels decreased and heart failure symptoms improved in seven of the subjects, but the effect was transient: autoantibodies and symptoms were restored to baseline 75 days after completion of immunoadsorption.304 In a companion pilot trial, nine patients were subjected to daily immunoadsorption treatments for 5 days using Therasorb-Ig.305 Patients received 35 g of intravenous polyclonal immunoglobulin after the final procedure. Once again, plasma levels of β1-adrenoreceptor autoantibody were substantially reduced and significant improvement in hemodynamic parameters (including cardiac output, mean arterial pressure, mean pulmonary arterial pressure, left ventricular filling pressure, and systemic vascular resistance) was demonstrated, thus providing objective preliminary evidence of the clinical benefit of antibody removal by immunoadsorption.305 Immunoadsorption was also shown to diminish histologic evidence of myocardial inflammation in another cohort of 25 patients with DCM.306 In a randomized, prospective but nonblinded trial, 18 patients with DCM and advanced heart failure were randomly assigned to either best medical therapy (control group) or best medical therapy with the addition of immunoadsorption.307 During the first course of immunoadsorption, procedures were performed on 3 consecutive days and patients received 0.5 g/kg of polyclonal IgG by intravenous infusion. Three subsequent courses of immunoadsorption, performed on 2 consecutive days at 4-week intervals were also followed by infusions of intravenous IgG at the same dose. Imunoadsorption was performed using the Therasorb-Ig column. After the first course of immunoadsorption/IgG, left ventricular ejection fraction, cardiac index, stroke volume index, and systemic vascular resistance improved significantly in the treatment group and these changes remained evident after the final series. Improvement in symptoms and functional status paralleled the hemodynamic changes. The control group demonstrated no hemodynamic improvement at the end of the 3-month study. β-adrenergic receptor antibodies decreased by 80% after the first course of immunoadsorption but tended to rise between monthly courses of treatment.

Although the studies described above demonstrated a response to immunoadsorption in patients with DCM and severe heart failure, subjects in those studies received a cumulative dose of intravenous IgG equal to that used in another study in which patients with DCM and advanced heart failure received a single course of 2 g/kg of intravenous IgG and showed persistent hemodynamic and functional improvement for a year of follow-up.308 Two randomized, blinded, placebo-controlled trials yielded conflicting results regarding IgG infusion in DCM. One trail, in which patients initially received a total of 2 g/kg followed by 0.4 g/ kg monthly for 5 months, demonstrated significant improvement in left ventricular ejection fraction in the treatment group but not the placebo group.309 A second trial, in which patients received a single course totaling 2 g/kg demonstrated no effect of intravenous IgG on left ventricular ejection fraction after 6 or 12 months (although both the treatment and the placebo group showed improvement).310 In a pilot study of four patients with DCM who received immunoadsorption with Immunosorba columns, procedures were performed daily for 5 consecutive days and no intravenous IgG was given.311 Total IgG decreased by 95% and IgG3 decreased by 61%. Functional parameters including mean left ventricular ejection fraction, left ventricular endsystolic and diastolic volumes, and global end-systolic strain improved but not to a statistically significant degree. However, a quality of life assessment demonstrated significant improvement at 6 months.311 ASFA has designated DCM a Category P (pending) indication for immunoadsorption apheresis.1,6

Selective Extraction of Low-Density Lipoproteins Background Familial hypercholesterolemia (FH), a dominantly inherited disorder, is a major cause of death or early disability resulting from premature atherosclerotic heart and peripheral vascular disease.312,313 Autosomal dominant FH is caused by mutations in the gene for the LDL receptor, with frequencies of 1:500 for heterozygotes and 1:1,000,000 for homozygotes.313 Clinical features including xanthomas, xanthelasmas, corneal arcus, and the occurrence of coronary heart disease, stroke, and death are common in the fourth or fifth decade of life.312,313 A serum LDL-cholesterol level below 100 mg/dL (achieved through diet, lifestyle modification, and 3-hydroxy-3methylglutaryl coenzyme A reductase inhibiting drugs or statins) can lower cardiovascular morbidity and mortality in high-risk patients, but a sizable minority of high-risk patients fail to meet LDL-cholesterol-lowering goals by this approach.314,315 Manual, then automated, plasmapheresis was successfully employed as an adjunct to lipid-lowering therapy beginning over 40 years ago. However, the kinetics of restoration of plasma lipid levels and the unwanted lowering of essential plasma proteins (eg, fibrinogen, albumin) rendered this approach somewhat impractical for long-term therapy.312,316 The development of devices that permit a more selective removal of plasma LDLcholesterol and related substances has provided a practical apheresis-based approach to managing statin-resistant patients

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in conjunction with statins and other medical therapies.316 Two apheresis systems for selective removal of LDL-cholesterol are FDA-approved for use in the United States.317,318 ASFA has designated FH a Category I indication for LDL apheresis in homozygotes and a Category II indication for LDL

Table 43-11. FDA-Approved Indications for Use of LDL Apheresis to Acutely Lower Plasma LDL-Cholesterol Levels in Certain High-Risk Patient Populations317,318 Patient Group

Description*

Group A

Functional hypercholesterolemic homozygotes with plasma level of LDL-cholesterol 500 mg/dL Functional hypercholesterolemic heterozygotes with plasma level of LDL-cholesterol 300 mg/dL Functional hypercholesterolemic heterozygotes with plasma level of LDL-cholesterol 200 mg/dL and documented coronary heart disease†

Group B Group C

*Stated LDL-cholesterol levels defined as the mean value of three serum samples obtained during a 2- to 4-week period after a 6-month trial of an American Heart Association Step II diet (or equivalent)314 and maximum tolerated combination drug therapy defined as an adequate trial of drugs from at least two separate classes of hypolipidemic agents (eg, anionic exchange resins/bile acid sequestrants, HMG-CoA reductase inhibitors (statins), fibric acid derivatives, or niacin/nicotinic acid). †

Documented coronary heart disease defined as having one or more of the following: ● Prior documented myocardial infarction ● Prior coronary artery bypass graft surgery ● Prior percutaneous transluminal coronary angioplasty with or without atherectomy or coronary artery stent placement ● Significant angina pectoris documented by thallium or other heart stress test LDL
low-density lipoprotein; HMG-CoA
3-hydroxy-3 methylglutaryl coenzyme A.

apheresis in heterozygotes.1,6 The eligibility criteria for patients referred for LDL apheresis, according to the conditions of FDA approval of the devices, are presented in Table 43-11.317,318 A regimen that combines medical therapy with LDL apheresis on a biweekly schedule can effectively lower LDL-cholesterol by 60% to 80% in otherwise treatment-resistant patients, improve the physical stigmata of hypercholesterolemia such as xanthomas and xanthelasma, improve myocardial perfusion and coronary artery patency, and favorably affect other markers of cardiovascular risk (eg, triglycerides, fibrinogen, homocysteine, C-reactive protein, adhesion molecules).312,319-324 A summary of LDL apheresis systems is provided in Table 43-12.

LDL Apheresis Devices Approved by the FDA The Liposorber LA-15 system317 (Kaneka Pharma America, New York, NY) uses dextran sulfate bound to cellulose to selectively extract LDL-cholesterol from plasma. In this system, plasma is initially separated from the cellular components of blood by filtration through a disposable semipermeable polysulfone hollow fiber column, and the separated plasma is then perfused over a disposable adsorption column that contains 150 mL of dextran sulfate. The dextran sulfate has strong affinity for lipoproteins that contain apolipoprotein B [eg, LDL-cholesterol, VLDL-cholesterol, lipoprotein (a)] and adsorbs these from the plasma. The system has two adsorption columns connected in parallel to the tubing set, each capable of binding 7.1 g of LDL. One column is used to process 500 mL of plasma, and then the plasma is diverted to the second column, allowing for regeneration of the first column with a hypertonic saline solution that desorbs the bound lipoproteins from the dextran sulfate. The alternate use and regeneration of the two adsorption columns is controlled by a microprocessor. The H.E.L.P. system318 (B. Braun Medical, Bethlehem, PA) employs a 0.55-micron hollow fiber column to separate the plasma from the cellular elements of the blood. The plasma is

Table 43-12. LDL Apheresis Devices317,318,323-326 Device

Principle of Operation

Anticoagulation

*Liposorber LA-15 (Kaneka)

Electrostatic adsorption of apolipoprotein-B-containing lipoproteins to negatively charged immobilized dextran sulfate

Heparin

H.E.L.P. (B Braun)

Precipitation of apolipoprotein-B-containing lipoproteins from acidified plasma by addition of heparin

Heparin

*Direct Adsorption of Lipoproteins (DALI, Fresenius)

Adsorption of apolipoprotein-B-containing lipoproteins directly from whole blood perfused over negatively charged polyacrylate-coated polyacrylamide beads

Heparin and/or ACD-A

*Liposorber D (Kaneka)

Adsorption of apolipoprotein-B-containing lipoproteins directly from whole blood perfused over dextran sulfate covalently bound to cellulose

ACD-A

Therasorb-Ig (Miltenyi Biotech)

Adsorption of plasma lipoproteins using sheep antihuman-apolipoprotein B immobilized on Sepharose gel

Heparin

Lipopak (Pocard)

Adsorption of plasma lipoproteins using sheep antihuman-Lp(a) bound to Sepharose

Heparin

*Perfusion of plasma over negatively charged surfaces may result in excessive bradykinin generation and atypical or anaphylactoid-reactions in patients who are taking angiotensin-converting enzyme inhibitor drugs.327-329

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Chapter 43: Specialized Therapeutic Hemapheresis and Phlebotomy

acidified with 0.3M sodium acetate buffer and heparin is added to precipitate LDL-cholesterol. The LDL-cholesterol/heparin precipitate is filtered from the plasma using a 0.45-micron polycarbonate filter, excess heparin is adsorbed from the filtered plasma with a DEAE cellulose membrane filter, and the filtered plasma is then restored to physiologic pH by bicarbonate hemodialysis.

Rheopheresis Rheopheresis is a term that describes the therapeutic application of double filtration plasmapheresis to clinical disorders whose pathophysiology is believed to involve compromised microcirculatory blood flow.330

Background Rheopheresis is an apheresis technique in which whole blood is first pumped through a polyethylene hollow fiber membrane column [Plasmaflo OP-05W(L), Asahi Kasei Medical, Tokyo, Japan] which separates plasma from the formed elements of the blood. The plasma is then passed through a hollow fiber membrane column (SR-20 polysulfone, or AR-2000 cellulose diacetate, both from Asahi Kasei Medical) that filters large molecules, including LDL-cholesterol, fibrinogen, α2-macroglobulin, IgM, and von Willebrand factor from the plasma, resulting in a 50% to 60% decrease in their plasma levels after one plasma volume is processed.330,331 Clinical conditions in which rheopheresis therapy is under investigation include critical limb ischemia,332 sudden sensorineural hearing loss,333 and ischemic diabetic foot syndrome.334 Rheopheresis has also been used to remove excess plasma phytanic acid in patients with Refsum’s disease, an autosomal recessive disorder caused by mutations of phytanolyl/pristanoyl-CoA-hydroxylase.335 But, arguably, the most important application of rheopheresis, at present, is in the treatment of dry age-related macular degeneration.331,336 Rheopheresis in Dry Age-Related Macular Degeneration In 2002 the World Health Organization considered 161 million people worldwide to be visually impaired, 37 million of whom were blind. Age-related macular degeneration (AMD) was identified as the primary cause of blindness in developed countries, accounting for at least 50% of blindness in North America, northern Europe, and Australia, and the third leading cause worldwide.337 Based on demographic data from the 2000 US Census, over 50% of blindness among Americans of European heritage was a result of AMD, a proportion expected to increase as the American population ages.338 The phrase “age-related” reflects the increasing risk of developing AMD after age 60. Over 13 million Americans above age 40 have signs or symptoms of macular degeneration, and 10% have the advanced “wet” stage of the disease.339,340 Current hypotheses regarding the pathophysiology of AMD consider diffusion of nutrients across Bruch’s membrane, and the rheologic characteristics of blood flow through the choriocapillaris, to be deficient.341,342 In part, these circumstances might result from a failure in the regulation of inflammation, as suggested by the strong association between macular degeneration

and inherited polymorphisms in the gene for complement factor H,342-347 and the association of macular degeneration with elevations of C-reactive protein.348,349 Therefore, rheopheresis would both improve microcirculatory flow in the choriocapillaris and across Bruch’s membrane, and remove proinflammatory complexes from the blood, in the treatment of AMD.342 Based on promising early results from pilot studies in Germany350 and the United States,341,342 a properly powered prospective, randomized, multicenter, double-masked, placebo-controlled, Phase III trial of rheopheresis in dry AMD was conducted in 13 clinical centers in the United States.351,352 The Multicenter Investigation of Rheopheresis for Age-Related Macular Degeneration (MIRA-1) trial randomly assigned patients to receive eight actual or sham rheopheresis treatments over a 10-week treatment period and tracked the change in best corrected visual acuity through 12 months after enrollment. At the conclusion of the study, 37% of study subjects in the treatment group, and 29% in the placebo group, were found to either not have met enrollment criteria or to have significantly violated the study protocol after enrollment, resulting in the absence of a statistically significant treatment effect in the final analysis of study outcome data.352 Although a post hoc analysis that excluded the protocol violators indicated a significant treatment effect, the size of the study population evaluable for that analysis did not provide sufficient statistical power for conclusive interpretation of the results.352 The study’s sponsor, Occulogix, Inc., a Toronto-based company, obtained permission from the FDA to conduct a follow-up study, RHEO-AMD, in order to definitively settle the issue of the efficacy of rheopheresis in AMD.353 In 2007 Occulogix suspended the RHEO-AMD trial because of financial considerations; thus, the future development of the technology for the treatment of dry AMD in the United States is uncertain.354 Nonetheless, dry AMD is designated by ASFA as a Category P (pending) indication for double filtration plasmapheresis.1,6

Conclusion The continued growth of therapeutic apheresis as a treatment option in diverse clinical conditions depends on an understanding of the pathophysiology of the disorders in question and the acquisition of evidence, from properly conducted clinical studies, of the efficacy of apheresis therapies in their management.

Disclaimer The author has disclosed no conflicts of interest.

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337. Resnikoff S, Pascolini D, Etya’ale D, et al. Global data on visual impairment in the year 2002. Bull World Health Organ 2004;82:844-51. 338. Congdon N, O’Colmain B, Klaver CC, et al. Causes and prevalence of visual impairment among adults in the United States. Arch Ophthalmol 2004;122:477-85. 339. Friedman DS, O’Colmain BJ, Muñoz B, et al. Prevalence of agerelated macular degeneration in the United States. Arch Opthalmol 2004;122:564-72. 340. Klein R, Klein BE, Knudtson MD, et al. Fifteen-year cumulative incidence of age-related macular degeneration: The Beaver Dam Eye Study. Opthalmology 2007;114:253-62. 341. Pulido JS, Sanders D, Klingel R. Rheopheresis for age-related macular degeneration: Clinical results and putative mechanism of action. Can J Opthalmol 2005;40:332-40. 342. Pulido J, Sanders D, Winters JL, et al. Clinical outcomes and mechanism of action for rheopheresis treatment of age-related macular degeneration (AMD). J Clin Apher 2005;20:185-94. 343. Klein RJ, Zeiss C, Chew EY, et al. Complement factor H polymorphism in age-related macular degeneration. Science 2005;308:385-9. 344. Haines JL, Hauser MA, Schmidt S, et al. Complement factor H variant increases the risk of age-related macular degeneration. Science 2005;308:419-21. 345. Edwards AO, Ritter R 3rd, Abel KJ, et al. Complement factor H polymorphism and age-related macular degeneration. Science 2005;308:421-4. 346. Conley YP, Thalamuthu A, Jakobsdottir J, et al. Candidate gene analysis suggests a role for fatty acid biosynthesis and regulation of the complement system in the etiology of age-related maculopathy. Hum Mol Genet 2005;14:1991-2002. 347. Narayanan R, Butani V, Boyer DS, et al. Complement factor H polymorphism in age-related macular degeneration. Opthalmology 2007;114:1327-31. 348. Seddon JM, Gensler G, Milton RC, et al. Association between Creactive protein and age-related macular degeneration. JAMA 2004;291:704-10. 349. Schaumberg DA, Christen WG, Kozlowski P, et al. A prospective assessment of the Y402H variant in complement factor H, genetic variants in C-reactive protein, and risk of age-related macular degeneration. Invest Opthalmol Vis Sci 2006;47:2336-40. 350. Brunner R, Widder RA, Walter P, et al. Influence of membrane differential filtration on the natural course of age-related macular degeneration: A randomized trial. Retina 2000;20:483-91. 351. Pulido JS. Multicenter prospective randomized double-masked, placebo-controlled study of rheopheresis to treat nonexudative age-related macular degeneration: Interim analysis. Trans Am Ophthalmol Soc 2002;100:85-108. 352. Pulido JS, Winters JL, Boyer D. Preliminary analysis of the final Multicenter Investigation of Rheopheresis for Age Related Macular Degeneration (AMD) Trial (MIRA-1) results. Trans Am Ophthalmol Soc 2006;104:221-31. 353. The Rheopheresis Treatment for Dry Age-Related Macular Degeneration Study (RHEO-AMD Study). Occulogix, Inc., Mississauga, ON Canada. [Available at http://www.rheo.com> resource library>current studies (accessed May 2, 2008).] 354. OccuLogix Suspends RHEO(TM) System Clinical Development Program. Occulogix, Inc., Mississauga, ON Canada. [Available at http://cnrp.ccnmatthews.com/client/occulogix/release.jsp?action For
787504 (accessed November 23, 2007).]

IV

Hazards of Transfusion

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PART I

44

Hemovigilance

Overview of Hemovigilance Dorothy Stainsby,1 Jean-Claude Faber,2 & Jan Jørgensen3 1

National Medical Co-ordinator (Retired), Serious Hazards of Transfusion, Manchester Blood Centre, Manchester, United Kingdom 2 Director Blood Transfusion Service (Retired), Luxembourg Red Cross, Luxembourg, Luxembourg 3 Director Blood Transfusion Centre (Retired), Aarhus University Hospital, Aarhus, Denmark

The term “hemovigilance” has become widely used over the past decade to describe the systematic surveillance of adverse transfusion reactions and events, encompassing the whole transfusion chain and aimed at improving the safety of the transfusion process, from donor to recipient, “vein to vein.”1 The term was coined in France in the early 1990s, has been developed and adopted internationally, and is now an integral part of transfusion practice. Hemovigilance in its broader sense encompasses other important aspects of transfusion safety; surveillance of donors to ascertain the residual risks of transfusion-transmitted infections, traceability of transfused blood components, monitoring of blood utilization, epidemiology of transfused patients, and monitoring of the hazards of blood conservation. Compilation of this information, together with clinical assessment of transfused patients, has the potential to provide much-needed evidence of the outcomes, risks and benefits of blood transfusion, and transfusion alternatives. Plasma derivatives may be included in hemovigilance or may be monitored according to pharmacovigilance. More recently, the concept of “biovigilance”2 emphasizes the need for similar systems for donated tissues and stem cells. This chapter focuses on surveillance systems, their design, what has been learned from them, how this information has been used to improve practice, and how information from other sources can contribute to the interpretation of these data.

Requirements for an Effective Hemovigilance Scheme A World Health Organization guideline on adverse event reporting and learning systems3 emphasized that the effectiveness of an adverse event reporting system is measured not only by Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

accurate collection and analysis of data, but also by its use to make recommendations that improve patient safety. The guideline outlined the following core concepts: ● The fundamental role of patient safety reporting systems is to enhance patient safety by learning from failures of the healthcare system. ● Reporting must be safe. Individuals who report incidents must not be punished or suffer other ill effects from reporting. ● Reporting is of value only if it leads to a constructive response. At a minimum, this entails feedback of findings from data analysis. Ideally, it also includes recommendations for changes in health-care procedures and systems. ● Meaningful analysis, learning, and dissemination of lessons learned require expertise and other human and financial resources. The agency that receives reports must be capable of disseminating information, making recommendations for changes, and informing the development of solutions. These concepts are directly relevant to hemovigilance systems and are applicable both locally and nationally. At the hospital level, there is a need for a point of contact for clinical staff to report reactions and adverse events. This may be a transfusion practitioner or safety officer, hematologist, transfusion medicine specialist, or blood bank manager. There must be a “reporting and learning culture” within which events are used as learning opportunities, but also within a framework of professional competence and accountability; hemovigilance reporting should be an integral part of the risk management or clinical governance framework of the institution. Hemovigilance reporting may be mandatory, as developed in France, or voluntary, as exemplified by the Serious Hazards of Transfusion (SHOT) scheme in the United Kingdom (UK). In both models, active participation is encouraged by educational feedback, and by confidence that centrally submitted data will be used effectively to build an evidence base for recommendations aimed at improvement. Confidentiality of patients, donors, and reporters must be ensured and the scheme must comply with legislative requirements for data protection. At regional or national level, the hemovigilance scheme must be seen as impartial, independent, supportive, and professionally

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credible. Data should be validated, analyzed, and reported within a predictable timeframe, and published in a format that can be used to support education and training. Active involvement and “ownership” by professional bodies will help to ensure that recommendations are incorporated into clinical practice. Collection of complete data on adverse reactions and events requires local awareness and vigilance, and a robust reporting system that is easily understood and user friendly. The scope of the system must be clear to reporters, definitions must be unambiguous, and the system must be sufficiently flexible to respond to developments and changes in transfusion practice.

The Scope of Hemovigilance In discussing the scope of hemovigilance, the terms agreed upon by the Haemovigilance Working Group of the International Society of Blood Transfusion (ISBT) (P Robillard, personal communication) are used here. An adverse event is an undesirable and unintended occurrence before, during, or after transfusion of blood or blood components that may be related to the administration of the blood or component. It may be the result of an error or an incident and it may or may not result in an adverse reaction in the recipient. An incident is when the patient receives a blood component that did not meet all the requirements for a suitable transfusion for that patient, or was intended for another patient. It thus includes transfusion errors and deviations from standard operating procedures or hospital policies. It may or may not result in an adverse reaction. A near miss is an error or deviation from standard procedure or policy that is discovered before the start of the transfusion. An adverse reaction is an undesirable response or effect in a patient temporally associated with the administration of blood or blood components. It may be the result of an incident or an interaction between the recipient and blood, a biologically active material. It is then necessary to determine the severity of the reaction, and the imputability, ie, the degree to which, on completion of investigation, the transfusion is considered to be the cause of the reaction. The ISBT Working party scales of severity and imputability may be found in Appendix 44-1 at the end of this chapter. Adverse reactions occurring during or within 24 hours after transfusion are likely to be recognized as being transfusion related, but the longer the interval between the transfusion and the reaction, the less likely the reaction is to be identified and the causation determined. Continuing education of clinicians responsible for transfused patients is needed to improve awareness of transfusion reactions and ensure that they are investigated and reported appropriately.

reactions they wish to have reported to them. There is some commonality; all established systems would expect to receive reports of severe allergic or anaphylactic reactions, acute and delayed hemolytic reactions, transfusion-transmitted infections (TTIs), transfusion-related acute lung injury (TRALI), transfusion-associated graft-vs-host disease (TA-GVHD), and posttransfusion purpura (PTP). These severe reactions are uncommon, and a hemovigilance scheme such as SHOT that restricts its scope to serious reactions receives approximately 10 such reports per 100,000 blood components issued by the blood services.4 Most schemes invite reports of transfusion-associated circulatory overload (TACO), the frequency and importance of which are increasingly widely recognized.5 Many hemovigilance systems also encompass less severe reactions, eg, febrile nonhemolytic transfusion reactions (FNHTRs), and mild allergic reactions presenting with only mucocutaneous symptoms and signs. Some systems also receive reports of alloimmunization, ie, the finding of a new clinically significant red cell antibody in a previously transfused patient but without clinical or laboratory signs of hemolysis. Minor reactions are common and, although they may be distressing for the patient, they require minimal clinical intervention. However, they produce a large workload if they are required to be reported to the hemovigilance system.6 It may be argued that such comprehensive reporting is excessively time-consuming and cannot be clinically justified; however, becasuse the observed rate of FNHTRs is known to be 0.5% to 1% of red cell transfusions overall,7 the number of these reports from an individual institution can provide a useful indicator of active participation in hemovigilance, and enable benchmarking between hospitals (P Robillard, personal communication). Although clear definitions of adverse reactions are necessary to ensure consistent reporting and enable comparisons, it must also be possible to report previously unrecognized complications that may not fit into one of the recognized definitions.

Adverse Incidents These events, sometimes referred to as “incorrect blood component transfused” (IBCT), result from avoidable system failures throughout the transfusion chain. Experience from the SHOT scheme over a period of 8 years has indicated that the observed rate of such events is 7:100,000 blood components issued, with a fatal outcome in 0.7:100,000.4 Other studies, using different methods, have calculated an error rate of 1:19,0008 and a risk of death caused by an ABO-incompatible transfusion of 1:1,800,000 allogeneic red cell transfusions.9 Detailed analysis of these incidents reveals that in approximately 50% of events there are multiple errors in the transfusion process, that approximately 70% of errors occur in clinical areas (the most frequent error being failure to ensure at the bedside that the right blood is being given to the right patient), and that 30% happen in hospital laboratories.10

What Should Be Reported? Adverse Reactions As will be described in the following section, national hemovigilance systems vary in their scope and in the type and severity of adverse

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Near Misses Near misses include those occasions an error was detected, either by chance or as a result of quality checks, and an incident

Chapter 44: Overview of Hemovigilance

prevented. As in other fields (eg, aviation), the study of near misses in health care can highlight high-risk areas and may also identify effective recovery systems. Near misses are approximately 10 times more frequent than incidents, and again, the work required for comprehensive reporting must be balanced against the potential benefit. These events may enable identification of previously under-recognized errors. For instance, “wrong blood in tube” (ie, pretransfusion blood sample taken from the wrong patient or labeled with the wrong patient’s details) is an infrequent cause of IBCT. However, sampling errors are, in fact, common, accounting for 56% of near-miss reports.4 A structured system for medical event reporting in transfusion medicine (MERS-TM) can identify high-risk areas and processes requiring system change.11 A 3-year pilot project of near-miss reporting in Ireland using this approach demonstrates the value of such an approach.12

Blood Management With the impending threat of blood supply shortages and increasing awareness of the known and unknown risks of transfusion, much emphasis has been placed on alternatives to allogeneic transfusion and conservative transfusion policies. If hemovigilance is to provide a true picture of the risks and benefits of transfusion, then the hazards of blood conservation should also be included. Few systems currently record the risks of blood recovery and reinfusion or adverse reactions to pharmacologic agents. Transfusion safety encompasses the appropriate use of blood components, but requires assessment by clinical audit.13 It is not within the scope of hemovigilance.

Biovigilance A number of commonalities can be recognized between blood transfusion and transplantation of tissues and hemopoietic stem cells. Infectious complications, system failures, and other adverse events may occur in both situations. Although the clinical situations and stakeholder groups may differ, the basic principles are the same, and experience in hemovigilance is of value when designing adverse event reporting and learning systems for tissues and cells. In Europe, legislation for tissues mirrors that for blood, and the development in the United States of new and comprehensive systems for “biovigilance” is awaited with great interest.2

Passive Reporting vs Active Surveillance Passive reporting systems, such as those described here, capture adverse events occurring within a short time following transfusion, provided clinical suspicion is high and an effective reporting mechanism is in place. Active surveillance of transfusion recipients may reveal a different picture, as illustrated by the difference in the incidence of TRALI reported by SHOT as 0.6 per 100,000 blood components issued4 but reported by a single hospital with high clinical awareness as 1 in 8000 blood components issued.14 Similarly, a prospective study of bedside errors found

major errors (including misidentification) in 15 of 3485 units transfused.15 Recognition of delayed complications may also require a different approach. Evidence for transfusion transmission of variant Creutzfeld-Jakob disease (vCJD) has been obtained only by rigorous implementation of active surveillance, involving the tracing of recipients of blood components from donors who were subsequently identified as developing vCJD, and retrospective tracing of blood donors who provided components to patients who developed vCJD.16

Traceability Two-way traceability, from donor to recipient and reverse, is essential for good manufacturing practice and for hemovigilance, and is a requirement of the European Union (EU) Blood Directive17 and the United States Food and Drug Administration (FDA) regulations.18 The ISBT 128 identification system for blood components provides a global standard for identification of human blood, tissue, and organ products to individual donors across international borders and disparate health-care systems, and has been designed and perfected over a 12-year period.19 Blood collectors must be able to trace blood from the donor, through component processing and testing, to the point of issue to the hospital blood bank; thereafter, the hospital blood bank or transfusion service is responsible for documenting that a specific blood component was transfused to an individual recipient, or its final fate if not transfused. Forward immediate traceability is essential if a donor becomes ill with a TTI within the shelf-life of the donation, when prompt treatment of the recipient may preempt the development of clinical illness. In the case of a blood pack fault or test kit failure, a robust traceability system will facilitate rapid recall of components and minimize risk to patients. In the longer term, full two-way traceability is essential for look-back, eg, when a new screening test is introduced20 or a new transfusion-transmissible agent recognized.21

Models of Hemovigilance in Different Countries History Pharmacovigilance was the first surveillance system in the arena of health care, covering medicinal products of all kinds. Hemovigilance, as a separate surveillance system, was first implemented in France in 1994, as required by the updated French regulation on blood in a response to a “blood scandal.” In the UK, anticipation of forthcoming European legislation, together with concerns regarding transfusion safety, led to the establishment of SHOT in 1996. By the end of the 1990s, the concept of hemovigilance was developing across Europe but there was a lack of commonality of definitions, terminology, structure, and scope of reporting. In many countries, development was hindered by organizational

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difficulties, lack of funding, unclear mandates, undefined responsibilities, inadequate training, and inability to make changes in response to reported findings. At the turn of the millennium, hemovigilance was established and functioning in several countries across Europe, although not all were national systems. In some countries hemovigilance was required by law, in some it was officially established but not functioning, and in still others it was established as a voluntary system. Table 44-1 shows the situation in the 15 “old” member states of the EU in 2004. The two national systems in France and the UK represented polarities in European hemovigilance. In France, the hemovigilance system was from the start centralized and nationwide, with a legal requirement for written notification of each and every side effect in relation to a blood transfusion (grades 1 to 4). Between 7000 and 7500 reports are generated annually, with an estimated 2.5 million blood components transfused every year in France.6 In the UK, where hemovigilance was designed as a national, voluntary, professionally led scheme covering transfusion reactions of grades 2 to 4, and IBCT regardless of outcome, the number of notifications is much lower than in France, with 2630 reports received in 8 years, 70% of which were IBCT.4 Subsequently, hemovigilance systems were established in nearly all of the 15 “old” member states of the EU. The Republic of Ireland contributed to SHOT until 1998, when the Irish National Haemovigilance Office was established, again in response to “blood scandals” relating to hepatitis C virus. The Irish program is largely modeled on SHOT, and has included a pilot study of near misses.12 In Germany, blood components are considered by law to be medicinal products. As such they fall under German Drug Law, and pharmacovigilance applies to component production.

For the clinical aspects of transfusion (the users of blood components), hemovigilance applies and there is a major challenge in linking these surveillance activities together. In Austria, the situation is comparable to Germany, although surveillance of collection, preparation, and transfusion of blood components are part of hemovigilance. In the Benelux countries (Belgium, the Netherlands, and Luxembourg), small surveillance systems were established; initially these were fragmented and lacking in coordination. The Transfusion Reactions in Patients (TRIP) scheme in the Netherlands is now well established and produced its first annual report in 2003.22 A major contribution of the Benelux countries lies in their leading role in creating and developing the European Haemovigilance Network (EHN). In Denmark, hemovigilance was initially established on a local basis, until the creation of the Danish Registration of Transfusion Risks (DART).23 The Danish system was the first to introduce structured donor hemovigilance, and the importance of donor adverse reactions was emphasized from the outset. The Danish model has largely influenced donor hemovigilance in other European countries, as now required by the EU Blood Directives. In Greece, there is a long tradition of hemovigilance and, despite the complex structure of the Greek transfusion system because of geographic and political factors, these surveillance activities have generated interesting data.24 In Spain, impressive progress has been made recently, showing that, even in a complicated political environment, professionals from the field who have a strong will to bring hemovigilance forward can achieve significant results in a short period. For the 12 “new” member states (joining the EU in May 2004), little is known about national hemovigilance activities, but this will change quite rapidly as these countries work toward compliance with the EU Blood Directive.

Table 44-1. Hemovigilance in Europe in 2004 before Implementation of the European Union Blood Directive Member State

Status of Hemovigilance

Mandatory or Voluntary

Responsible Organization

Austria Belgium Denmark Finland France Germany Greece Ireland Italy Luxembourg Netherlands Portugal Spain Sweden United Kingdom

Started in 2003 Established/functioning Established/functioning Established/functioning Established/functioning Established/functioning Established/functioning Established/functioning In development Established/functioning Started in 2003 In development Started in 2004 In development Established/functioning

M M V V M M V V V/M V V M? V V/M V

Österreischiches Bundes-Institut fur Gesundheit Red Cross Danish Registry for Adverse Reactions in Transfusion Red Cross Agence Français de Sécurité Sanitaire des Produits de Santé Paul Ehrlich Institute Hellenic Co-ordinating Haemovigilance Centre National Haemovigilance Office Instituto Superiore de Sanita ⫹ Regions Red Cross ⫹ Ministry of Health Transfusion Reactions in Patients Instituto Portugues do Sangue ⫹ Ministry of Health Programa Estatal de Hemovigilancia ⫹ Regulator Swedish Association for Transfusion Medicine ⫹ Hospitals Serious Hazards of Transfusion

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The EU Blood Directives and the Future of European Community Hemovigilance Blood Directives The key role of hemovigilance in blood transfusion safety is reflected in the EU Blood Directive 2002/98/EC.17 This Directive of the European Parliament and of the Council of Europe sets standards of quality and safety for the collection, testing, processing, storage, and distribution of human blood and blood components and amends Directive 2001/83/EC. An entire chapter is dedicated to hemovigilance, and encompasses traceability and notification of serious adverse events and reactions. According to the Directives on blood, hemovigilance is defined as “a set of organized surveillance procedures relating to serious adverse or unexpected events or reactions in donors or recipients, and the epidemiologic follow-up of donors.” It is stated that “it is important to introduce a set of organized surveillance procedures to collect and evaluate information on the adverse or unexpected events or reactions resulting from the collection of blood or blood components in order to prevent similar or equivalent events or reactions from occurring thereby improving the security of transfusion by adequate measures. To this end a common system of notification of serious adverse events and reactions … should be established in member states.” The legal framework on blood at the European Community level comprises four Directives: ● 2002/98/EC (Directive on principles and organization) ● 2004/33/EC (Directive on donations, donors, and blood components) ● 2005/61/EC (Directive on traceability and notification— hemovigilance) ● 2005/62/EC (Directive on a quality management system). The Directives lay down standards of quality and safety of human blood and of blood components, in order to ensure a high level of human health protection. These are minimum standards, and do not require harmonization of activities related to blood transfusion in the member states of the EU. In the context of blood matters, including hemovigilance, it is important to understand that the European Treaty (treaty of Amsterdam, establishing the European Community) sets limits in Article 152 that restrict the Directives on blood to collection and testing of human blood and blood components, whatever their intended purpose, and their processing, storage, and distribution when intended for transfusion. However, the Directives do not apply to clinical activities. Hence, although hemovigilance (including rapid alert) should encompass the whole blood chain from donor to recipient and vice versa, including production and clinical use, current legislation can cover only the producers (the blood establishments) and to some extent the hospital blood banks. It cannot include users and their clinical activities, which are the exclusive responsibility of the individual member states. This obviously creates a complex situation in Europe with regard to hemovigilance systems. In Chapter V of Directive 2005/61/EC, on hemovigilance, there are legally binding requirements for traceability (Article 14) and for the notification of serious adverse events and

reactions (Article 15). In the definition of “hemovigilance” it is stated that the organized surveillance procedures should relate to serious adverse or unexpected events or reactions in donors or recipients, and to the epidemiologic follow-up of donors. The Directive requires reporting of only serious adverse or unexpected events or reactions. A “serious adverse event” is defined in the Directive as any untoward occurrence associated with the collection, testing, processing, storage and distribution of blood and blood components that might lead to death or life-threatening, disabling, or incapacitating conditions for patients or that results in, or prolongs, hospitalization or morbidity. A “serious adverse reaction” is defined in the Directive as an unintended response in a donor or patient associated with the collection or transfusion of blood or blood components that is fatal, life-threatening, disabling, incapacitating, or that results in, or prolongs, hospitalization or morbidity. In the definition of “hemovigilance” it is required that organized surveillance cover both donors and recipients. The principle of subsidiarity allows other issues to be determined by individual EU member states, which may adopt regulations in addition to the provisions in the Blood Directives. However, the Directives must be transposed into national legislation by the member states.

European Hemovigilance Some years ago the need for EU-wide cooperation was anticipated when several countries decided to create a network for hemovigilance. The EHN was founded in 1998 by representatives from Belgium, France, Luxembourg, the Netherlands, and Portugal; these countries were later joined by Austria, Denmark, Finland, Greece, Ireland, Malta, Slovenia, Spain, and United Kingdom. The boundaries were more recently extended to include Canada, Croatia, Iceland, New Zealand, Norway, Singapore, and Switzerland. Further expansion is likely in future. The EHN is conceptually a bottom-up organization of professionals from the field and includes different specialties: transfusion medicine, hematology, internal medicine, and civil servants. The aim of the EHN is to develop and maintain in Europe a common structure with regard to safety of blood and blood components and hemovigilance in blood transfusion and transfusion medicine. To this end, the EHN pursues the following objectives: ● Favor exchange of valid information between members. ● Increase rapid alert and early warning between members. ● Encourage joint activities between members. ● Undertake educational activities relating to hemovigilance. To fulfill these objectives, the EHN has: ● Developed and maintained a website (www.ehn-org.net). ● Established an on-line system for rapid alert and early warning. ● Worked toward standard definitions relevant to hemovigilance, in collaboration with ISBT. ● Initiated standardization of processes and forms. ● Started to compile, analyze, and compare data generated by the national hemovigilance systems. ● Organized annual European Haemovigilance Seminars. 687

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The Rapid Alert system (RAS) was established and has been maintained by the EHN as a high priority from the outset, providing a framework for the international transmission of information on unexpected adverse reactions or equipment defects. It is especially useful for those countries where no previous system existed. A designated official contact person (OCP) in each country is responsible for the transmission and receipt of information, and coordinates with the blood services regarding appropriate corrective or preventive action to maintain or improve safety. The EHN-RAS is a network of OCPs who are nominated or nationally agreed-upon individuals, responsible for entering, retrieving, and filtering information, and onward transmission if appropriate. The EHN-RAS has been used to disseminate information on newly recognized transfusion reactions (eg, red-eye syndrome), adverse reactions in donors (eg, allergic or anaphylactic reactions associated with apheresis devices), defects in disposables, problems with equipment or information technology, and deficiencies in reagents. In the first 5 years more than 30 alerts were launched and disseminated to participating countries and organizations, and the RAS has developed with increasing cooperation of manufacturers. There remains scope for further development. In Europe it was recognized quite early that hemovigilance has an important role. With the impact of the Directives on blood, national hemovigilance systems in Europe will converge to some degree, but will remain autonomous. The development of EHN has demonstrated that international cooperation at a professional expert level is possible, and can be effective in improving the safety and quality of blood transfusion. It is encouraging that newly established hemovigilance schemes worldwide are seeking to maintain and further develop established professional links, and EHN works closely with the Haemovigilance Working Group of the ISBT.

Development of Hemovigilance Worldwide An international forum in 2006 indicated the extent to which hemovigilance is becoming an integral and important part of transfusion practice worldwide.25 Twenty-four countries responded to a survey; all but a few had a full hemovigilance system in place. The Japanese Red Cross has had a comprehensive hemovigilance system since 1993, and data are now coming from New Zealand, Russia, the Czech Republic, Slovakia, Poland, South Africa, and Brazil, as well as established systems in Europe, Scandinavia, and Canada. Work is in progress to develop hemovigilance in the US, Australia, and North Africa. Comparisons of observed incidence of adverse reactions between countries with different practices can assist risk/benefit analysis and policy decision making. For example, in Norway, where there is 100% use of solvent/detergent-treated plasma (Octaplas, Octapharma, Lachen, Switzerland), the frequency of adverse events observed with plasma is low in comparison with that of other countries using Fresh Frozen Plasma (FFP).25

688

If comparative data are to be useful, like must be compared with like or, at the very least, differences in methodology must be recognized and understood. To this end the Haemovigilance Working Group of the ISBT is developing consensus definitions of adverse incidents in recipients and donors. Denominator data are needed for interpretation of hemovigilance findings in the context of transfusion practice. The number of reported incidents is meaningful only when expressed against a denominator of blood components transfused. However, in many countries, including the UK, the only accurate figure available is the number of components issued by the blood collectors, with little or no reliable data on the number of components actually transfused, the number of transfusion episodes, or transfused patients. The question also arises as to whether the denominator should include blood components issued to hospitals that do not report incidents, as is done by SHOT in the UK, or should be restricted to components issued to actively participating hospitals, as in Canada (P Robillard, personal communication). Epidemiologic studies are beginning to cast light on the clinical situations in which blood is used and the demographic profile and survival of transfused patients.26-28 This will enable the future development of more sophisticated denominator data, identification of high-risk patients and situations, and estimation of the risks of transfusion in an individual patient.

Hemovigilance Related to Blood Donors and Blood Donation Systematic surveillance of the first part of the transfusion chain—the collection of blood from the donor—is an essential element of hemovigilance, and aims to secure and improve the safety of both the donor and the recipient. It should include registration of unexpected adverse events in whole blood and component donors and the action taken as a result. These events may be adverse reactions or complications resulting from donation, or adverse events related to the selection and management of donors, which may directly harm the donor or influence the quality of the product, thereby harming the recipient. Provision of information about the risks of donation based on evidence from surveillance, together with good clinical management of any complications, indicates a high professional standard of the blood collection facility and its care of the wellbeing of the donors. This, in turn, will improve donor confidence and satisfaction, making it more likely that the donor will return, thus benefitting the national supply of blood.29,30 In 2004, awareness of the importance of donor complications was raised by presentations of preliminary data from a pilot study.31,32 A joint working group from the ISBT and EHN was established, and has proposed a classification and a set of definitions of complications related to blood donation (Appendix 44-2), to form the basis for a registry. It is hoped that the use of this registry will enable development of an evidence base to support improvements in donor care and selection.

Chapter 44: Overview of Hemovigilance

Complications Related to Regular Whole Blood Donation Data on the occurrence of these complications are sparse, because most hemovigilance systems have not hitherto included donor adverse events. Large studies have been reported from single blood centers in the United States and in Greece33-38; comprehensive national data have been reported from Denmark39; and data collected with the use of the definitions agreed-upon have been presented at several meetings (Table 44-2). The rate of reported donor complications has varied according to the severity and range of reactions included. Preliminary data registered according to the ISBT classification suggest that the overall rate of complications may be as high as approximately 1 per 100 donations. The most common complications related directly to the bleeding process are vasovagal reactions (2/3) and injuries of soft tissues, vessels (1/3), nerves, or tendons caused by the inserted needle.

Vasovagal Reactions Vasovagal reaction is the most common donor complication (488 per 100,000 donations). The predominant symptoms are general discomfort, weakness, anxiety, dizziness, nausea, sweating, vomiting, pallor, and hyperventilation, associated with hypotension and bradycardia. The two last symptoms are essential for the diagnosis. Most vasovagal reactions are mild and transient, but some donors may lose consciousness (vasovagal syncope). In the more severe cases this may result in an accident, if the donor faints and falls, or may be associated with convulsions and incontinence. It is important to reassure the donor that these reactions do not indicate a predisposition to true epilepsy. Vasovagal reactions are generated by the autonomic nervous system stimulated by psychologic factors, and by the volume of blood removed relative to the donor’s total blood volume. Therefore, donor age, weight, and donation status have been found to be significant variables.40 The risk can be reduced by fluid replacement,41 and by careful management of the donors during their stay in the blood facility. Some vasovagal reactions (⬃10%)39 occur after the donor has left the donation area, so-called delayed reactions. These reactions are potentially dangerous, as the donor may be at risk of a serious accident, and occasional deaths have been reported anecdotally. In order to lower the risk of delayed reactions, and of serious outcome of these, it is essential to ensure that the donor feels completely well before leaving the donation area. Many facilities permanently defer donors following a severe delayed vasovagal reaction. Donors with hazardous occupations where they or others could be put at risk (eg, pilots) should not return to work within 24 hours of donation. Hematomas Hematoma is the second most common complication related to blood donation (275 per 100,000 donations). The symptoms are bruising, swelling, and pain at the venipuncture site. A hematoma may occur if the needle punctures small arterial vessels, or if blood leaks from the vein during or after venipuncture. Blood in the soft tissues behind the biceps tendon will initially spread behind the tendon and may not produce any

visible swelling or pain. As the hematoma increases in size, it tracks along the blood vessels, nerves, and tendons to the forearm, and may cause paresthesiae in the fingers because of compression or irritation of the median nerve. Accidental puncture of a large artery in the antecubital fossa carries a high risk of a hematoma and of delayed bleeding, and other rare but serious complications (Appendix 44-2). Donor care staff should be trained to recognize and correctly manage arterial puncture, so as to avoid extremly rare, but very severe, complications such as compartment syndrome of the forearm. Injury to the median nerve in the antecubital fossa accounts for only 8% of all immediate adverse reactions, but gives rise to nearly all serious long-term complications.39 In approximately one-third of these cases the median nerve is directly damaged by the venipuncture needle.39 The donor may experience immediate severe shooting pain, radiating down the forearm, sometimes associated with paresthesiae in the median nerve distribution of the hand. Indirect nerve injury may be caused by pressure from an increasing hematoma, with symptoms of pain and paresthesiae developing later. Anatomical studies have shown marked individual variation in the arrangement of nerves and blood vessels in the antecubital fossa, and injuries may occur despite good venipuncture technique.42 It is essential however that venipuncturists are familiar with normal anatomy of the region and are trained in correct techniques of needle insertion, together with prompt recognition and correct management of complications. To reduce the risk of direct nerve injury, the needle should be inserted only once and, if unsuccessful, no further attempts should be made. Hematoma should be managed by stopping the donation immediately if the donor complains of symptoms, applying pressure to the venipuncture site, and recommending that the donor rest the arm and avoid manual work for 24 hours. The donor should return to the blood center or seek medical treatment if there is persistent bleeding from the venipuncture site or if the swelling increases.

Long-Term Effects of Regular Whole Blood Donation and Apheresis The surveillance of donors should also include the long-term effects of regular whole blood donation and repeated apheresis. Those of most concern are depletion of iron, calcium, and plasma proteins. The surveillance of repeat donors of whole blood should include at least the measurement of the hemoglobin concentration on each occasion. Programs of intensive plasmapheresis should include monitoring of plasma albumin concentrations.43

Learning from Hemovigilance Effective hemovigilance requires not only accurate and complete collection of data, but also interpretation of the data in the context of what is known of transfusion epidemiology and practice, and use of the data to improve patient safety, both locally and nationally.

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690

Table 44-2. Occurrence of Complications Related to Donation of Whole Blood Donor Complications (serious)†

Occurrence Shown as Number of Cases/100,000 Donations

Donor Complications (all grades)

Study

Donations Surveilled (comments)

Vasovagal Reaction

Hematoma

Nerve Injury

Total

Newman33

1,000 (interview 3 weeks after donation)

6400

1700

900

9000

Newman33

1,000 (information obtained at donation)

2100

324

16

2440

Khan35

5,536 (random)

1210

nd

nd

Newman

34

949 (random)

2600

nd

nd

Newman

32

419,000 (random)

nd

nd

16

Zervou37

12,173 (one center)

879

nd

nd

Sorensen38

2,575,246 (national register* serious complications)

Sorensen38

41,274 (county register* all complications)

Caffrey‡

2,332,439 (national data,* England and N.Wales)

Hauser40

2,196,900 (national data, France)*

Okazaki‡

3,758,712 (national data, Japan)*

Vasovagal Reaction

Hematoma

Nerve Injury

nd

nd

5

7

1

11

19

14

478

274

70

822

1399

36

10

445

9

3

2

5

1

0.4

736

93

7

836

5

2

3

*Data collected in accordance with Standard for Collecting and Presentation of Data on Complications Related to Blood Donation (Version 2007). † Complications with symptoms that lasted for more than a year or that needed medical treatment were graded as serious. ‡ Personal communications.

Total

6 10

Chapter 44: Overview of Hemovigilance

Early SHOT data focused attention on avoidable system failures in the transfusion chain, with the potential for serious patient harm. Acute intravascular hemolysis caused by major ABO incompatibility is the most feared outcome, but transfusion errors can cause patient harm in other ways, such as failure to provide blood of the correct specification for the patient, eg, irradiated cellular components for immunosuppressed patients at risk of TA-GVHD, or D sensitization of D-negative females of childbearing potential. Volume overload or unnecessary exposure to blood components can result from 1) incorrect decision making; 2) prescribing errors such as volume miscalculation; or 3) transfusing based on a misleading hemoglobin level from a sample diluted by intravenous infusion, from the wrong patient, or from a laboratory result that is incorrect or wrongly documented. Over a 10-year period, during which time 30 million blood components were issued from the UK blood services, SHOT received 3770 reports, of which 2717 (72%) were of IBCT (see Table 44-3 and Fig 44-1). Ninety-five percent of these patients survived with no serious effects, but 24 deaths were attributed wholly or in part to avoidable transfusion errors, and 100 patients suffered major morbidity.44 Detailed analysis of these incidents is necessary if lessons are to be learned from them. For example, the SHOT questionnaire

on IBCT elicits the stage(s) of the transfusion chain where the error(s) occurred, and has thus been able to identify that 70% of errors occur in clinical areas, the most frequent error being failure to correctly perform the pretransfusion patient identification check at the bedside.10 Approximately 30% of errors occur in hospital transfusion laboratories10 and detailed knowledge of the circumstances can help to support improvements in standards of practice. Errors in blood sampling for pretransfusion testing are usually detected in the laboratory if the patient is previously known. Data on near misses from SHOT and from the National Haemovigilance Office in Ireland12 have revealed the true frequency of such errors and highlighted the importance of correct patient identification at the point of blood sampling. Information on the time and place of transfusion, and the level of staff involved, is useful—not to apportion blame, but to target education and training and to direct efforts toward improving standards of practice. Such detail will be forthcoming only if reporters are assured that information will be used positively and constructively, and in total confidence. Reports to SHOT, when interpreted in the context of epidemiologic data, suggest that night-time transfusion carries an increased risk45 compared to those administered during the day

Table 44-3. SHOT Cumulative Morbidity/Mortality 1996 to 2006 All Causes

IBCT

ATR*

HTR*

PTP

TA-GVHD

TRALI

TTI

Death definitely attributed to transfusion (imputability 3)

47

7

2

6

1

13

8

10

Death probably attributed to transfusion (imputability 2) Death possibly attributed to transfusion (imputability 1) Subtotal 1

15

4

4

1

0

0

6

0

47

13

7

1

1

0

25

0

109

Major morbidity† probably or definitely attributed 315 to transfusion reaction Minor or no morbidity as a result of transfusion 3324 reaction Outcome unknown 15 Subtotal 2 3654 TOTAL 3763‡

24

13

8

2

13

39

10

100

17

29

13

0

118

38

2582

387

280

31

0

38

6

11 2693 2717

3 107 420

0 310 318

0 44 46

0 0 13

0 156 195

0 44 54

*The HTR category was formerly DTR (delayed transfusion reactions); from 2006, all hemolytic reactions are included in HTR. Major morbidity is classified as the presence of one or more of the following:

†

‡

●

Requirement for intensive care and/or mechanical ventilation.

●

Dialysis or renal impairment.

●

Minor hemorrhage from transfusion-induced coagulopathy.

●

Intravascular hemolysis.

●

Potential D sensitization in a female of childbearing potential.

Excludes seven unclassified cases from 1998/1999.

SHOT ⫽ serious hazards of transfusion; IBCT ⫽ incorrect blood component transfused; ATR ⫽ acute transfusion reaction (included acute hemolytic reactions until 2006); HTR ⫽ hemolytic transfusion reaction (pre-2006 this included only delayed hemolytic transfusion reactions); PTP ⫽ posttransfusion purpura; TA-GVHD ⫽ transfusionassociated graft-vs-host disease; TTI ⫽ transfusion-transmitted infection.

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Section IV: Part I

IBCT (72.1%) 13 195 46

54

ATR (11.1%)

7

HTR (8.4%)*

318 420

PTP (1.2%) TA-GVHD (0.3%) TRALI (1.4%) TTI (0.2%)

2717 3770 cases reviewed

Unclassified (0.2%) *Formerly DTR

Figure 44-1. Analysis of reports to SHOT over the 10-year period 1996. SHOT⫽Serious hazards of transfusion; IBCT⫽Incorrect blood component transfused; ATR⫽Acute transfusion reaction (included acute hemolytic reactions until 2006); HTR⫽ Hemolytic transfusion reaction (pre-2006 this included only delayed hemolytic transfusion reactions); PTP⫽Post transfusion purpura; TA-GVHD⫽Transfusion-associated graftvs-host disease; TTI⫽Transfusion-transmitted infection.

and that transfusion to pediatric patients is more likely to result in an adverse outcome than adult transfusion.46 Successive SHOT reports have led to a number of national safety initiatives including professional guidelines on blood administration,47 a nursing guideline,48 a Safer Practice Notice from the National Patient Safety Agency relating to bedside pretransfusion checks,49 and a Quality Standard on transfusion.50 Hemovigilance data can be enormously valuable for monitoring changes in practice, such as the implementation of universal leukocyte reduction, which in France was noted to result in a reduction in the incidence of FNHTRs,9 and in the UK was found to reduce, but not eliminate, TA-GVHD and alter the pattern of PTP.51 Hemovigilance data have also been used to monitor measures to reduce the risk of bacterial contamination of platelets, and strategies to reduce the incidence of TRALI. Cumulative SHOT data on transfusion-related mortality and morbidity4 revealed the importance of TRALI as the leading cause of such outcomes. These findings are consistent with those from other countries, although hemovigilance data almost certainly underestimate the risk of this controversial and complex complication of transfusion. In institutions with a high index of suspicion of TRALI, its incidence has been estimated as 1:2000 to 1:8000 plasma-containing components.14,52 Reports of TRALI to SHOT remained level at an average of 14 (range 11-19) per year for the first 5 years, but rose to 26 in 2001/2002, and to 36 in 2003, with increasing awareness of the condition. This led, in 2003, to a policy implemented by the UK blood services to obtain plasma, as far as possible, from male donors, to minimize the risk of TRALI caused by passive transfer of HLA or granulocyte antibodies arising in female donors as a result of pregnancy. The success of this policy has been measured by ongoing hemovigilance reporting; the number of cases of TRALI with high imputability reported to SHOT fell from 22 in 2003 to 13 in 2004, to six in 2005, and to three in 2006.44

692

Careful collection and analysis of hemovigilance data in the context of accurate denominator data has enabled risk estimation of transfusion in the developed world, as exemplified by Canada.53 Such data must be made available, in comprehensible format, to doctors prescribing blood and patients receiving it, so that informed decisions can be made regarding risks and benefits, in the short and long term. Hemovigilance systems must be flexible, forward looking, responsive to developments in transfusion practice, and, above all, in touch with clinicians prescribing blood, and the requirements of patients.

Disclaimer The authors have disclosed no conflicts of interest.

References 1. Faber JC. Worldwide overview of existing hemovigilance systems. Transfus Apher Sci 2004;31:99-110. 2. AuBuchon JP, Whitaker BI. America finds hemovigilance! Transfusion 2007;47:1937-42. 3. WHO guidance on adverse event reporting and learning systems. Geneva, Switzerland: World Health Organization, 2005. [Available at http://www.who.int/patientsafety/events/05/Reporting_Guidelines. pdf (accessed May 27, 2008).] 4. Stainsby D, Jones H, Asher D, et al. Serious hazards of transfusion: A decade of haemovigilance in the UK. Transfus Med Rev 2006;20:273-82. 5. Popovsky MA. Circulatory overload. In: Popovsky MA, ed. Transfusion reactions, 3rd ed. Bethesda, AABB Press, MD: 2007:331-9. 6. Rebibo D, Hauser L, Slimani A, et al. The French haemovigilance system: Organisation and results for 2003. Transfus Apher Sci 2004;31:145-53.

Chapter 44: Overview of Hemovigilance

7. Heddle NM. Febrile nonhemolytic transfusion reactions. In: Popovsky MA, ed. Transfusion reactions, 3rd ed. Bethesda, MD: AABB Press, 2007:57-103. 8. Linden JV, Wagner K, Voytovitch AE, et al. Transfusion errors in New York State: An analysis of 10 years’ experience. Transfusion 2000;40:1207-13. 9. Andreu G, Morel P, Forestier F, et al. Hemovigilance network in France: Organization and analysis of immediate transfusion incident reports from 1994 to 1998. Transfusion 2002;42:1356-64. 10. Stainsby D, Russell J, Cohen H, et al. Reducing adverse events in blood transfusion. Br J Haematol 2005;131:8-12. 11. Callum JL, Merkley LL, Coovadia AS, et al. Experience with the medical event reporting system for transfusion medicine (MERSTM) at three hospitals. Transfus Apher Sci 2004;31:133-43. 12. Lundy D, Laspina S, et al. Seven hundred and fifty-nine chances to learn: A 3-year pilot project to analyse transfusion-related near-miss events in the Republic of Ireland. Vox Sang 2007;92:233-41. 13. Wallis JP, Stainsby D, McClelland DBL. Audit of red cell transfusion. Transfus Med 2002;12:1-9. 14. Wallis JP, Lubenko A, Chapman CE, et al. Single hospital experience of TRALI. Transfusion 2003;43:1053-9. 15. Baele PL, De Bruyere M, Deneys V, et al. Bedside transfusion errors. A prospective survey by the Belgian SAnGUIS Group. Vox Sang 1994;66:117-21. 16. Llewellyn CA, Hewitt PE Knight RS, et al. Possible transmission of variant Creutzfeld-Jakob disease by blood transfusion. Lancet 2004;363:417-21. 17. EU Blood Directive 2002/98/EC. (February 8, 2003) Official Journal of European Union, 2003. [Available at http://eur-lex.europa.eu (accessed May 13, 2008).] 18. Food and Drug Administration. Current good manufacturing practice and related regulations for blood and blood components; and “lookback” requirements. Rockville, MD: CBER Office of Communication, Training, and Manufacturers Assistance, 2002. [Available at http://www. fda.gov/ohrms/dockets/98fr/oc02254.pdf (accessed May 13, 2008).] 19. ISBT 128—an introduction 3rd edition. York, PA: ICCBBA, 2006. [Available at http://iccbba.org (accessed August 15, 2008).] 20. English National Blood Service HCV Lookback Collaborators. Probability of receiving testing in a national lookback program. Transfusion 2002;42:1140-5. 21. Hewitt PE, Llewelyn CA, Mackenzie J, Will RG. Creutzfeldt-Jakob disease and blood transfusion: Results of the UK Transfusion Medicine Epidemiological Review Study. Vox Sang 2006;91:221-30. 22. TRIP Annual Report 2003. Den Haag, Netherlands, 2003. [Available at http://www.tripnet.nl/pages/nl/documents/TRIPrapport2003.pdf (accessed May 13, 2008).] 23. DART Annual Report 2006. Arhaus, Denmark, 2006. [Available at http://www.hemovigilance.dk/index_eng.htm (accessed May 13, 2008).] 24. Politis C. International Forum on Haemovigilance. Vox Sang 2006; 90:221-2. 25. Engelfriet CP, Reesink HW. International Forum on Haemovigilance. Vox Sang 2006;90;207-41. 26. Wells AW, Mounter PJ, Chapman CE, et al. Where does blood go? Prospective, observational study of red cell transfusion in north England. Br Med J 2002;325: 803-4. 27. Wallis JP, Wells AW, Chapman CE. Changing indications for red cell transfusion from 2000 to 2004 in the North of England. Transfus Med 2006;16:411-17.

28. Wallis JP, Wells AW, Matthews JNS, et al. Long-term survival after blood transfusion: A population based survey in the north of England. Transfusion 2004;44;1025-32. 29. Popovsky MA. Vasovagal donor reactions: An important issue with implications for the blood supply (editorial). Transfusion 2002;42:1534-6. 30. France CR, France JL, Roussos M, Ditto B. Mild reactions to blood donation predict a decreased likelihood of donor return. Transfus Apher Sci 2004;30:17-22. 31. Samuelsen B, Aagaard B, and Jørgensen J. Occurrences of unusual events in blood donation (abstract). Vox Sang 2004;87(Suppl 3): S2-16. 32. Aagaard B, Samuelsen B, Jørgensen J, et al. Risk and prognosis of blood donation related complications—a nationwide prospective Danish study (abstract). Vox Sang 2004;87(Suppl 3):S17-92. 33. Newman BH, Waxman DA. Blood donation-related neurologic needle injury: Evaluation of 2 years’ worth of data from a large blood bank. Transfusion 1996;36:213-15. 34. Newman BH, Pichette S, Pichette D, Dzaka E. Adverse effects in blood donors after whole-blood donation: A study of 1000 blood donors interviewed 3 weeks after whole-blood donation. Transfusion 2003;43:598-603. 35. Newman BH. Vasovagal reaction rates and body weight: Findings in high- and low-risk populations. Transfusion 2003;43:1084-8. 36. Khan W, Newman BH. Comparison of donor reaction rates in highschool, college, and general blood drives (abstract). Transfusion 1999;36(Suppl):31S. 37. Newman BH, Satz SL, Janowiicz NM, Siegfried BA. Donor reactions in high-school donors: The effect of sex, weight, and collection volume. Transfusion 2006;46:284-8. 38. Zervou EK, Ziciadis K, Karabini F, et al . Vasovagal reactions in blood donors during or immediately after blood donation. Transfus Med 2005;15:389-94. 39. Sorensen B, Johnsen SP, and Jørgensen J. Complications related to blood donation: A population-based study. Vox Sang 2008; 94:132-7. 40. Trouern-Trend JJ, Cable RG, Badon SJ, et al. A case-controlled multicenter study of vasovagal reactions in blood donors: Influence of sex, age, donation status, weight, blood pressure, and pulse. Transfusion 1999;39:316-20. 41. Hanson SA, France CR. Predonation water ingestion attenuates negative reactions to blood donation. Transfusion 2004;44: 924-8. 42. Horowitz SH. Venipuncture-induced causalgia: Anatomic relations of upper extremity superficial veins and nerves, and clinical considerations. Transfusion 2000;40:1036-40. 43. Bechtloff S, Tran-My B, Haubelt H, et al. A prospective trial on the safety of long-term intensive plasmapheresis in donors. Vox Sang 2005;88:189-95. 44. SHOT Steering Group Annual Report 2006. Manchester, UK: SHOT Office, 2007. [Available at http://www.shotuk.org (accessed May 27, 2008).] 45. Tinegate HN, Thompson CL, Stainsby D. Where and when is blood transfused? An observational study of the timing and location of red cell transfusions in the north of England. Vox Sang 2007;93:229-32. 46. Stainsby D, Jones H, Wells AW, et al. Adverse outcomes of blood transfusion in children; analysis of UK reports to the SHOT scheme 1996-2005. Br J Haematol 2008;141:73-9.

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47. British Committee for Standards in Haematology Transfusion Taskforce. The administration of blood and blood components and management of the transfused patient. Transfus Med 1999;9:227-38. 48. Right patient, right blood, right time. London, UK: Royal College of Nursing, 2007. [Available at http://www.rcn.org.uk/_data/assets/ pdf_file/0009/78615/002306.pdf (accessed May 13, 2008).] 49. Safer Practice Notice 14. Right patient, right blood: Advice for safer blood transfusions. London, UK: National Patient Safety Agency, 2006. [Available at http://www.npsa.nhs.uk/patientsafety/alerts-anddirectives/notices/blood-transfusions/ (accessed May 13, 2008).] 50. Clinical standards for blood transfusion. Edinburgh, UK: NHS Quality Improvement Scotland, 2006. [Available at http://

www.nhshealthquality.org/nhsqis/files/BLOODTRANS_STNF_ SEP06.pdf (accessed May 27, 2008).] 51. Williamson LW, Stainsby D, Jones H et al. The impact of universal leukodepletion of the blood supply on hemovigilance reports of posttransfusion purpura and transfusion-associated graft-versushost disease. Transfusion 2007;47:1455-69. 52. Kopko PM. Transfusion-related acute lung injury. Br J Haematol 1999;105:322-9. 53. Kleinman S, Chan P, Robillard P. Risks associated with transfusion of cellular blood components in Canada. Transfus Med Rev 2003;17:120-62.

Appendix 44-1. ISBT Criteria for Severity and Imputability of Transfusion Reactions Grade/Score Severity 1 (non-severe)

Definition

3 (life-threatening) 4 (death)

The recipient may have required medical intervention (eg, symptomatic treatment) but lack of such would not result in permanent damage or impairment of a body function. The recipient required in-patient hospitalization or prolongation of hospitalization directly attributable to the event. OR The adverse event resulted in persistent or significant disability or incapacity. OR The adverse event necessitated medical or surgical intervention to preclude permanent damage or impairment of a body function. The recipient required major intervention following the transfusion (vasopressors, intubation, transfer to intensive care) to prevent death. The recipient died following an adverse transfusion reaction.*

Imputability† Definite (certain) Probable (likely) Possible Unlikely (doubtful) Excluded

When there is conclusive evidence beyond reasonable doubt that the adverse event can be attributed to the transfusion. When the evidence is clearly in favor of attributing the adverse event to the transfusion. When the evidence is indeterminate for attributing the adverse event to the transfusion or an alternate cause. When the evidence is clearly in favor of attributing the adverse event to causes other than the transfusion. When there is conclusive evidence beyond reasonable doubt that the adverse event can be attributed to causes other than the transfusion.

2 (severe)

*Grade 4 should be used only if death is possibly, probably, or definitely related to transfusion. If the patient died of another cause, the severity of the reaction should be graded as 1, 2, or 3. † Once the investigation of the adverse transfusion event has been completed, this is the assessment of the strength of relation to the transfusion of the event.

Appendix 44-2. Standard for Collecting and Presenting of Data on Complications Related to Blood Donation In order to facilitate international benchmarking, it is recommended that this standard be used when data on complications related to blood donation are presented. The Standard consists of ● Descriptions. A list with the categories grouped according to a code system. ● Data collection, Part 1. A list with description of the categories and definition of the grades of severity. ● Data collection, Part 2. An addendum with a form suitable for collecting data according to the standard. The categories are divided in two main groups according to etiology and localization in the body. Some rare, but important, complications have been included in order to secure reports on occurrence. For the sake of simplicity they are grouped together under 300—Rare, important complications.The description of the categories includes definition, symptoms, complications, and grades of severity. Medical terms are defined as indicated in common medical dictionaries. The severity is graded according to duration of symptoms and of requirements for medical treatment. The level of imputability is not included except for some of the complications in group 300—Rare, important complications. If some of the symptoms essential for the definition of a category are missing, the complication can be registered in a more common or broad category. For example, if a donor complains of pain in the arm, the complication belongs to one of the following categories depending on when the symptoms occurred: When needle was inserted Direct needle injury of a nerve (121). After needle was inserted Indirect needle injury of a nerve by a haematoma (122). However, if this information has not been registered the complication can be categorized as a nerve injury without specification (120).

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Chapter 44: Overview of Hemovigilance

Complications Related to Blood Donation List of categories, numeric code, description, and grading (Version Madrid 2007) 100 Local Reactions Related to Needle Insertion Code

Category

Definition

Symptoms

Grades

110 111

Vessel injuries Haematoma

Collection of blood in the tissues

Swelling, pain (local)

112

Arterial puncture

Puncture of brachial artery

113

Thrombophlebitis

120

Nerve injuries

121

Injury of a nerve

Direct nerve injury by the needle

Severe, immediate pain when the needle was inserted which radiates down the forearm, often associated with paraesthesiae

122

Injury of a nerve by a haematoma

Neurological symptoms caused by pressure from a haematoma

Pain and paraesthesia as 121 but Mild symptoms ⬍ 2 weeks symptoms develop some time after Moderate symptoms ⬎ 2 the needle was inserted weeks but ⬍ 1 year

Moderate subjective symptoms Severe required medical treatment Swelling, pain (local) Moderate observed (with or Restricted movements of the elbow without symptoms) Severe required medical treatment Swelling of the vein, pain (local) Severe required medical Redness of the skin treatment Mild symptoms ⬍ 2 weeks Moderate symptoms ⬎ 2 weeks but ⬍ 1 year Severe symptoms ⬎ 1 year or required medical treatment

Severe symptoms ⬎ 1 year or required medical treatment 130

Other complications (related to needle insertion)

131

Tendon injury

132

Allergic reaction (local)

133

Infection (local)

Direct tendon injury by needle

Severe, local pain when needle was inserted At the venipuncture site – rash swelling and itching Swelling, redness and pain (local)

Severe required medical treatment Severe required medical treatment Severe required medical treatment

200 General Reactions 210

Vasovagal reactions

211

Immediate type

Symptoms occurred before donor left the donation site

Subjective symptoms: dizziness, nausea Objective symptoms: Sweating, vomiting, pallor, hyperventilation, convulsions, loss of consciousness

Mild: Subjective symptoms only Moderate: Also objective symptoms Severe: Convulsions and/or Loss of consciousness and/or Required medical treatment

212

Delayed type

Symptoms occur after donor left the donation site but within 24 hours of leaving

Subjective symptoms: Discomfort, weakness, anxiety, dizziness, nausea Objective symptoms: sweating, vomiting, pallor, hyperventilation, convulsions, loss of consciousness

Mild: Subjective symptoms only Moderate: Also objective symptoms. Severe: Convulsions and/or Loss of consciousness and/or Required medical treatment

(Continued)

695

Section IV: Part I

List of categories, numeric code and description (Version Madrid 2007) 300 Rare, Important Complications (The diagnosis must be confirmed by a medical doctor. Include only those complications which required medical treatment) 310

Related to vessel injury

311

Brachial artery pseudoaneurysm

312

Arteriovenous fistula

313

Compartment syndrome

314

Axillary vein thrombosis

320

Accidents

321

Accidents related to vasovagal syncope (immediate and delayed)

322

Other accidents which occurred at the donation site

330

Cardiovascular reactions (symptoms commence within 24 hours of donor leaving donation site)

331

Angina pectoris

332

Myocardial infarct

333

Acute neurological condition (TIA, stroke)

340

Related to apheresis procedures

341

Diffuse allergic reaction

342

Anaphylaxis

343

Haemolysis

344

Air embolus

350

Death – From any cause within 7 days of donation

360

Other

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Chapter 44: Overview of Hemovigilance

Form that could be used for collection of data (Version Madrid 2007) DATA COLLECTION (part 1) Country:

Period:

Procedure:

Number of procedures:

Code

Category

Severity

Number

100 Local Reactions to Needle Insertion 110

Vessel injuries

111

Haematoma

moderate severe Sub total

112

Arterial puncture

moderate severe Sub total

113

Thrombophlebitis

severe

Injury of a nerve by the needle

mild moderate severe Sub total mild moderate severe Sub total

(111 ⫹ 112 ⫹ 113) Total 120 121

Nerve injuries

122

Injury of a nerve by a haematoma

(121⫹122) Total 130

Other complications (related to needle insertion)

131 132 133

Tendon injury Allergic reaction (local) Infection (local)

severe severe severe

(131⫹132⫹133) Total

210 211

212

200 General Reactions Vasovagal reactions Immediate type

Delayed type

mild moderate severe Sub total mild moderate severe Sub total

(210 ⫹ 220) Total

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Section IV: Part I

DATA COLLECTION (part 2) Code

Category 300 Rare, Important Complications

310

Related to vessel injury

311 312 313 314

Brachial artery pseudoaneurysm Arteriovenous fistula Compartment syndrome Axillary vein thrombosis

(311⫹312⫹313⫹314⫹315) Total 320

Accidents

321 322

Accidents related to vasovagal syncope Other kinds of accidents

(321⫹322) Total 330

Cardiovascular reactions

331 332 333

Angina pectoris Myocardial infarct Acute neurological condition (TIA, stroke)

(331⫹332⫹333) Total 340

Related to apheresis procedures

341 342 343 344

Diffuse allergic reaction Anaphylaxis Haemolysis Air Embolus

(341⫹342⫹343⫹344) Total 350

Death

360

Other(s)

*Developed jointly by a joint working group of the International Society of Blood Transfusion and the European Haemovigilance Network.

698

Number

45

Immunomodulatory and Proinflammatory Effects of Allogeneic Blood Transfusion Eleftherios C. Vamvakas,1 José O. Bordin,2 & Morris A. Blajchman3 1

Vice-Chair and Director of Clinical Pathology, Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA 2 Associate Professor, Division of Hematology and Transfusion Medicine, Universidade Federal de Sao Paulo, Sao Paulo, Brazil 3 Professor Emeritus, Departments of Pathology and Medicine, McMaster University; Medical Director, Canadian Blood Services, Hamilton, Ontario, Canada

As knowledge about the mechanisms of immune responsiveness and tolerance evolves, and as new tools to measure alterations in immunity become available, additional immunologic consequences of allogeneic blood transfusion (ABT) are detected. The lingering question has been whether these observations represent no more than laboratory curiosities, or whether they reflect some clinically relevant alteration in the recipient’s immune function—the so-called “immunomodulatory” effect of ABT.1 The constellation of all such ABT-associated laboratory and clinical findings is known as transfusion-related immunomodulation (TRIM). Initially, TRIM encompassed effects attributable to ABT by means of immunologic mechanisms only; however, more recently, the term has been used more broadly, to encompass additional effects that could be related to ABT by means of both immunomodulatory and proinflammatory rather than only immunomodulatory mechanisms.2 ABT may either cause alloimmunization or induce tolerance. ABT introduces a multitude of foreign antigens into the recipient, including HLA-DR antigens found on the donor’s dendritic antigen-presenting cells (APCs). The presence or absence of autologous HLA-DR antigens on the donor’s white blood cells (WBCs) plays a decisive role in whether alloimmunization or immune suppression will ensue following ABT.3 Transfusions sharing at least one HLA-DR antigen with the recipient will induce tolerance, while fully HLA-DR-mismatched transfusions lead to alloimmunization.4 In addition to the degree of HLA-DR compatibility between donor and recipient, the immunogenicity of cellular or soluble HLA antigens found in transfused blood components depends on the viability of the donor dendritic APCs and the presence of the required costimulatory signals for the presentation of the donor antigens to the recipient’s T cells. Nonviable APCs and/or absence of the requisite costimulatory signals result in T-cell unresponsiveness.5-7 Thus, when a multitude of antigens

is introduced into the host by ABT, the host response to some of these antigens is often decreased, and immune tolerance (or TRIM) ensues.8 Several immune-function alterations have been documented in association with ABT (Table 45-1).9,10 All these ABT-related laboratory immune alterations could potentially be associated with clinical effects. Evidence from a variety of sources indicates that ABT enhances the survival of renal allografts11; may increase the recurrence rate of resected malignancies and the incidence of postoperative bacterial infections; may reduce the recurrence rate of Crohn’s disease12 and the risk of fetal loss in women with recurrent spontaneous abortions (RSAs); may activate infections with cytomegalovirus (CMV) or human immunodeficiency virus (HIV); and/or may increase short-term (up to 3 months after transfusion) mortality from all causes.13,14 Different biologic mechanisms may be involved in each of these purported clinical manifestations of TRIM, and the clinical evidence supporting each of the aforementioned hypotheses should be examined on its own merits.13,15 The specific constituent(s) of allogeneic blood that mediate(s) the TRIM effect(s) also remain(s) unknown, and published literature has

Table 45-1. Documented Immune Function Alterations in Association with Allogeneic Blood Transfusion ● ● ● ● ● ● ● ● ● ● ●

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

● ●

Decreased T-helper (CD4) cell count Decreased helper/suppressor (CD4/CD8) T-lymphocyte ratio Decreased lymphocyte response to mitogens Reduction in delayed-type hypersensitivity Decreased natural killer (NK) cell function B-cell activation T-cell activation Hypergammaglobulinemia Decreased cytokine (interleukin-2, interferon-γ) production Suppression of lymphocyte blastogenesis Decreased monocyte/macrophage phagocytic function Increased production of anti-idiotypic antibodies Increased production of anti-clonotypic antibodies

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Section IV: Part I

Purportedly caused by:

Trim effects:

1. Increased recurrence rate of resected malignancies 2. Increased incidence of postoperative bacterial infections

Soluble HLA peptides circulating in allogeneic plasma

1. Autologous transfusion

Soluble, WBCderived mediators accumulating in the supernatant fluid of stored RBCs

1. Fresh autologous blood obtained by ANH, IBR, or PBR

?

?

3. Activation of endogenous CMV or HIV infections 4. Increased shortterm (up to 3month) mortality

Prevented by:

2. Prestorage WBC reduction

? Allogeneic mononuclear cells

1. Autologous transfusion 2. Prestorage WBC reduction 3. Poststorage WBC reduction

suggested that TRIM effects may be mediated by: 1) soluble HLA Class I peptides that circulate in allogeneic plasma; 2) soluble biologic response modifiers released in a time-dependent manner from WBC granules or membranes into the supernatant fluid of Red Blood Cell (RBC) or platelet concentrates during storage; and/or 3) allogeneic mononuclear cells (Fig 45-1).15

Beneficial Clinical TRIM Effects Enhanced Survival of Renal Allografts The only clearly established TRIM effect is beneficial—not deleterious: it is the enhanced survival of renal allografts in patients who have received pretransplant ABT.11,17 Patients who receive allogeneic blood have been shown to have a significantly better renal allograft survival than untransfused patients, regardless of the number of HLA-A, HLA-B, and HLA-DR locus mismatches between recipient and donor.11,18 This is true even when there is a common HLA haplotype, or shared HLA-B and HLA-DR antigens between donor and recipient.19 The TRIM effect has also been reported with allografts between HLA-identical siblings.20 The ABT-related enhancement of renal allograft survival has been confirmed by animal data and clinical experience worldwide. In fact, it used to be standard policy in many renal transplant units to deliberately expose patients on transplant waiting lists to one or more allogeneic RBC transfusions. Subsequently, the beneficial effect of pretransplant ABT was thought to be less important—after cyclosporine and other potent immunosuppressive drugs were introduced. As a consequence, many centers discontinued its use. However, a multicenter observational study, reporting on 58,036 renal allografts from cadaveric donors after

700

Figure 45-1. TRIM effects, postulated mediators of TRIM, and preventive strategies that could be effective if the TRIM effects were mediated by each corresponding mediator. The purported deleterious TRIM effects are mediated by an unknown constituent(s) of allogeneic blood that may (or may not) include one (or more) of the mediators shown. Stored autologous blood obtained by preoperative autologous blood donation is replete with white blood cell (WBC)derived soluble mediators, because both autologous and allogeneic WBCs deteriorate equally during storage, releasing such mediators. Fresh autologous blood, transfused within hours of collection and processing, is free of WBC-derived soluble mediators and can be procured by acute normovolemic hemodilution (ANH), intraoperative blood recovery (IBR), or postoperative blood recovery (PBR). WBC-reduction filters do not retain WBC-derived soluble mediators, accounting for the difference between prestorage and poststorage WBC reduction in abrogating the TRIM effects mediated by such mediators. Modified with permission from Vamvakas.16 CMV ⫽ cytomegalovirus; HIV ⫽ human immunodeficiency virus.

the advent of cyclosporine, indicated that patients who received ABT were still more likely to have a successful renal allograft than those who did not.21 This study reported that the 1-year renal allograft survival of patients receiving pretransplant ABT was 3% to 5% better than that of those who did not receive ABT. Similar results were also reported for patients who received renal allografts from livingrelated donors.22 The beneficial effect of pretransplant ABT in the outcome of cadaveric renal allografts was confirmed by a randomized controlled trial (RCT) conducted at 14 transplant centers.23 Patients were randomly assigned to receive either three pretransplant, non-WBC-reduced RBC transfusions or no ABT. The renal allograft survival was significantly higher in the 205 transfused patients than in the 218 untransfused subjects (90% vs 82% at 1 year, p ⫽ 0.02; 79% vs 70% at 5 years, p ⫽ 0.025). The beneficial effect of ABT was found to be independent of age, gender, underlying disease, prophylaxis with lymphocyte antibodies, or the presence of preformed lymphocytotoxins.23 Two RCTs24,25 have compared types of pretransplant ABTs given to prolong graft survival. Both studies were small, enrolling 52 and 144 patients, respectively. The first RCT24 compared nonWBC-reduced and WBC-reduced RBCs and found no difference in graft survival. The other RCT25 compared recipients of one HLA-DR-mismatched ABT, one HLA-DR-matched ABT, and no ABT. There was no difference in graft survival at 1 year or at 5 years. Recipients of non-WBC-reduced whole blood or RBCs have better 1-year cadaveric allograft survival than patients given WBCreduced blood components such as frozen-thawed-deglycerolized RBCs. These data indicate that allogeneic WBCs are involved in eliciting this beneficial TRIM effect.26 However, the mechanism(s) involved in the ABT-related enhancement of renal allograft

Chapter 45: Immunomodulatory and Proinflammatory Effects of Allogeneic Blood Transfusion

survival remain(s) to be elucidated, and many questions about the optimal use of ABT in such patients remain to be answered. The latter include: the optimal number of ABTs, the volume of RBCs with each ABT, and the timing of the ABTs to produce the optimal clinical effect; and whether the TRIM effect of pretransplant ABTs poses any hazard(s) in addition to conferring benefit. ABT administered during the actual operation for renal transplantation has not been shown to affect subsequent allograft survival.17 Patients who receive more than 10 RBC units have a better 1-year allograft survival than patients who receive only 1 or 2 RBC units. However, patients who receive more than 10 RBC units appear to have a poorer overall allograft survival than those who receive fewer than 10 RBC units.27 Such data suggest that multitransfused patients often develop cytotoxic antibodies and thus are at greater risk for earlier and more severe allograft rejection episodes.22 Along these lines, Solheim28 has reported another potential benefit from pretransplant ABTs, which is especially relevant in settings where there is a shortage of organs for transplantation. When several prospective recipients on a renal transplant list produce crossmatch-negative results with an available organ, pretransplant ABTs could help identify high-responder patients (ie, patients most likely to form cytotoxic antibodies in response to pretransplant ABTs, and also most likely to reject a transplanted kidney because of formation of cytotoxic antibodies after a negative crossmatch). Transplant surgeons could thus channel the scarce organ away from such patients and give it to a patient in whom it is most likely to survive.28 An experimental animal model has suggested that the beneficial effect of donor-specific ABT might be related to the type of transplanted organ. Whereas ABT appears to lead to permanent acceptance of all renal allografts, this benefit was not observed with pancreas, skin, or heart allografts.29 Although additional data are required to understand how ABT induces its beneficial effect in renal transplantation, the authors believe that the use of pretransplant, non-WBC-reduced ABT remains a potentially useful intervention in the management of selected patients scheduled for renal transplantation even today; however, this benefit may not apply to settings where universal WBC reduction has been implemented.

components have included pooled buffy coats, single-donor buffy coats, or RBC suspensions containing WBCs. The American Society for Reproductive Immunology (ASRI) conducted a worldwide collaborative individual-patient-data (IPD) meta-analysis to examine the efficacy of immunotherapy with allogeneic WBCs in patients with a history of RSAs32 and concluded that such treatment was effective. The effect was small, with only 8% to 10% of affected women benefiting from the treatment. In contrast to the ASRI data, a recent multicenter RCT33 that enrolled 183 women with a history of three or more spontaneous abortions reported a nonsignificant decrease in livebirth rate in patients randomly assigned to immunotherapy as compared with controls (36% vs 48%). However, the size of this RCT was likely inadequate to establish a clinically relevant treatment effect. Thus, the efficacy of transfusions of allogeneic WBCs for the treatment of patients with RSAs remains to be established.

Reduced Risk of Recurrence of Crohn’s Disease Several observational studies have examined whether postoperative recurrence in patients with Crohn’s disease—an immune-mediated disease—can be reduced by the perioperative administration of ABT. Pooled data from the available studies suggest that the recurrence rate in transfused and untransfused patients is similar: 37.5% vs 40.5%.12,34-37 However, such integration of the reported data may be inappropriate, because the available studies are observational and medically heterogeneous (ie, using different follow-up periods and surgical interventions). Nonetheless, an IPD meta-analysis of 622 patients with Crohn’s disease found no effect of perioperative ABT on subsequent need for surgical intervention, independent of age, gender, disease location, or extent of the resection.38 Because many factors affect the risk of recurrence in patients with Crohn’s disease, a large RCT is needed to clarify the role of ABT, if any, in modulating disease activity in patients with this disorder.

Deleterious Clinical TRIM Effects Increased Recurrence of Resected Malignancies

Reduced Risk of Recurrent Spontaneous Abortions Transfusion of allogeneic WBCs has been proposed as a form of immunotherapy for the prevention of RSAs.30,31 In this setting, the fetus represents a semi-allogeneic graft to its mother, and maintenance of a pregnancy depends on immunologic equilibrium between the implanted fetus and the maternal immune response to the fetus. When the genetic parents share HLA antigens, this balance may be altered and maternal blocking antibodies may not be formed, predisposing the pregnant woman to RSAs. Different transfusion protocols of allogeneic WBCs have been used at various centers to reduce the risk of RSAs, employing WBCs obtained from either sexual partners or thirdparty donors. Such WBCs have been administered intravenously, intracutaneously, or as an intradermal injection of mononuclear cells obtained by gradient separation. WBC-containing blood

If ABT has a beneficial effect in renal transplantation, where immunosuppression is beneficial because it may prevent allograft rejection, ABT could also have deleterious effects in situations where impairment of the recipient’s immune function can be detrimental. In 1981, 8 years after the first report of the beneficial effect of pretransplant ABT on renal allograft survival,11 Gantt39 voiced his concern that perioperative ABT for curative resection of a malignancy might provoke recurrence of cancer. Thus, if the host’s immune response to a tumor contributes to controlling the tumor’s growth, one would expect that impairment of the host’s immunity, caused by ABT, would impair this defense mechanism and facilitate tumor growth. More than 100 observational studies of ABT and cancer recurrence have been reported,40 and their unadjusted results (ie, their findings before adjustment for the effects of confounding factors)

701

Section IV: Part I

were subjected to three meta-analyses.40-42 When the available results were integrated for seven cancer sites, there was agreement between the three overviews on the magnitude and statistical significance of the risk of cancer recurrence, death from cancer recurrence, or overall mortality in transfused compared with untransfused patients. A statistically significant adverse clinical outcome was found among transfused (compared with untransfused) patients for all cancer sites evaluated except for cervix.40 A statistically significant ABT effect in the observational studies that had presented multivariate analyses adjusting for the effects of confounding factors was found in 24 studies, including 11 studies of colorectal cancer, four studies of head and neck cancer, one study of breast cancer, two studies of gastric cancer, four studies of lung cancer, and two studies of prostate cancer.40 This ABT effect has not been confirmed by the available RCTs of perioperative ABT and cancer recurrence that are discussed later in this chapter (see TRIM effects mediated by allogeneic mononuclear cells).

Increased Risk of Postoperative Bacterial Infection None of the purported adverse TRIM effects of ABT has been established by RCTs, and the debate over the existence of clinical deleterious TRIM effects has been long and sometimes acrimonious.43-48 Initially, the debate focused on differing interpretations of the findings of approximately 40 observational studies that had compared the risk of postoperative bacterial infection between transfused and untransfused patients undergoing gastrointestinal surgery, orthopedic operations, cardiac surgery, or various other surgical procedures. These studies tended to indicate that patients receiving perioperative ABT (compared with those not receiving ABT) almost always had a higher risk of developing postoperative bacterial infection.43 The studies also indicated that patients receiving transfusion generally differed from those not receiving transfusion in several prognostic factors that predisposed to adverse clinical outcomes.44 Based on these two sets of observations, some authors concluded that ABT has a direct deleterious effect on the recipient, causing an increased risk of postoperative bacterial infection.43 Other investigators concluded that clinical need for ABT can be a surrogate marker for a variety of adverse prognostic factors and that the other variables that generated the need for ABT in the published studies also determined the subsequent clinical outcome.44 Today, the controversy over TRIM is focused on differing interpretations45-48 of the findings of the available RCTs of perioperative ABT and postoperative infection. There have been 22 RCTs49-70 that compared the risk of postoperative infection and/ or mortality between patients randomly assigned to receive nonWBC-reduced allogeneic vs autologous or WBC-reduced allogeneic RBCs. Three of these RCTs50,52,71 also compared the risk of cancer recurrence between the two randomization study arms. Nineteen RCTs were conducted in the perioperative setting; three more RCTs enrolled HIV-seropositive patients,62 all hospitalized patients,65 or trauma patients.70 Based on an integration of the results of all nine RCTs published or reported through 200249,52,54,58-60,63,66,67 and comparing the risk of postoperative infection between patients randomly

702

assigned to receive non-WBC-reduced vs WBC-reduced ABT in the event that they needed perioperative transfusion, two meta-analyses45,46 concluded that non-WBC-reduced ABT is associated with postoperative infection. In contrast, a third metaanalysis47 that integrated the findings of all 12 RCTs published or reported through 200549,52,54,58-60,63,66-70 found no association between non-WBC-reduced ABT and postoperative infection. Similarly, no association between ABT and postoperative infection was detected when the findings of RCTs50,51,55,56,64 comparing recipients of allogeneic and autologous RBCs were integrated.72 The reasons for the disagreements between the three metaanalyses45-47 that compared recipients of non-WBC-reduced vs WBC-reduced ABT have been discussed.48 There were two main reasons for the disagreements: 1) the inclusion in the analysis of all 12 RCTs available today vs the nine initially published RCTs; and 2) the integration (or not) of the results of all 12 (or all nine) RCTs despite extreme medical heterogeneity. Medical heterogeneity refers to differences in study design with respect to such factors as the RBC product transfused to the non-WBC-reduced arm (ie, non-buffy-coat-reduced vs buffy-coat-reduced allogeneic RBCs or whole blood), the RBC product transfused to the WBC-reduced arm (ie, WBC-reduced RBCs or whole blood filtered before or after storage), the transfusion dose, the surgical setting (gastrointestinal, cardiac, or other), the types of postoperative infections evaluated, the criteria for diagnosing postoperative infection, and the frequency of postoperative infection recorded in each study.48 One would expect more of a TRIM effect in association with the transfusion of non-buffy-coat-reduced (compared with buffycoat-reduced) allogeneic RBCs because the buffy-coat-reduced RBCs used in Europe contain only about one-third of the donor WBCs found in the non-buffy-coat-reduced RBCs used in North America. Second, one would expect a greater reduction of a TRIM effect(s) attributable to the transfusion of prestorage (compared with poststorage) filtered WBC-reduced allogeneic RBCs, because of the removal of WBC-derived soluble mediators through prestorage (but not poststorage) filtration (Fig 45-1). Third, one would expect more of a TRIM effect in cardiac (compared with other) surgical settings, because in cardiac surgery allogeneic mononuclear cells and/or WBC-derived soluble mediators might serve as a second inflammatory insult that compounds the diffuse inflammatory response to the extracorporeal circuit.73 Therefore, one would not expect all 12 RCTs available today (or all nine initially published RCTs) to have targeted the same TRIM effect. In contrast, depending on their design, these 12 (or nine) RCTs probably targeted TRIM effects that varied in magnitude and/or nature, making the integration of all 12 (or all nine) RCTs by a meta-analysis inappropriate. For example, RCTs administering RBCs filtered before storage to the WBCreduced arm52,58,60,63,66-70 investigated TRIM effects mediated by both WBC-derived soluble mediators and allogeneic mononuclear cells (Fig 45-1). However, RCTs transfusing RBCs filtered after storage to the WBC-reduced arm49,54,58,59 investigated only those TRIM effects mediated by allogeneic mononuclear cells. Meta-analyses should integrate results only from subsets

Chapter 45: Immunomodulatory and Proinflammatory Effects of Allogeneic Blood Transfusion

of RCTs that are medically sufficiently homogeneous to justify the assumption that all combined studies have targeted a TRIM effect that is biologically similar.48 Results from meta-analyses of such homogeneous subsets of RCTs are presented later in this chapter where TRIM effects mediated by WBC-derived soluble mediators or allogeneic mononuclear cells are discussed. Blumberg et al46 had earlier attributed the disagreements between the three meta-analyses45-47 to the reliance on the intention-to-treat principle in the meta-analysis that did not detect an adverse TRIM effect of ABT47 vs the use of results from “astreated” analyses in the two meta-analyses that did detect a deleterious TRIM effect.45,46 Intention-to-treat analyses often have reduced statistical power to detect a treatment effect compared with “as-treated” analyses.46 However, both intention-to-treat and “as-treated” analyses demonstrated a deleterious TRIM effect when the analysis integrated the findings of all nine initially published RCTs. Neither the intention-to-treat nor the “as-treated” analysis showed an association between non-WBC-reduced ABT and postoperative infection when the analysis integrated the results of all 12 RCTs available today (Table 45-2).48

Increased Risk of Short-Term Mortality An association between non-WBC-reduced ABT and short-term (up to 3 months after transfusion) mortality from all causes was described in the RCT of van de Watering et al.58 This RCT had been designed to investigate an association between non-WBC-reduced ABT and postoperative infection and, instead of that association, it observed an association between non-WBC-reduced ABT and mortality. The association between ABT and mortality was reported as a data-derived hypothesis,58 and the authors postulated

that non-WBC-reduced ABT may predispose to multiple-organ failure (MOF), which might—in turn—predispose to mortality. These investigators undertook another RCT that confirmed the association between ABT and mortality but did not find an association between non-WBC-reduced ABT and increased MOF.63 Several preclinical74-78 and clinical73,79-89 observations have supported the hypothesis that ABT in general, and non-WBCreduced ABT in particular, may be associated with MOF. The mechanisms underlying the development of MOF are unclear, but most evidence suggests that tissue injury is mediated by reactive oxygen species and proteolytic enzymes released from activated neutrophils.90-92 Silliman et al74 proposed that ABT may exercise a neutrophil-priming effect mediated by bioactive lipids that accumulate during storage. They postulated that rapidly deteriorating WBCs in stored RBC units release cytotoxic enzymes that may act on fragmented red cell membranes to produce mediators that are responsible for neutrophil priming and endothelial-cell activation (Fig 45-2). These investigators74-76 demonstrated that plasma obtained from stored RBC units primes neutrophils for superoxide production and enhanced cytotoxicity, and also activates pulmonary endothelial cells in a dose- and age-dependent fashion. The length of RBC storage was important in these studies, because no evidence of neutrophil priming was obtained when plasma stored for short periods was used. Silliman et al77 also showed that lipids from the plasma supernatant of RBCs stored for 42 days cause acute lung injury in isolated pulmonary models. Similarly, Chin-Yee et al78 reported that plasma supernatant from stored RBCs activates neutrophils. In that study, WBC reduction of the RBC units abrogated the effect.78

Table 45-2. Meta-Analyses of RCTs of Non-WBC-Reduced Allogeneic Blood Transfusion and Postoperative Infection: Impact of the Method of Analysis and the Number of RCTs Included in the Analysis48 Method of Analysis Intention-to-Treat*

“As-Treated”*

Number of RCTs included in the Analysis†

Number of Patients Analyzed

Summary Odds Ratio

95% Confidence Interval

Number of Patients Analyzed‡

Summary Odds Ratio

95% Confidence Interval

9 RCTs49,52,54,58-60,63,66-67 published or reported through 200245,46

5017

1.38

1.03-1.85§

3265 (65.1%)

1.56

1.06-2.31§

12 RCTs49,52,54,58-60,63,66-70 published or reported through 200547

6290

1.24

0.98-1.56

4460 (70.1%)

1.31

0.98-1.75

*The intention-to-treat analyses included all patients randomly assigned preoperatively to receive non-WBC-reduced or WBC-reduced allogeneic blood in the event that they needed perioperative transfusion. The “as-treated” analyses retained only those patients from each randomization arm who ended up receiving transfusion during or after surgery. † Integration of all nine (or all 12) RCTs, as shown in this table, is inappropriate because of the extreme medical heterogeneity of the studies (see text). Therefore, readers should resist the temptation to assign a medical or biological meaning to the figures presented in the table. Instead, readers are referred to Figs 45-4 and 45-5, which depict the results obtained when medically homogeneous subsets of these RCTs49,52,54,58-60,63,66-70 were integrated. ‡ The percentage of all randomized patients that was included in the “as-treated” analyses is given within parentheses. § Statistically significant adverse ABT-related immunomodulatory effect (p ⬍0.05).

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Section IV: Part I

Donor unit Deteriorating WBCs in stored RBC units release enzymes

That act on red cell membranes to produce bioactive lipids responsible for: Recipient

Prestorage WBC reduction

Endothelial-cell activation

Neutrophil priming

Superoxide production

Enhanced cytotoxicity

Based on this observation that ascribes a neutrophil-priming effect to ABT (Fig 45-2), it is possible that the recently reported58,63 association between ABT and short-term mortality could, in fact, reflect a “proinflammatory” rather than an “immunomodulatory” effect of ABT. However, for the time being and until these effects can be clearly defined, the authors believe that the association between ABT and MOF or mortality should be covered under the general overarching concept of adverse “TRIM” effects.2 In the study of Johnston et al,80 patients receiving allogeneic RBCs had a significantly higher risk of MOF than recipients of polymerized hemoglobin. Neutrophils obtained from recipients of RBCs demonstrated priming, as evidenced by increased beta-2 integrin expression, superoxide production, and elastase release. Neutrophils obtained from recipients of polymerized hemoglobin showed no evidence of priming. Studies investigating the benefits obtained from placing a WBC reduction filter in the arterial line of the cardiopulmonary bypass circuit81-83 suggested that non-WBC-reduced ABT may provoke cardiac and/ or pulmonary failure. Furthermore, associations between ABT and prolonged mechanical ventilation84,85 or MOF79,86-89 were reported by some, but not all,93,94 observational studies. Recently, on the basis of an observational study of 248 patients, Netzer et al95 reported that ABT in patients with acute lung injury was associated with increased in-hospital mortality. Following adjustment for confounding factors, ABT given after the onset of acute lung injury was associated with a 13% increase in mortality per unit of transfused RBCs (p ⬍0.0001), but ABT given before the onset of acute lung injury was not associated with any higher risk. Both non-WBC-reduced and WBC-reduced ABT were associated with a significant (p ⬍0.001) increase in mortality, although the magnitude of the increase in risk was greater when non-WBCreduced (rather than WBC-reduced) RBCs had been used. Along the same lines, Murphy et al96 linked the United Kingdom population register with the clinical, intensive-care-unit (ICU), hematology, and blood bank databases of 8516 patients who had undergone cardiac surgery over 8 years (1996-2003). When transfused and

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Figure 45-2. Proposed mechanism leading from the accumulation of bioactive lipids in the supernatant fluid of stored non-WBC-reduced Red Blood Cells (RBCs) to the development of multiple-organ failure in the recipient.74-78

untransfused patients were compared after adjustment for confounding factors, ABT was found to be associated with a higher risk of postoperative infection, ischemic postoperative morbidity, and early (0 to 30 days) as well as late (31 days to a year) mortality. An association between non-WBC-reduced ABT and prolonged mechanical ventilation or MOF has not yet been reported by any RCT. Eleven RCTs, comparing recipients of non-WBCreduced vs WBC-reduced allogeneic RBCs, and reporting on cancer recurrence, postoperative infection, or mortality as the primary outcome have presented information on shortterm (up to 3 months after transfusion) mortality from all causes.54,57,58,60,63,65-70 Because WBC reduction filters do not retain soluble mediators, if ABT exercised the described WBCdependent neutrophil-priming effect mediated by bioactive lipids that accumulate during storage (Fig 45-2), allogeneic RBCs WBC-reduced before storage should abrogate this effect; but allogeneic RBCs WBC-reduced after storage should confer no benefit. Despite this theoretical prediction, no increase in mortality in association with non-WBC-reduced ABT was detected either across the subset of RCTs transfusing RBCs filtered before storage to the WBC-reduced arm, or across the subset of RCTs transfusing RBCs filtered after storage to the WBC-reduced arm.47 However, across five RCTs conducted in cardiac surgery58,63,66,68,69 that had transfused RBCs filtered before storage to the non-WBC-reduced arm, non-WBC-reduced ABT was associated with a 72% increase in postoperative mortality [summary odds ratio, 1.72; 95% confidence interval (CI), 1.05–2.81; p ⬍0.05—Fig 45-3). As already discussed, the TRIM effect seen across these studies may be associated with factors prevalent in the setting of patients undergoing cardiac surgery. For example, one can speculate that bioactive lipids accumulating during the storage of non-WBC-reduced RBCs and/or allogeneic mononuclear cells may represent a second inflammatory insult. That insult may compound the diffuse inflammatory response associated with the cardiopulmonary bypass circuit that may predispose recipients to MOF that may—in turn—predispose to mortality.73

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Figure 45-3. Postoperative mortality in cardiac-surgery patients. Randomized controlled trials (RCTs) investigating the association of non-WBC-reduced allogeneic blood transfusion (ABT) with short-term (up to 3 months after transfusion) mortality from all causes and conducted in the setting of cardiac surgery.58,63,66,68,69 For each RCT, the figure shows the odds ratio (OR) of shortterm mortality in recipients of non-WBC-reduced vs WBC-reduced allogeneic Red Blood Cells, as calculated from an intention-to-treat analysis; and the summary OR across the depicted RCTs, as calculated from a meta-analysis.47 A deleterious ABT effect (and thus a benefit from WBC reduction) is demonstrated by an OR ⬎1, provided that the effect is statistically significant (p ⬍0.05; ie, provided that the associated 95% CI does not include the null value of 1).

Effect of the Length of RBC Storage If bioactive lipids or other soluble mediators accumulating in a time-dependent manner during storage were associated with adverse outcomes, observational studies should be reporting an association between prolonged storage of transfused nonWBC-reduced allogeneic RBCs and increased risk of occurrence of these adverse outcomes (because the longer the non-WBCreduced allogeneic RBCs are stored, the higher the level of such soluble mediators they will contain). Also, the red cell storage lesion (Chapter 4) occurs in both the non-WBC-reduced and the WBC-reduced units, generating considerable numbers of nonfunctioning red cells. Such nonfunctioning red cells are removed from the recipient’s circulation within 24 hours of the transfusion. Removal of large numbers of nonfunctioning red cells places a considerable burden on the reticuloendothelial system of a multitransfused recipient—a burden that could interfere with the host’s response to bacteria and other challenges.

Nine observational studies have reported on the association between prolonged storage of transfused allogeneic RBCs and increased risk of morbidity or mortality. Four studies97-100 reported on the association of prolonged storage of transfused RBCs with postoperative infection; one study79 on the association between prolonged storage of transfused RBCs and MOF; and three studies101-103 on the association between prolonged storage of transfused RBCs and increased mortality. The earlier studies79,97-102 found prolonged storage of transfused non-WBC-reduced allogeneic RBCs to be associated with an increased risk of these adverse outcomes but van de Watering et al103 found no association between prolonged storage time of transfused RBCs and increased mortality or longer stay in the ICU. Recently, in the setting of cardiac surgery, Gorman Koch et al104 compared 2872 patients given RBCs stored for 14 days or less with 3130 patients given RBCs stored for more than 14 days. Prolonged storage was associated with an increase in the riskadjusted rate of a composite of complications (p ⫽ 0.03), while at 1 year mortality was significantly increased in patients given blood stored for more than 14 days (11.0% mortality) compared with patients given blood stored for 14 days or less (7.4% mortality). The effect of the length of storage on non-WBC-reduced RBCs on clinical outcomes has not yet been evaluated in RCTs.

“Before-and-After” Studies In the late 1990s, Canada and many western European countries implemented universal WBC reduction of cellular blood components by means of prestorage filtration. Once universal WBC reduction is implemented, the opportunity to conduct RCTs to establish adverse TRIM effects attributable to allogeneic WBCs is removed. It is, however, possible to compare the risk of infection or mortality in recipients of non-WBC-reduced RBCs before implementation of WBC reduction with the risk of infection or mortality in recipients of WBC-reduced RBCs after implementation of WBC reduction. Such “before-and-after” studies are observational, and—as such—they cannot establish causal relationships. Five before-and-after studies have reported data on the risk of infection,105-109 and five such studies have reported data on the risk of short-term mortality.105,106,108-110 In Canada, Hébert et al108 conducted a large study including 9525 patients undergoing cardiac surgery, 1731 patients undergoing orthopedic surgery, and 3530 patients admitted to the ICU. These investigators observed a statistically significant (p ⫽ 0.04) decrease in short-term mortality (from 7.0% to 6.2%) after WBC reduction was introduced, without a concomitant reduction in the risk of postoperative infection. They offered the hypothesis that the observed decrease in the number of deaths might not have been mediated through suppression of the recipient’s immune function, but through a proinflammatory microvascular effect of transfused WBCs that affects several organ systems. This hypothesis was buttressed by the findings of a companion before-and-after study in premature infants.109 In that setting, the implementation of universal WBC reduction coincided with a reduction in several secondary morbidity outcomes from

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several organ systems (ie, bronchopulmonary dysplasia, retinopathy of prematurity, necrotizing enterocolitis)—an observation consistent with a diffuse proinflammatory microvascular effect of allogeneic WBCs. However, when all before-and-after studies were considered together in a meta-analysis,111 and the findings of the unadjusted analyses from five studies105-109 were integrated, there was an unadjusted association of WBC reduction with a decreased risk of postoperative infection. This association did not persist when findings from the multivariate analyses of the observational studies that had adjusted for the effects of confounding factors106,108,109 were integrated. There was neither an unadjusted105,106,108-110 nor an adjusted106,108,109 association of WBC reduction with decreased short-term mortality.111

TRIM Effects Mediated by Soluble Molecules Circulating in Allogeneic Plasma Soluble HLA Molecules Soluble HLA proteins and immunoreactive HLA peptides remain important candidates as mediators of the TRIM effect(s). It has been suggested that nonpolymorphic peptides derived from HLA Class I molecules induce antigen-nonspecific immunosuppression, while polymorphic HLA Class I peptides have antigen-specific immunomodulatory effects.112-114 It also seems possible that allogeneic plasma containing soluble HLA antigens may enter the recipient’s thymic circulation, producing clonal deletion of the recipient’s T cells that are directed against the allogeneic donor antigens.115,116

normal donors), Hansen et al119 demonstrated a 500-fold or greater increase in the concentration of complexed IL-6/autoantibody to IL-6, as compared with free IL-6 detectable in the patients’ plasma before the transfusion. When these autoantibodies reach a certain level, they may render a donor (or the recipient of plasma from such a donor) cytokinedeficient, but overt clinical sequelae of such a cytokine deficiency have not been reported. Hansen et al119 christened this phenomenon transfusion-related inhibition of cytokines (TRICK). Depending on the cytokine or growth factor involved, TRICK could conceivably increase the transfusion recipient’s susceptibility to infection or delay hematopoietic recovery after stem cell transplantation.

Evidence from RCTs The authors know of only one RCT66 whose design permitted the investigators to examine the hypothesis that soluble plasma molecules circulating in allogeneic plasma may mediate TRIM effects. Wallis et al66 randomly assigned 597 patients undergoing cardiac surgery to receive plasma-reduced, buffy-coat-reduced, or WBCreduced allogeneic RBCs. In terms of their WBC content, plasmareduced RBCs are equivalent to the buffy-coat-rich RBCs used in North America. The highest risk of postoperative infection was observed in the plasma-reduced study arm, in which the incidence of postoperative infection was 17.1%, as compared with 10.8% in the buffy-coat-reduced arm and 11.3% in the WBC-reduced arm (p ⫽ 0.20). Although the difference between the three study arms was not significant, it appeared as though plasma removal did not confer a benefit with regard to the prevention of TRIM, or—by extension—that allogeneic plasma did not mediate TRIM.

Factor VIII Concentrates In-vitro studies have indicated that low-molecular-weight components found in Factor VIII concentrates may inhibit the proliferative responses of peripheral blood mononuclear cells to phytohemagglutinin.117 In these studies, high-purity Factor VIII concentrates have been shown to reduce the induction of T-cell-activation molecules such as the interleukin (IL)-2 receptor (CD25), the transferrin receptor (CD71), CD38, the CD11a/CD18 ratio, and HLA-DR antigen expression.117 Moreover, evidence has been provided that this inhibitory action of Factor VIII concentrates was at least partly as a result of their contamination by transforming growth factor (TGF)-β.118

Autoantibodies For some unknown reason, some people spontaneously produce large amounts of neutralizing autoantibodies to a number of growth factors (eg, granulocyte-macrophage colony-stimulating factor) or cytokines (eg, IL-1, IL-6, interferon-α).119 The autoantibodies in question are detectable in immunoglobulin preparations and correspondingly in plasma for transfusion. Therefore, if such donors donate blood, large amounts of neutralizing autoantibodies to growth factors or cytokines may be transferred to recipients by transfusion. In the plasma of transfusion recipients who had received plasma from donors with high titers of high-affinity neutralizing autoantibodies to IL-6 (0.1% of

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TRIM Effects Mediated by WBC-Derived Soluble Mediators Mediators Originating in WBC Granules Biologic response modifiers accumulating in blood components during storage have been implicated in the pathogenesis of TRIM.120 These mediators are contained in intracellular WBC granules, and are released in a time-dependent manner as the cells deteriorate. 121 Nielsen et al121 reported that the concentration of histamine, eosinophil cationic protein, eosinophil protein X, myeloperoxidase, and plasminogen activator inhibitor-1 increase 3- to 25-fold in the supernatant fluid of RBC components between Days 0 and 35 of storage. Histamine, eosinophil cationic protein, and eosinophil protein X have been shown to inhibit neutrophil function, thereby contributing to the development of immunosuppression and tissue damage.122,123

Soluble HLA Molecules and Fas Ligand Soluble HLA molecules are present in the serum or plasma of healthy individuals. The liver is the main source of soluble HLA molecules found in the circulation. High levels of these molecules have been found in the serum or plasma of transplant recipients and patients with a variety of conditions, including

Chapter 45: Immunomodulatory and Proinflammatory Effects of Allogeneic Blood Transfusion

inflammatory, autoimmune, and infectious diseases. Soluble HLA molecules are also found in the supernatant fluid of stored RBCs and platelets, in direct proportion to the length of storage and the number of cells present. The biologic significance of these molecules has not been fully established, although it has been reported that they may be involved in the downregulation of the immune response and/or induction of tolerance. Ghio et al124 and Puppo et al125 found soluble Fas-ligand (sFasL) and soluble HLA Class I molecules in the supernatant plasma of RBC and whole-blood-derived platelet units. The sFasL content of either 30-day stored RBCs or 5-day stored platelets was approximately 20 ng/mL. The infusion of sFasL in transfused blood components may bind the Fas molecule expressed on the natural killer (NK) and cytotoxic T cells of the recipient, thus preventing the binding of the Fas molecule on these immune cells to the Fas-ligand on virus-infected cells. Therefore, the infusion of sFasL in transfused blood components may impair the function of NK and cytotoxic T cells in the recipient, thus preventing apoptosis of virus-infected cells.126,127 Ghio et al124 and Puppo et al125 demonstrated the functional capacity of sFasL molecules in stored blood components by culturing Jurkat cells in the presence of plasma supernatant from stored RBCs. Jurkat cells express Fas, and are thus susceptible to the effects of sFasL present in transfused blood components. In this in-vitro experiment, sFasL from the plasma supernatant of stored RBCs triggered apoptosis of the Jurkat cells, which was measured by flow cytometry. These authors124,125 also documented the accumulation of soluble HLA Class I molecules in stored RBCs and platelets, although the concentrations achieved were only 4 ng/mL in 30day stored RBCs and 5-day stored platelets. Furthermore, stored supernatant plasma was shown to exercise an immunosuppressive effect in functional experiments, in that it inhibited the cytotoxic activity of lymphocytes known to be cytotoxic for cells infected with Epstein-Barr virus (EBV). This was not a nonspecific effect, because the cytotoxic activity of lymphocytes was restored after the stored supernatant plasma was depleted of soluble HLA Class I molecules. However, only supernatant plasma from stored nonWBC-reduced (as opposed to WBC-reduced) cellular blood components inhibited the cytotoxic activity of lymphocytes directed against EBV-infected cells. Similarly, prestorage WBC reduction prevented the accumulation of sFasL in stored RBCs.

anaphylatoxins131 have also been reported during storage, but their significance in the context of TRIM is uncertain.

Apoptotic WBCs

Evidence from RCTs

Innerhofer et al128,129 reported that impaired proliferative T-cell responses, decreased CD3⫹ counts, and a state of inappropriate immune activation, along with a diminished cytolytic response, occur even after transfusion of WBC-reduced RBCs containing a median residual WBC count of 0.03 ⫻ 106 WBCs/unit (ie, a count far below the 5 ⫻ 106 limit that qualifies a cellular blood component as “WBC-reduced”). If that were the case, it would appear that not only transfused intact, immunologically competent WBCs, but also transfused apoptotic or necrotic WBCs, could be important in provoking TRIM responses. Furthermore, activation of complement components130 and formation of

With respect to infection, a recent theory133,134 attributes the purported susceptibility of transfused patients to infection to a sustained inhibition of neutrophil chemotaxis caused by TGF-β. TGF-β renders neutrophils insensitive to chemotactic stimulation. Inhibition of chemotaxis is caused by both exogenous TGF-β, contained in the supernatant of transfused blood components,133 and endogenous TGF-β produced by the recipient’s neutrophils in response to sFasL and soluble HLA molecules found in the transfused supernatant.134 However, the results of meta-analyses of medically homogeneous subsets of RCTs that reported on postoperative infection

Evidence from Animal Models If soluble biologic response modifiers and remnants of apoptotic or necrotic WBCs accumulating in blood components during storage were shown to be responsible for some of the adverse TRIM effects of ABT, WBC reduction procedures intended to prevent such TRIM effects should be performed before storage, before WBC deterioration, and before the release of soluble biologic response modifiers from cell membranes or granules (Fig 45-1). The available WBC reduction filters do not retain soluble biologic response modifiers and are also ineffective in removing cell fragments. Therefore, both biologic response modifiers and the remnants of apoptotic or necrotic cells can be expected to persist in a blood component subjected to WBC filtration after storage. The importance of the timing of WBC reduction as regards the TRIM effect has been demonstrated in experimental animals.132 Bordin et al132 showed that ABT promotes growth of established animal tumors and that the tumor-growth promoting effect of ABT can be ameliorated by prestorage (but not by poststorage) WBC reduction. These authors132 used outbred New Zealand White (NZW) rabbits with established tumors as blood recipients, and outbred California Black rabbits as allogeneic blood donors. “Syngeneic” donor blood was collected from NZW rabbits who were littermates, or siblings, of the transfusion recipients. Non-WBC-reduced allogeneic, poststorage WBCreduced allogeneic, prestorage WBC-reduced allogeneic, or syngeneic RBCs were transfused on Days 4 and 9 after the infusion of syngeneic epithelial tumor cells. All rabbits were killed 28 days after the infusion of the tumor cells, and the number of pulmonary tumor nodules was counted. Rabbits that received nonWBC-reduced allogeneic, poststorage WBC-reduced allogeneic, prestorage WBC-reduced allogeneic, or syngeneic RBCs had a median of 50.0, 39.0, 20.0, and 17.5 pulmonary nodules, respectively. The difference between non-WBC-reduced allogeneic and prestorage WBC-reduced allogeneic or syngeneic transfusion was highly significant (p ⬍0.0001), but the difference between nonWBC-reduced allogeneic and poststorage WBC-reduced allogeneic transfusion was only marginally significant (p ⫽ 0.06).

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Does not support WBC reduction Figure 45-4. Risk of postoperative infection after transfusion of prestoragefiltered WBC-reduced RBCs. Randomized controlled trials (RCTs) of ABT and postoperative infection administering prestorage-filtered allogeneic RBCs to the WBC-reduced study arm.52,58,60,63,66-70 The figure shows the odds ratio (OR) of postoperative infection in recipients of non-WBC-reduced vs WBC-reduced allogeneic RBCs, as calculated from an intention-to-treat analysis of each study; and the summary OR across the depicted RCTs, as calculated from a meta-analysis.47 A deleterious ABT effect (and thus a benefit from WBC reduction) is demonstrated by an OR ⬎1, provided that the effect is statistically significant (p ⬍0.05; ie, provided that the associated 95% CI does not include the null value of 1).

contradicted the theory that attributes the TRIM effect to WBCderived soluble mediators that accumulate during storage.47,48 Across nine RCTs transfusing RBCs WBC-reduced before storage to the WBC-reduced study arm,52,58,60,63,66-70 no TRIM effect was detected (summary odds ratio, 1.06; 95% CI, 0.91-1.24; p ⬎0.05).47 If the TRIM effect were mediated by WBC-derived soluble mediators, prestorage filtration should have abrogated an increased infection risk associated with non-WBC-reduced ABT, because it would have removed the allogeneic WBCs from the components given to the WBC-reduced arm of the studies before WBCs could release mediators into the supernatant fluid. Accordingly, a deleterious TRIM effect associated with nonWBC-reduced ABT would have been expected in this analysis, but the meta-analysis detected no such effect (Fig 45-4). In contrast, across four RCTs49,54,58,59 that transfused RBCs filtered after storage to the WBC-reduced arm, there was a more than twofold increase in the risk of infection in association with non-WBC-reduced ABT (summary odds ratio, 2.25; 95% CI, 1.12-4.25; p ⬍0.05—Fig 45-5). If the TRIM effect were mediated by WBC-derived soluble mediators, poststorage filtration should not have abrogated an increased infection risk associated with

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Figure 45-5. Risk of postoperative infection after transfusion of poststoragefiltered WBC-reduced RBCs or whole blood. Randomized controlled trials (RCTs) of ABT and postoperative infection administering poststorage-filtered allogeneic RBCs or whole blood to the WBC-reduced study arm.49,54,58,59 The figure shows the odds ratio (OR) of postoperative infection in recipients of non-WBC-reduced vs WBC-reduced allogeneic RBCs or whole blood, as calculated from an intentionto-treat analysis of each study; and the summary OR across the depicted RCTs, as calculated from a meta-analysis.47 A deleterious ABT effect (and thus a benefit from WBC reduction) is demonstrated by an OR ⬎1, provided that the effect is statistically significant (p ⬍0.05; ie, provided that the associated 95% CI does not include the null value of 1).

non-WBC-reduced ABT, because it would not have removed such mediators from the supernatant fluid of the stored RBCs given to the WBC-reduced arm of the studies. Thus, the large TRIM effect detected in this analysis (Fig 45-5) may be the result of the inclusion of three early RCTs49,54,59 that had reported an unusually large TRIM effect.48 These RCTs involved blood components no longer used in Western Europe or North America (allogeneic whole blood,49 poststorage-filtered allogeneic whole blood,49 or poststorage-filtered allogeneic RBCs54,59).

TRIM Effects Mediated by Allogeneic Mononuclear Cells The only established TRIM effect (ie, the beneficial effect of pretransplant ABT on renal allograft survival) appears to require viable WBCs. Patients awaiting renal transplantation derive less immunologic benefit from pretransplant RBC transfusions that are WBC-reduced, washed, or frozen-thawed than from

Chapter 45: Immunomodulatory and Proinflammatory Effects of Allogeneic Blood Transfusion

non-WBC-reduced ABT. Mincheff et al135 implicated the dendritic APCs of the allogeneic donor in the induction of a state of anergy in the recipient, proposing that during refrigeration APCs lose their ability to deliver costimulation. These investigators hypothesized that, following ABT, the recipient’s T cells are stimulated by allogeneic donor APCs in the absence of costimulation, and this interaction induces a state of anergy in the recipient’s T cells.

Evidence from Animal Models Animal data suggest that the TRIM effects are most likely mediated by transfused allogeneic mononuclear cells.136 Kao137 induced immune suppression in mice receiving donor WBCs free of plasma and platelets. A recent theory138 proposes that donor dendritic cells expressing both alloantigen and the OX-2 (CD200) costimulatory molecule are required for the production of the TRIM effect. CD200 is a transmembrane protein of the immunoglobulin superfamily that is expressed on various cell types, including a subpopulation of dendritic cells and some T and B cells.139 Its receptor (CD200R or OX-2R) appears only on myeloid dendritic cells and some T cells. The interaction between CD200 and its receptor provokes a tolerance signal that leads to suppression of classical T-cell-mediated responses and the generation of γδ-suppressor T cells. Thus, CD200-deficient mice have reduced ability to downregulate activation of APCs. The absence of downregulatory signals in such mice results in exaggerated inflammatory responses and increased susceptibility to autoimmune encephalitis and collagen-induced arthritis. The interaction between CD200 and its receptor suppresses macrophage function, prolongs allograft survival, and prevents allogeneic fetal loss in a mouse model of cytokine-triggered abortions.140 Clark et al138 demonstrated ABT-induced tumor growth in a murine model that employed BALB/c mice as allogeneic donors and C57B1/6 mice as blood recipients. The recipient mice received a tail vein injection of syngeneic FSL10 fibrosarcoma cells, followed by transfusion of 50 to 200 µL of allogeneic blood. Pulmonary tumor nodules were counted 3 weeks after the FSL10 cell infusion. There was a dose-response relationship between the volume of transfused allogeneic blood and the number of pulmonary tumor nodules, along with proliferation of TGF-β-positive suppressor T cells in the spleen. The tumor-growth-promoting effect of ABT was mediated by donor myeloid dendritic cells that expressed both CD11c and CD200 on their surface, because it could be blocked by monoclonal antibodies to either CD11c or CD200. (The effect could not be blocked by antibodies to CD200R, or by antibodies to other molecules participating in these interactions, an observation that implicated the subset of donor myeloid dendritic cells expressing both CD11c and CD200 in the pathogenesis of TRIM.) The interaction between the donor CD200 and its receptor on the recipient’s T cells induced proliferation of γδ-suppressor T cells that released cytokines, especially TGF-β. Physiologic concentrations of TGF-β stimulated proliferation of FSL10 fibrosarcoma cells in vitro. Because TGF-β can also suppress host

defenses against infectious agents,141 it could be the basis of the TRIM effect with regard to both postoperative infections and tumor growth, at least as regards sarcomas. Bordin and Blajchman142 reviewed the findings of animal models of ABT and cancer recurrence and reported that 17 published models had found stimulation of tumor growth by ABT, as compared with three models that had reported inhibition of tumor growth and four models that had found no effect. Data from both inbred and outbred animal models have indicated that ABT accelerates tumor growth and enhances formation of metastatic nodules.132,143-146 Allogeneically transfused mice inoculated intramuscularly with either syngeneic malignant melanoma (B16) or mastocytoma (P815) cells developed larger tumors than did syngeneically transfused mice.143 Similar results were obtained when syngeneic B16 tumor cells were infused intravenously and the numbers of pulmonary nodules enumerated.143,144 Experiments performed to investigate the effect of the tumor-cell dose showed that the ABT effect was only evident when small numbers (1.25–2.5 ⫻ 105) of tumor cells were inoculated into the host animal. The effect was not evident when large numbers of tumor cells were inoculated, suggesting that the tumor burden had a strong bearing on whether the ABT effect became manifest. The influence of the timing of ABT in enhancing pulmonarynodule formation has also been examined. Studies in both inbred (mice) and outbred (rabbits) animals have shown that ABT has a tumor-growth-promoting effect when administered before the infusion of syngeneic tumor cells.132,145 In the murine model, male C57Bl/6J mice (MHC type H-2b) were blood recipients; Balb/c mice (MHC type H-2d) were allogeneic donors; and the tumor cells were syngeneic (H-2b) methylcholanthrene-induced fibrosarcoma cells.145 As already discussed, to better replicate the situation seen clinically, the enhancement of tumor growth by ABT has been investigated in animals (mice and rabbits) that received such syngeneic and allogeneic transfusions subsequent to the inoculation of the tumor cells, and the data indicated that ABT enhanced tumor growth also in animals with established tumors.132 Using inbred animals (mice) only, another series of investigations provided similar evidence indicating that ABT given after tumor-cell engraftment enhanced tumor growth.146 Is was also shown that animals with either nonestablished or established tumors receiving non-WBC-reduced ABT developed significantly larger numbers of pulmonary nodules than did animals given WBC-reduced ABT.132,145 Finally, the tumor-growth-promoting effect of ABT can be adoptively transferred to naive animals by spleen cells harvested from allogeneically transfused animals.145 In these experiments, the number of pulmonary nodules observed in animals that had received spleen cells from allogeneically transfused animals was significantly higher than that observed in animals that had received spleen cells from animals transfused with syngeneic blood. However, the ABT effect could not be adoptively transferred to naive animals that received spleen cells derived from animals transfused with prestorage-WBC-reduced allogeneic blood.

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The clonal deletion seen in recipients of ABT refers to the removal of lymphocytes that promote the clearance of transfused alloantigens. Interactions between Fas and FasL are involved in the clonal deletion of T cells and the downregulation of cytotoxic T-cell activity. In a murine model, Hashimoto et al147 investigated the possibility of splenic-lymphocyte deletion secondary to ABT-related augmentation of apoptosis. These investigators demonstrated that non-WBC-reduced ABT upregulated the expression of Fas and FasL on CD4⫹ as well as CD8⫹ splenic T cells and could thereby promote their apoptosis. The ABT-related immune alterations could be partially prevented by WBC reduction of the transfused blood, as CD8⫹ splenic cells from mice receiving non-WBC-reduced ABT showed higher expression of Fas and FasL than cells from mice receiving WBC-reduced ABT. The data regarding the TRIM effect and infection in animal models are contradictory. Moreover, a variety of experimental conditions such as anesthesia, shock, trauma, type of surgery, blood volume, as well as timing and transfusion frequency have all been reported to have an impact on the results.148-152 In a series of studies in experimental animal, Waymack et al148-151 have demonstrated that allogeneically transfused animals had immune impairment and a poorer response to a septic challenge than did syngeneically transfused animals. In a burn model, these investigators observed that rats given ABT had higher mortality than did rats given syngeneic blood or saline.152 In a rat bacterial-peritonitis model, a significant adverse effect on survival was associated with ABT.149 In another study, ABT was associated with marked immune impairment to a bacterial challenge immediately after the transfusion.153 Moreover, ABT and, in particular, transfused allogeneic WBCs adversely affected host resistance to a gut-derived infection with Escherichia coli in a murine model.154 In addition, in a cecal ligation and puncture murine model, ABT greatly increased susceptibility to infection. These studies also indicated that spleen cells of allogeneically transfused mice produced increased quantities of the Th-2 cytokines (IL-4 and IL-10 and lesser amounts of IL-2), probably leading to increased antibody production and a decreased cell-mediated response.155 In contrast, in murine experiments using a bacterial-peritonitis model that compared syngeneic with allogeneic transfusion, the latter was shown not to influence overall survival of animals challenged with E. coli.156 Similarly, while a clear negative effect of shock was detected, no adverse effect of transfusions, either syngeneic or allogeneic, was observed in a rat model.157

Microchimerism HLA compatibility between donor and recipient may result in the persistence of allogeneic donor WBCs, including dendritic APCs, in the recipient. Such long-term engraftment and survival of small numbers of donor cells (microchimerism) has been proposed as a possible mechanism of TRIM.158 Microchimerism could cause the downregulation of the recipient’s immune response, resulting in tolerance to donor alloantigens and allograft survival. Microchimerism results in the release of IL-4,

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IL-10, and TGF-β from T-helper type 2 (Th-2) lymphocytes.159 These cytokines have been shown to inhibit the production of T-helper type 1 (Th-1) cells and to deactivate cytotoxic T cells, thereby suppressing allograft rejection. Along similar lines, ABT was shown to cause a shift in peripheral T-cell cytokine secretion toward that of the Th-2 phenotype, and to downregulate Th-1 cytokine secretion. Impairment of Th-1 cytokine secretion results in impairment of various functions of cellular immunity (including antigen processing, macrophage activation, the T-cell cytotoxic function, and the neutrophil and monocyte cytocidal activity) that are supported by Th-1 cytokines, such as IL-2, IL-12, and interferon-γ.160 In 1995, Lee et al161 employed quantitative allele-specific polymerase chain reaction methods to demonstrate a thousandfold expansion of allogeneic donor WBCs in the recipient’s circulation 3 to 5 days following transfusion in otherwise healthy adults undergoing elective orthopedic surgery. The allogeneic WBCs were cleared from the circulation within 2 weeks. The finding was verified in a canine transfusion model and—as expected—irradiation of blood products abrogated the allogeneic donor WBC expansion phase. However, in 1999, the same group documented high-level and long-lasting WBC microchimerism among selected victims of traumatic injury who had received a large number of very fresh units of blood during resuscitation.162 In some of these trauma patients, up to 3% to 4% of circulating WBCs were of donor origin as long as 2 years following transfusion. Analysis of lymphocyte subsets using immunomagnetic bead enrichment showed that both lymphoid and myeloid lineages were represented. Long-term transfusion-associated microchimerism appears to be a common, albeit only recently recognized, complication of ABT163 that has hitherto been demonstrated only in trauma patients.162 Injury produces an immunosuppressive and inflammatory milieu in which very fresh blood components, containing WBCs capable of replication, are often transfused in large quantities. Transfusion-associated microchimerism is present in approximately half of transfused, severely injured patients at hospital discharge.163 In approximately 10% of the patients, the chimerism associated with a single blood donor may increase in magnitude over months to years, representing up to 2% to 5% of circulating WBCs.162,163 Nonetheless, in other patient populations, such as those infected with HIV, ABT-induced microchimerism appears to be transient.164 If microchemism does lead to the downregulation of the host’s immune response, it is important to note that microchimerism is detected also following administration of WBCreduced allogeneic RBCs.165 Utter et al166 examined a subgroup of the trauma patients enrolled in the RCT of Nathens et al70 that had randomly assigned patients to receive non-WBC-reduced or WBC-reduced allogeneic RBCs filtered before storage. Nine of 32 (28%) patients in the non-WBC-reduced group developed microchimerism, as compared with 13 of 35 (37%) patients in the WBC-reduced group (p ⫽ 0.43).166 Several months after the transfusion, subjects with transfusion-associated

Chapter 45: Immunomodulatory and Proinflammatory Effects of Allogeneic Blood Transfusion

microchimerism were no more likely than subjects without transfusion-associated microchimerism to have at least one symptom suggestive of chronic graft-vs-host disease (64% vs 76%, respectively), indicating that transfusion-associated microchimerism is prevalent in this patient population but unlikely to be associated with symptoms.166 Similarly, Fresland et al167 reported that microchimerism after ABT could be induced by transfusion of RBCs that were WBCreduced by prestorage filtration. Moreover, in that study,167 microchimerism was not dose dependent, and it could be induced even by RBC units that had been stored for more than 3 weeks.

1

0.1

10

Busch et al 1.0

Houbiers et al

1.0

Heiss et al 2.0

Evidence from RCTs The authors know of only one RCT62 that had been specifically designed to test a possible TRIM effect of allogeneic mononuclear cells. The Viral Activation Transfusion Study (VATS)62 transfused unmodified allogeneic RBCs stored for ⬍2 weeks (and thus containing relatively undamaged allogeneic mononuclear cells) to the non-WBC-reduced arm. Also, the two study arms were controlled for comparable duration of storage of the transfused RBCs. There was no difference between the study arms in the HIV or CMV viral load or the length of survival. Median survival was 13.0 months in recipients of prestorage-filtered allogeneic RBCs, as compared with 20.5 months in recipients of non-buffycoat-reduced allogeneic RBCs (p ⫽ 0.12). Thus, the VATS results have impugned the theory attributing the TRIM effect to allogeneic mononuclear cells. It should be noted, however, that no other RCT transfusing fresh components to the non-WBC-reduced study arm has been reported, and the effect of fresh components has not been studied in the context of more “traditional” TRIM effects (ie, cancer recurrence or postoperative infection). Despite the convincing evidence provided by animal models for a relationship between transfusion of allogeneic mononuclear cells and tumor recurrence, no RCT of ABT and cancer recurrence has transfused fresh non-WBC-reduced RBCs to the nonWBC-reduced study arm to test for the effect of immunologically competent allogeneic mononuclear cells seen in animal models. Moreover, no RCT of ABT and cancer recurrence has enrolled patients with sarcomas—tumors whose growth is stimulated by TGF-β138—or patients with tumors for which the immune response plays a major role. (These include skin tumors—such as melanomas, keratoacanthomas, squamous and basal-cell carcinomas—and certain virus-induced tumors—notably Kaposi’s sarcoma and certain lymphomas.168) Instead, the three available RCTs50,52,71 of ABT and cancer recurrence enrolled patients with colorectal cancer—a tumor that is not sufficiently antigenic to render an impairment of the host’s immunity capable of facilitating tumor growth, and whose cells have not been shown to be stimulated by TGF-β. Thus, these three RCTs50,52,71 permit very limited inference with regard to the biologic significance of the TRIM mediators discussed in this chapter. No adverse TRIM effect of ABT on cancer recurrence is detected across the three

Summary odds ratio

Does not support WBC reduction or autologous transfusion

1.0

Supports WBC reduction or autologous transfusion

Figure 45-6. Cancer recurrence after transfusion. Randomized controlled trials (RCTs) investigating the association of non-WBC-reduced allogeneic blood transfusion (ABT) with cancer recurrence.50,52,71 For each RCT, the figure shows the odds ratio (OR) of cancer recurrence in recipients of non-WBC-reduced vs WBC-reduced allogeneic or autologous RBCs or whole blood, as calculated from an intention-to-treat analysis; and the summary OR across the depicted RCTs, as calculated from a meta-analysis.167 Each OR is surrounded by its 95% confidence interval (CI). A deleterious effect of ABT (and thus a benefit from autologous transfusion or WBC reduction) is demonstrated by an OR ⬎1, provided that the effect is statistically significant (p ⬍0.05; ie, provided that the associated 95% CI does not include the null value of 1).

studies (Fig 45-6). The summary odds ratio of cancer recurrence in recipients of non-WBC-reduced allogeneic compared with autologous or WBC-reduced allogeneic RBCs is 1.04 (95% CI, 0.81-1.35; p ⬎0.05).169

Summary and Conclusions TRIM encompasses the laboratory immune aberrations that occur after ABT (Table 45-1) and their established or purported clinical effects. TRIM is a real biologic phenomenon resulting in at least one established beneficial clinical effect in humans (the enhanced survival of renal allografts), but the existence of deleterious clinical TRIM effects has not yet been confirmed. Initially, TRIM encompassed effects attributable to ABT by immunomodulatory mechanisms (eg, cancer recurrence, postoperative infection, or virus activation); more recently, TRIM has also included effects attributable to ABT by proinflammatory mechanisms (eg, multiple-organ failure or mortality).

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The mechanism(s) of the TRIM effect(s) remain(s) elusive, and it is possible that a large number of biologic mechanisms may underlie these effect(s). The infusion of foreign antigen in either soluble or cell-associated form has been shown to induce immune suppression and anergy, as well as clonal deletion in studies in experimental animals. However, most studies evaluating proposed mechanisms have been conducted in rodents, and the findings may not be applicable to the human immune system.170 Dzik171 suggested that there may be two categories of a TRIM effect: one that is HLA-dependent and directed against adaptive immunity and another that is nonspecific and directed against innate immunity. The nonspecific effect might result from the infusion of WBCs that undergo apoptosis during refrigerated storage. Immunosuppression resulting from the infusion of apoptotic cells may be linked to TGF-β. Support for the theory that TRIM is caused by the allogeneic WBCs has come mainly from data from animal models. These have shown that animals receiving allogeneic buffy-coat leukocytes develop significantly more pulmonary tumor nodules than do animals given either plasma or prestorage-WBC-reduced whole blood.132 It is possible that prestorage WBC reduction may prevent the accumulation of soluble mediators that are actively synthesized and released by WBCs during RBC storage, and that such WBC-derived soluble mediators are involved in the immunomodulation observed following ABT. However, storage lesions of RBCs and platelets occur even when the units have been WBC-reduced before storage (WBC removal only slightly improves these storage lesions—see Chapters 4 and 12). The totality of the evidence from RCTs does not demonstrate the kind of deleterious TRIM effect that would justify universal WBC reduction specifically for prevention of this effect (ie, a TRIM effect manifest across all clinical settings and transfused RBC products), although universal WBC reduction may be justified on the basis of other WBC-related adverse effects.136 Non-WBC-reduced ABT is associated with an increased risk of short-term (up to 3 months after transfusion) mortality from all causes specifically in cardiac surgery. Even in this setting, the reasons for the excess deaths attributed to non-WBC-reduced ABT remain elusive. The initial hypothesis suggested that non-WBC-reduced ABT may predispose to MOF which, in turn, may predispose to mortality.58 However, no cardiac-surgery RCT has demonstrated an association between non-WBC-reduced ABT and MOF. The TRIM effect seen in cardiac surgery deserves further study to pinpoint the cause(s) of the excess deaths, but—now that the majority of transfusions in Western Europe and North America are WBC-reduced—the undertaking of further RCTs comparing recipients of non-WBC-reduced vs WBC-reduced RBCs in cardiac surgery is difficult. Until further studies are conducted, whether to opt for WBC reduction of all cellular blood components transfused in cardiac surgery (in the absence of information on the specific cause or causes of death that can be ascribed to non-WBC-reduced ABT) is a policy decision that will have to be made on the basis of available data (Fig 45-3). The authors believe that WBC reduction of all cellular blood

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components transfused in cardiac surgery is appropriate based on the accumulated evidence. It must be emphasized that, at least in the case of some adverse TRIM effects, the evidence for the existence of such effects may not be available because the requisite studies have not been conducted. An effect of the transfusion of allogeneic mononuclear cells would be expected to occur on tumor recurrence for example, based on the convincing findings from experimental animals. However, no available RCT has transfused fresh non-WBC-reduced RBCs to the non-WBC-reduced study arm to specifically study the effect of allogeneic mononuclear cells. Moreover, no available RCT has enrolled patients with a tumor whose growth would be expected to be stimulated by ABT. A possible adverse TRIM effect of allogeneic mononuclear cells has similarly not been adequately investigated in the areas of postoperative infection and mortality. Indeed, in many cases, the preclinical studies were conducted and the hypotheses about mechanisms formulated after clinical studies (including RCTs) had already presented data-derived hypotheses to account for unexpected ABT effects. Because it has not been possible to conduct further RCTs after the hypotheses about TRIM mediators (Fig 45-1) were crystallized, whether some adverse TRIM effects exist (or not) in humans may remain a mystery. Moreover, it is possible that the available RCTs have targeted outcomes that did not capture the true nature of the ABT effect. If this effect were “proinflammatory” rather than “immunomodulatory,” it would have been expected to result not in clinical impairment of the recipient’s immunity, but in multiple-organ dysfunction. MOF and related outcomes were not studied in most completed RCTs. New data continue to appear,172-177 and the controversy on the purported deleterious TRIM effects continues to persist.45-48,178-182 Because the use of WBC-reduced allogeneic RBCs and platelets is becoming widespread, the opportunity to conduct RCTs to establish the existence of adverse TRIM effects mediated by allogeneic WBCs is removed. Despite the widespread acceptance of WBC reduction as a standard of care in many countries, however, it remains unclear whether deleterious clinical TRIM effects of ABT truly exist, and—in the event that they do—whether they are mediated, directly or indirectly, by allogeneic WBCs. The uncertainties and hypotheses discussed here underline the need for further research!

Disclaimer The authors have disclosed no conflicts of interest.

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Chapter 45: Immunomodulatory and Proinflammatory Effects of Allogeneic Blood Transfusion

85. Vamvakas EC, Carven JH. Allogeneic blood transfusion and postoperative duration of mechanical ventilation: Effects of red cell supernatant, platelet supernatant, plasma components, and total transfused fluid. Vox Sang 2002;82:141-9. 86. Maetani S, Nishikawa T, Hirakawa A, et al. Role of blood transfusion in organ system failure following major abdominal surgery. Ann Surg 1986;203:275-81. 87. Sauaia A, Moore FA, Moore EE, et al. Early predictors of postinjury multiple-organ failure. Arch Surg 1994;129:39-45. 88. Trann DD, van Onselen EB, Wensink AJ, et al. Factors related to multiple-organ system failure and mortality in a surgical intensive care unit. Nephrol Dial Transplant 1994;9(Suppl.4):172S-8S. 89. Vincent JL, Baron JF, Reinhart K, et al. Anemia and blood transfusion in critically ill patients. JAMA 2002;288:1499-507. 90. Vedder NB, Fouty BW, Winn RK, Harlan JM. Role of neutrophils in generalized reperfusion injury associated with resuscitation from shock. Surgery 1989;106:509-16. 91. Anderson BO, Harken AH. Multiple-organ failure: Inflammatory priming and activation sequences promote autologous tissue injury. J Trauma 1990;30:S44-S9. 92. Botha AJ, Moore FA, Moore EE, et al. Postinjury neutrophil priming and activation states: Therapeutic challenges. Shock 1995;3:157-66. 93. Santamaria F, Villa MP, Werner B, et al. The effect of transfusion on pulmonary function in patients with thalassemia major. Pediatr Pulmonol 1994;18:139-43. 94. Hébert PC, Blajchman MA, Cook DJ, et al. Do blood transfusions improve outcomes related to mechanical ventilation? Chest 2001;119:1850-7. 95. Netzer G, Shah CV, Iwashyna TJ, et al. Association of RBC transfusion with mortality in patients with acute lung injury. Chest 2007;132:1116-23. 96. Murphy GJ, Reeves BC, Rogers CA, et al. Increased mortality, postoperative morbidity, and cost after red-blood-cell transfusion in patients having cardiac surgery. Circulation 2007;116:2544-52. 97. Vamvakas EC, Carven JH. Transfusion and postoperative pneumonia in coronary artery bypass graft surgery: Effect of the length of storage of transfused red cells. Transfusion 1999;39:701-10. 98. Mynster T, Nielsen HJ. The impact of storage time of transfused blood on postoperative infectious complications in rectal cancer surgery. Scand J Gastroenterol 2000;35:212-7. 99. Offner PLJ, Moore EE, Biff WL, et al. Increased rate of infection associated with transfusion of old blood after severe injury. Arch Surg 2002;137:711-6. 100. Leal-Noval SR, Jara-Lopez I, Garcia-Garmendix JL, et al. Influence of erythrocyte concentrate storage time on postsurgical morbidity in cardiac surgery patients. Anesthesiology 2003;98:815-22. 101. Purdy FR, Tweeddale MG, Merrick PM. Association of mortality with age of blood transfused in septic ICU patients. Can J Anaesth 1997;44:1256-61. 102. Basran S, Frumento RJ, Cohen A, et al. The association between duration of storage of transfused red blood cells and morbidity and mortality after reoperative cardiac surgery. Anesth Analg 2006;103:15-20. 103. van de Watering LMG, Lorinser J, Verseegh M, et al. Effects of storage time on the prognosis of coronary artery bypass graft patients. Transfusion 2006;46:1712-8. 104. Gorman Koch C, Li L, Sessler DI, et al. Duration of red cell storage and complications after cardiac surgery. N Engl J Med 2008;358:1229-31.

105. Baron JF, Gourdin M, Bertrand M, et al. The effect of universal leukodepletion of packed red blood cells on postoperative infections in high-risk patients undergoing abdominal aortic surgery. Anesth Analg 2002;94:529-37. 106. Llewelyn CA, Taylor RS, Todd AA, et al. The effect of universal leukoreduction on postoperative infections and length of stay in elective orthopedic and cardiac surgery. Transfusion 2004;44:489-500. 107. Volkova N, Klapper E, Pepkowitz SH, et al. A case-control study of the impact of WBC reduction on the cost of hospital care for patients undergoing coronary artery bypass graft surgery. Transfusion 2002;42:1123-6. 108. Hébert PC, Fergusson D, Blajchman MA, et al. Clinical outcomes following institution of the Canadian universal leukoreduction program for red blood cell transfusions. JAMA 2003;289:1941-9. 109. Fergusson D, Hébert PC, Lee SK, et al. Clinical outcomes following institution of universal leukoreduction of blood transfusions for premature infants. JAMA 2003;289:1950-6. 110. Blumberg N, Heal JM, Cowles JW, et al. Leukocyte-reduced transfusions in cardiac surgery: Results of an implementation trial. Am J Clin Pathol 2002;118:376-81. 111. Vamvakas EC. White-blood-cell containing allogeneic blood transfusion, postoperative infection, and mortality: A meta-analysis of observational, “before-and-after” studies. Vox Sang 2004;86:111-9. 112. Magee CC, Sayegh MH. Peptide-mediated immunosuppression. Curr Opin Immunol 1997;9:669-75. 113. Krensky AM, Clayberger C. Structure of HLA molecules and immunosuppressive effects of HLA derived peptides. Int Rev Immunol 1996;13:173-85. 114. Chueh SC, Tian L, Wang M, et al. Induction of tolerance toward rat cardiac allografts by treatment with allochimeric class I MHC antigen and FTY720. Transplantation 1997;64:1407-14. 115. Naji A. Induction of tolerance by intrathymic inoculation of alloantigen. Curr Opin Immunol 1996;8:704-9. 116. Chowdhury NC, Murphy B, Sayegh MH, et al. Acquired systemic tolerance to rat cardiac allografts induced by intrathymic inoculation of synthetic polymorphic MHC class I allopeptides. Transplantation 1996;62:1878-82. 117. Vermont-Desroches C, Rigal D, Blourde C, Bernaud J. Immunosuppressive property of a very high purity antihaemophilic preparation: A low-molecular-weight component inhibits an early step of PHA-induced cell activation. Br J Haematol 1992;80:370-7. 118. Wadhwa M, Dilger P, Tubbs J, et al. Identification of transforming growth factor-β as a contaminant in factor VIII concentrates: A possible link with immunosuppressive effects in hemophiliacs. Blood 1994;84:2021-30. 119. Hansen MB, Galle P, Salomo M, et al. Transfusion-related inhibition of cytokines: Experimental transfer of neutralizing autoantibodies to interleukin-6 by plasma transfusions. Vox Sang 2007;92:213-23. 120. Nielsen HJ. Detrimental effects of perioperative blood transfusion. Br J Surg 1995;82:582-7. 121. Nielsen HJ, Reimert CM, Pedersen AN, et al. Time-dependent, spontaneous release of white cell- and platelet-derived bioactive substances from stored human blood. Transfusion 1996;36:960-5. 122. Bury TB, Corhay JL, Radermecker MF. Histamine-induced inhibition of neutrophil chemotaxis and T-lymphocyte proliferation in man. Allergy 1992;47:624-9. 123. Peterson CG, Skoog V, Venge P. Human eosinophil cationic proteins (ECP and EPX) and their suppressive effects on lymphocyte proliferation. Immunobiology 1986;171:1-13.

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124. Ghio M, Contini P, Mazzei C, et al. Soluble HLA Class I, HLA Class II, and Fas ligand in blood components: A possible key to explain the immunomodulatory effects of allogeneic blood transfusion. Blood 1999;93:1770-7. 125. Puppo F, Contini P, Ghio M, et al. Soluble human MHC class I molecules induce soluble Fas-ligand secretion and trigger apoptosis in activated CD8⫹ Fas (CD95)⫹ T lymphocytes. Int Immunol 2000;12:195-203. 126. Pitti RM, Marster SA, Lawrence DA, et al. Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer. Nature 1998;396:699-703. 127. Ashkenazi A, Dixit VM. Death receptors: Signaling and modulation. Science 1998; 281:1305-8. 128. Innerhofer P, Luz G, Spotl L, et al. Immunologic changes following transfusion of autologous or allogeneic buffy-coated-poor versus leukocyte-depleted blood in patients undergoing arthroplasty. I. Proliferative T-cell responses and T-helper/T-suppressor cell balance. Transfusion 1999;39:1089-96. 129. Innerhofer P, Tilz G, Fuchs D, et al. Immunologic changes following transfusion of autologous or allogeneic buffy-coat-poor versus leukocyte-depleted blood transfusions in patients undergoing arthroplasty. II. Activation of T-cells, macrophages and cellmediated lympholysis. Transfusion 2000;40:821-7. 130. Hyllner M, Arnestad JP, Bengston JP, et al. Complement activation during storage of whole blood, red cells, plasma, and buffy coat. Transfusion 1997;37:264-8. 131. Schleuning M, Utz H, Heim M, Mempel W. Effects of leukocyte depletion on the formation of anaphylatoxins in stored whole blood. Infusionsther Transfusionmed 1992;19:242-4. 132. Bordin JO, Bardossy L, Blajchman MA. Growth enhancement of established tumors by allogeneic blood transfusion in experimental animals and its amelioration by leukodepletion: The importance of timing of the leukodepletion. Blood 1994;84:344-8. 133. Ghio M, Ottonello L, Contini P, et al. Transforming growth factorbeta-1 in supernatants from stored red blood cells inhibits neutrophil locomotion. Blood 2003;102:1100-7. 134. Ottonello L, Ghio M, Contini P, et al. Nonleukoreduced red blood cell transfusion induces a sustained inhibition of neutrophil chemotaxis by stimulating in vivo production of TGF-β-1 by neutrophils: Role of the immunoglobulin-like transcript 1, sFasL, and sHLA-I. Transfusion 2007;47:1395-404. 135. Mincheff MS, Meryman HT, Kapoor V, et al. Blood transfusion and immunomodulation: A possible mechanism. Vox Sang 1993;65:18-24. 136. Bordin JO, Heddle NM, Blajchman MA. Biologic effects of leukocytes present in transfused cellular blood products. Blood 1994;84:1705-21. 137. Kao KJ. Induction of humoral immune tolerance to major histocompatibility complex antigens by transfusions of UV-B irradiated leukocytes. Blood 1996;88:4375-82. 138. Clark DA, Gorczynski RM, Blajchman MA. Transfusion-related immunomodulation due to peripheral blood dendritic cells expressing the CD200 tolerance signaling molecules and alloantigen. Transfusion 2008;48:214-21. 139. Barclay AN, Wright GJ, Brooke G, Brown MH. CD200 and membrane protein interactions in the control of myeloid cells. Trend Immunol 2003;23:285-90. 140. Gorczynski RM, Hadidi S, Yu G, Clark DA. The same immunoregulatory molecules contribute to successful pregnancy and transplantation. Am J Reprod Immunol 2002;48:18-26.

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141. Reed SG. TGF-β in infections and infectious disease. Microbes Infect 1999;1:1313-25. 142. Bordin JO, Blajchman MA. Blood transfusion and tumor growth in animal models. In: Vamvakas EC, Blajchman MA, eds. Immunomodulatory effects of allogeneic blood transfusion. Bethesda, MD: AABB Press, 1999:29-42. 143. Shirwadkar S, Blajchman MA, Frame B, Singal DP. Effect of allogeneic blood transfusion on solid tumor growth and pulmonary metastases in mice. J Cancer Res Clin Oncol 1992;118:76-180. 144. Shirwadkar S, Blajchman MA, Frame B, et al. Effect of blood transfusions on experimental pulmonary metastases in mice. Transfusion 1990;30:188-90. 145. Blajchman MA, Bardossy I, Carmen R, et al. Allogeneic blood transfusion-induced enhancement of tumor growth: Two animal models showing amelioration by leukodepletion and passive transfer using spleen cells. Blood 1993;81:1880-82. 146. Francis DMA, Clunie GJA. Influence of the timing of blood transfusion on experimental tumor growth. J Exp Surg Res 1993;54: 237-41. 147. Hashimoto MN, Kimura EY, Yamamoto M, Bordin JO. Expression of Fas and Fas ligand on spleen T cells of experimental animals after unmodified or leukoreduced allogeneic blood transfusions. Transfusion 2004;44:158-63. 148. Waymack JP, George CD, Pietsch JD, et al. Effect of varying number and volume of transfusions on mortality rate following septic challenge in an animal model. World J Surg 1987;11:387-91. 149. Waymack JP, Warden GD, Alexander JW, et al. Effect of blood transfusion and anesthesia on resistance to bacterial peritonitis. J Surg Res 1987;42:528-35. 150. Waymack JP, Robb E, Alexander JW. Effect of transfusion on immune function in a traumatized animal model. II. Effect on mortality rate following septic challenge. Arch Surg 1987;122:935-9. 151. Waymack JP, Miskell P, Gonce S. Alterations in host defense associated with inhalation anesthesia and blood transfusion. Anesth Analg 1989;69:163-8. 152. Waymack JP, Gallon L, Barcelli U, Alexander JW. Effect of blood transfusions on macrophage function in a burned animal model. Curr Surg 1986;43:305-7. 153. Galandiuk S, George CD, Pietsch JD, et al. An experimental assessment of the effect of blood transfusion on susceptibility to bacterial infection. Surgery 1990;108:567-71. 154. Gianotti L, Pyles T, Alexander JW, et al. Identification of the blood component responsible for increased susceptibility to gut-derived infection. Transfusion 1993;33:458-65. 155. Babcock GF, Alexander JW. The effects of blood transfusion on cytokine production by Th-1 and Th-2 lymphocytes in the mouse. Transplantation 1996;61:465-8. 156. Goldman M, Frame B, Singal DP, Blajchman MA. Effect of blood transfusion on survival in a mouse bacterial peritonitis model. Transfusion 1991;31:710-12. 157. Beko KR, Tran HO, Hewitt CW, et al. Mechanisms of prior blood transfusion-cyclosporine-induced tolerance: A potential role for immune-cellular chimerism. Transplant Proc 1991;23:147-8. 158. Dzik WH. Mononuclear cell microchimerism and the immunomodulatory effect of transfusion. Transfusion 1994;34:1007-12. 159. Gafter U, Kalechman Y, Sredni B. Blood transfusion enhances production of T-helper-2 cytokines and transforming growth factor beta in humans. Clin Sci (Lond) 1996;91:519-23.

Chapter 45: Immunomodulatory and Proinflammatory Effects of Allogeneic Blood Transfusion

160. Kirkley SA, Cowles J, Pellegrini VD Jr, et al. Blood transfusion and total joint replacement surgery: T-helper 2 cytokine secretion and clinical outcome. Transfus Med 1998;8:195-204. 161. Lee TH, Donegan E, Slichter S, Busch M. Transient increase in circulating donor leukocytes after allogeneic transfusion in immunocompetent recipients compatible with donor cell proliferation. Blood 1995; 85:1207-14. 162. Lee TH, Paglieroni T, Ohto H, et al. Survival of donor leukocyte subpopulations in immunocompetent transfusion recipients: Frequent long-term microchimerism in severe trauma patients. Blood 1999;93:3127-39. 163. Reed W, Lee TH, Norris PJ, et al. Transfusion-associated microchimerism: A new complication of blood transfusions in severely ill patients. Semin Hematol 2007;44:24-31. 164. Kruskall MS, Lee TH, Assmann SF, et al. Survival of transfused donor white blood cells in HIV-infected recipients. Blood 2001;98:272-9. 165. Lee TH, Paglieroni T, Utter TH, et al. High-level long-term whiteblood-cell microchimerism after transfusion of leukoreduced blood components to patients resuscitated after severe traumatic injury. Transfusion 2005;35:1280-90. 166. Utter GH, Nathens AB, Lee TH, et al. Leukoreduction of blood transfusions does not diminish transfusion-associated microchimerism in trauma patients. Transfusion 2006;46:1863-9. 167. Flesland O, Ip LSK, Storlien AS, et al. Microchimerism in immune-competent patients related to the leukocyte content of transfused red-blood-cell-concentrates. Transfus Apher Sci 2004;31:173-80. 168. Lowry WS, Clark DA, Hanneman JH. Skin cancer and immunosuppression. Lancet 1973;i:1290-1. 169. Vamvakas EC. Transfusion-associated cancer recurrence and infection: Meta-analysis of the randomized controlled clinical trials. Transfusion 1996;36:175-86.

170. Goodarzi MO, Lee TH, Pallavicini MG, et al. Unusual kinetics of white cell clearance in transfused mice. Transfusion 1995;35:145-9. 171. Dzik WH. Apoptosis, TGF-beta, and transfusion-related immunosuppression: Biologic versus clinical effects. Transfusion Apheresis Sci 2003;29:127-9. 172. Fung MK, Rao N, Rice J, et al. Leukoreduction in the setting of open-heart surgery: A prospective cohort-controlled study. Transfusion 2004;44:30-5. 173. Spiess BD, Royston D, Levy JH, et al. Platelet transfusions during coronary artery bypass graft surgery are associated with serious adverse outcomes. Transfusion 2004;44:1143-8. 174. Rao SV, Jollis JG, Harrington RA, et al. Relationship of blood transfusion and clinical outcomes in patients with acute coronary syndromes. JAMA 2004;292:1555-62. 175. Fung M, Moore F, Ridenour M, et al. Clinical effects of reverting from leukoreduced to non-leukoreduced blood in cardiac surgery. Transfusion 2006;46:386-91. 176. Karkouti K, Wijeysundera DN, Yau TM, et al. Platelet transfusions are not associated with increased morbidity or mortality in cardiac surgery. Can J Anesth 2006;53:279-87. 177. Vamvakas EC. Platelet transfusion and postoperative infection in cardiac surgery. Transfusion 2007;47:352-4. 178. Corwin RL, AuBuchon JP. Is leukoreduction of blood components for everyone? JAMA 2003;289:1993-5. 179. Wallis JP. Leukoreduction vs. buffy-coat depletion and the safety of blood transfusion. JAMA 2003;290:1580. 180. Dzik W. Platelet transfusion and cardiac surgery: A cautionary tale. Transfusion 2004;44:1132-4. 181. Vamvakas EC. Platelet transfusion and adverse outcomes. Lancet 2004;264:1736-8. 182. Blumberg N. Deleterious clinical effects of transfusion immunomodulation: Proven beyond a reasonable doubt. Transfusion 2005;45(Suppl):33S-9S.

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PART II

46

Infectious Hazards of Transfusion

Transfusion-Transmitted Hepatitis Harvey J. Alter1 & Juan I. Esteban-Mur2 1

Chief, Clinical Studies, and Associate Director for Research and Department of Transfusion Medicine, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland, USA 2 Section Head, Liver Unit, Department of Medicine, Hospital Universitari Vall d’Hebron, Universitat Autonoma de Barcelona, Centro de Investigaciones Biomédicas en Red (Ciberehd), Instituto de Salud Carlos III, Barcelona, Spain

Hepatitis B virus (HBV) infection, originally called serum hepatitis, was definitively shown to be a serious, and sometimes fatal complication of blood transfusion when unscreened blood and plasma were massively transfused during World War II. Despite this clinical recognition, little was known of the causative agent until the 1960s when the serendipitous finding of an antigen in the serum of an Australian aborigine (Australia antigen)1 ultimately led to the linkage of this antigen to human hepatitis.2 Further investigation revealed that the Australia antigen represented the surface protein of a viral particle (Dane particle) that was later shown to be the hepatitis B virion. Once this relationship was understood, the Australia antigen was renamed the hepatitis B surface antigen (HBsAg) and tests to detect this antigen in blood donors were introduced in the United States in 1970. This screening measure, plus the universal adoption of an all volunteer donor system, resulted in a near 70% reduction in total posttransfusion hepatitis and a greater than 85% reduction in transfusion-associated hepatitis B.3 Improvements in test sensitivity for HBsAg and the addition of screening assays for the detection of antibody to the hepatitis B core (nucleocapsid) protein (anti-HBc) in 1987 further reduced transfusion-associated hepatitis B to a calculated incidence of approximately one infection in every 150,000 transfusions. Rarely do these infections result in clinical disease and
95% of HBV infections in adults are spontaneously cleared. Hence, the current disease burden of transfusion-transmitted hepatitis B infection is very small. Following the development of assays for HBsAg detection, it became apparent that HBV was not the primary causative agent of transfusion-associated hepatitis (TAH) and that some additional agent or agents accounted for at least 75% of cases.4 It was initially thought that some or all of these non-B cases might be caused by the hepatitis A virus (HAV), the only other human hepatitis virus known at that time. However, HAV was definitively

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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identified in 19735 and when prospectively followed non-B hepatitis cases were retrospectively tested for antibody seroconversion to HAV, it was shown that none were HAV related. Thus, it became apparent that there was a third human hepatitis virus and this received the exclusionary designation, the non-A, non-B (NANB) agent, pending its further characterization. Although the NANB agent was shown to be transmissible to chimpanzees and was partially characterized in that model, it took more than a decade before the true nature of the NANB agent was revealed by the sophisticated approaches of the emerging field of molecular biology. In 1988, Houghton and coworkers at Chiron Corporation cloned the primary agent of NANB hepatitis, unraveled its molecular sequence, and expressed epitopes that now serve as the basis for highly specific serologic assays routinely used for blood donor screening.6 The NANB agent was then renamed the hepatitis C virus (HCV) and was found responsible for at least 90% of cases of TAH.7 The introduction of screening assays to detect antibody to HCV and subsequently the detection of amplified HCV RNA has resulted in a dramatic reduction in the incidence of transfusion-associated hepatitis C that is now calculated to be approximately one infection per 1.5 to 2.0 million transfusions.8 In addition, virus inactivation procedures for pooled plasma products, including clotting factor concentrates, has rendered these formerly high-risk products virtually hepatitis-free. Efforts are now under way to develop virus inactivation procedures for cellular blood components. Although this will be more difficult, such an accomplishment would presage the end of TAH, as well as many other transfusion-transmitted infections.

Incidence of Transfusion-Associated Hepatitis The incidence of TAH in the United States was formerly based on data from prospective studies, but as the incidence decreased to rates less than 1%, it became logistically and economically unfeasible to conduct prospective studies with sufficiently large numbers of subjects to achieve a statistically valid incidence. The incidence of TAH is now derived from mathematical modeling

Chapter 46: Transfusion-Transmitted Hepatitis

based on 1) the incidence of HBV and HCV infections among repeat blood donors and estimates of the window period between exposure to these agents and the first detectable marker of infection in recipients (incidence-window period model) or 2) the yield of new infections based on detection of nucleic acid in the absence of antibody [nucleic acid amplification test (NAT)-yield model]. The need to utilize mathematical models is a reflection of the success of blood bank programs in interdicting donors with high-risk behavior and in detecting those with serologic or molecular markers of hepatitis virus infection. Before mathematical modeling, determination of the incidence of TAH required prospective studies to detect biochemical evidence of liver disease because 75% of cases of TAH are clinically inapparent and because fewer than 10% of overt cases are reported to the transfusion service. Before 1970, a prospective study of patients undergoing open-heart surgery at the National Institutes of Health (NIH)9 showed that the incidence of TAH exceeded 30%. The introduction of an all-volunteer donor system and first-generation assays for HBsAg dramatically reduced the incidence of TAH to the range of 10% to 12%.3 That incidence persisted through the 1970s despite increasingly sensitive tests for HBsAg, but declined from 10% to 6% from 1980 to 1987.9 By 1989, the cumulative effect of more stringent donor questioning regarding high-risk behaviors, the introduction of human immunodeficiency virus (HIV) and anti-HBc assays as surrogate markers for NANB hepatitis, the routine virus inactivation of plasma products, and the more judicious use of blood in the wake of the AIDS epidemic served to decrease the incidence of TAH to approximately 4%. The introduction of specific anti-HCV assays in 1990 led to a further marked decline in the incidence of TAH to approximately 1%.9 Testing of stored donor and recipient serum revealed that first-generation anti-HCV assays could have prevented 80% of cases of TAH that occurred before 1990 and that second-generation assays, introduced in 1992, could have prevented 90% of cases.7 On the basis of the prevalence of HCV in the donor population just before antiHCV testing, it is estimated that the introduction of serologic tests for anti-HCV prevented 40,500 cases of TAH per year (111 cases per day) in the United States alone. Mathematical modeling conducted before the implementation of routine NAT testing of donor blood estimated the risk of HCV transmission to be 1 case per 100,000 transfusions and the risk of HBV transmission to range from 1 in 65,000 to 1 in 150,000 transfusions.8 After the implementation of minipool (MP)-NAT testing, the calculated risk of HCV infection was reduced to 1 in 1.5 million transfusions.8 Individual donor (ID)-NAT testing could decrease that risk further, but the benefit would be marginal given the already low frequency with minipool donor testing. The current risk of transfusion-transmitted HBV infection is still calculated to be relatively high (approximately 1:150,000 to 1:175,000), but that is based on anti-HBc seroconversions and an adjustment for missed HBsAg detection and does not represent clinical or even biochemical hepatitis.8 Actual transfusion-associated hepatitis B cases appear to be quite

rare. Nucleic acid testing for HBV has not been implemented as of this writing, but individual NAT for HBV would decrease the current seronegative window by 7 to 25 days, depending on the sensitivity of the HBsAg assay, and might bring the risk of HBV infection into the same range as the risk of HCV infection.8 Blood transmission of hepatitis A or hepatitis E in the developed world is so rare as to reportable. It is still unclear whether there is a human hepatitis beyond the identified agents for hepatitis A to E. This is addressed later in this chapter.

Hepatitis B Virus Historical Perspective The first reported outbreak of percutaneously transmitted hepatitis was described more than a century ago, when 15% of German dockyard workers developed jaundice after smallpox vaccination with lymph derived from convalescent persons.10 Although HCV cannot be excluded, the most likely responsible agent was HBV. However, HBV infection probably has afflicted humankind since ancient times, judging by the extraordinary prevalence of the virus in Asia and Africa, where it has been perpetuated by perinatal and percutaneous transmission. In the third and fourth decades of the last century, with the widespread use of syringes, percutaneous spread of viral hepatitis regained notoriety. Reports followed describing jaundice among persons attending venereal disease11 and arthritis12 clinics. Because the disease seemed to be associated with the sharing of inadequately sterilized needles, it became known as “postinoculation jaundice.” Further support for a link between needle exposure and infection came from reports of severe hepatitis among children and British troops inoculated with convalescent measles and mumps sera13,14 and among recipients of yellow fever vaccine.15,16 During World War II, viral hepatitis research increased dramatically. Not only was “epidemic” hepatitis once again found to be common, but also the widespread use of blood transfusion led to the emergence of TAH first reported in 1943.17,18 Subsequent investigations, including experimental human transmission studies, initially pointed to the existence of two viruses, with different modes of transmission, incubation periods, severity, and outcome.19-21 Hepatitis B virus was held responsible for all cases of hepatitis following transfusion (“serum hepatitis” or “posttransfusion hepatitis”). The current terminology, hepatitis B, was proposed in the late 1940s and adopted by the World Health Organization (WHO) in the mid-1970s. Proof that HBsAg was associated with a transmissible infectious agent came from the earliest studies, employing an insensitive immunodiffusion test.22 Approximately 75% of recipients of HBsAg-positive donor blood were found to have either hepatitis or serologic markers of HBV infection, although hepatitis also was noted in recipients of HBsAg-negative donor blood. Testing for HBsAg evolved from agar gel diffusion to counterelectrophoresis, to radioimmunoassay and finally, to enzyme

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Section IV: Part II

immunoassay (EIA). Testing of all blood donations for this serum marker was made mandatory by federal regulation in July 1972 and third-generation HBsAg testing was mandated in 1975. Prospective studies of TAH conducted during the 1980s and 1990s in the Far and Middle East, Europe, Canada, and Australia23-27 showed that despite hepatitis frequencies ranging from 3% to 19%, few cases could be attributed to HBV. In the majority of studies, no cases of hepatitis B were identified, and hepatitis B accounted for 7% or fewer of total cases in three studies.23,26,28 Widespread implementation of more sensitive assays for HBsAg and adoption of all-volunteer donor programs in developed countries has virtually eliminated clinically significant transfusion-associated hepatitis B.

Molecular Virology Hepatitis B virus is a 42-nm DNA virus composed of an outer shell (7 nm in width) and an inner core (27 nm in diameter). It belongs to a novel genus, Hepadnavirus, within the Hepadnaviridae family; HBV is designated hepadnavirus type 1. The outer coat material (or envelope), is composed of three virus-encoded polypeptides, collectively designated the hepatitis B surface antigen, and hostderived lipid components. The HBV envelope proteins can be detected in serum, not only as a part of the intact virus, but also as separate 22-nm spherical or tubular subvirus particles, that circulate in amounts that outnumber complete HBV particles by a factor of 104, that do not contain DNA, and that are highly immunogenic, but noninfectious. They, in fact, constituted the source for the first generation of plasma-derived HBV vaccines. Clinically, HBsAg is detected in serum by serologic assays, visualized in the cytoplasm of hepatocytes by electron microscopy, and identified in liver tissue of infected persons by its groundglass appearance and by antigen-specific immunohistochemical staining. The inner icosahedral nucleocapsid core of HBV is composed of the nucleocapsid protein known as the hepatitis B core antigen (HBcAg), the viral genome, and the viral DNA polymerase. The genome of HBV is a relaxed circular, partially double-stranded DNA, with a complete negative DNA strand (3200 nucleotides) and an incomplete positive DNA strand. The genomic information of the virus, present on the long strand of the DNA, comprises four highly overlapping (67%) open reading frames (referred to as S, C, P, and X genes), encoding the envelope (pre-S/S), core (precore/core), polymerase, and X proteins, respectively (Fig 46-1). The S gene has three in-frame start codons and a common stop codon, dividing the gene into the preS1, preS2, and S regions encoding the large (L), middle (M), and small (S) envelope proteins, respectively, depending on where translation is initiated. The product of the S region is the HBsAg (major protein).29 The M and S envelope proteins are found in both virions and subvirus particles, whereas the L proteins are found mostly in complete virions. The C gene has two in-frame start codons. The core protein (HBcAg) is translated from the second start codon and expressed on the surface of the nucleocapsid. Translation from the precore

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start codon produces a precore polypeptide that is posttranslationally cleaved into a soluble nonparticulate protein known as the hepatitis B e antigen (HBeAg). The P gene, which encompasses 80% of the viral genome, encodes a single multifunctional protein with four distinct domains: a protein primer, a spacer, a reverse transcriptase (RT)/ DNA polymerase, and an RNAse H domain. The X gene encodes a multifunctional transactivator of transcription (HBxAg), of both viral and cellular genes.30,31 It has pleiotropic effects on the transcriptome of the infected cell, including signaling pathways, protein degradation, cell cycle checkpoints, cell death, and carcinogenesis.30 The HBV genome contains four promoters (preS1, preS2/S, C and X) that control transcription by the cell RNA polymerase II of six functionally co-terminal viral mRNAs.32 The C promoter has a basic core promoter (BCP) sequence that acts as a binding site for a variety of cellular transcription factors (ie, HNF3, HNF4, SP1).33,34 HBV infects hepatocytes by binding a domain in the large surface protein to an unknown receptor and enters the cytoplasm by endocytosis. Once delivered to the nucleus, the plusstrand DNA is completed by the DNA polymerase and the HBV genome is converted into a covalently closed circular DNA (cccDNA) that acts as the template for transcription of five viral RNAs. These comprise two 3.5 kb precore (translated into the precore polypeptide, which is further processed at both ends to the HBeAg) and pregenomic RNAs, two 2.4 and 2.1 kb mRNAs for the surface proteins and a 0.7 kb mRNA for the X protein. The pregenomic RNA, which functions as the messenger RNA for translation of the HBcAg and the polymerase, is also the template for reverse transcription. A stem-loop near the 5 end of the pregenomic RNA contains a packaging signal (ε) and is used by the RT domain of the viral polymerase to prime minus-strand DNA synthesis with concomitant packaging by the nucleocapsid protein into immature cores. Inside the immature cores, a new minus-strand HBV DNA is produced through reverse transcription of the pregenome followed by the synthesis of a new plusstrand DNA. Maturation of the nucleocapsid before plus-strand synthesis is complete results in the partially double-stranded genome. The cccDNA persists as a stable chromatinized episome in the cell nuclei, appears to have a very long half-life, and is very resistant to antiviral therapy. Its stability along with the long half-life of hepatocytes implies that once HBV infection has occurred it may continue for life, even after apparent resolution.35

Genetic Variation of HBV HBV can be classified into eight genotypes (A to H) based upon an inter-group divergence of
8% in the complete nucleotide sequence.36 There is growing evidence suggesting that HBV genotypes influence clinical outcomes, HBeAg seroconversion rates, mutational patterns in the precore and core promoter regions, and response to antiviral therapy.37,38 The prevalence of specific genotypes varies geographically, with genotype A found mainly

Chapter 46: Transfusion-Transmitted Hepatitis

Subtype determinants “a” determinant d/y w1-w4 100-170 r/w

HBV particle (Dane) 42 nm DNA (+) (-)

Viral envelope HBsAg

(-)

Large Middle Small P protein

Cell chaperones

preS2 55 aa

S1 pre aa 6 2 1

3,

5

pr ot ein

kb

RN

A

C—C

22 S 6 aa

Spacer

RT

rm in

al

HBcAg dimer

2,1 2,4 kb R N kb RN A A

l

Te

po

HBV DNA 3,2 kb

aa 149

RP

P 833 aa

RN

P

eC

in Northern Europe, North America, India, and Africa; genotypes B and C in Asia; and genotype D in Southern Europe, the Middle East, and India. All known genotypes have been found in the United States, with prevalence of genotypes A, B, C, and D of 35%, 22%, 31%, and 10%, respectively, while genotypes E to H represent 2%.39 The HBV viral polymerase lacks proofreading activity, resulting in an estimated error rate of 104 to 105 nucleotide substitutions per site/year. Combined with a high turnover rate (up to 1011 virions/day), this leads to the accumulation of a swarm of closely related genomic sequences, referred to as quasispecies. Although mutations occur randomly along the genome, the overlapping open reading frames limit the number and location of viable mutations.40-42 Mutants may become selected according to their relative fitness when environmental conditions change

eH As

pr

AAA AAAA A A A AA AA AA

aa 19

()

C HBcAg

HBeAg

(+) 185 aa

Figure 46-1. Genomic structure and translated proteins of HBV. The inner circles represent the full-length minus-strand DNA, with the terminal protein attached to its 5 end, and the incomplete plus-strand DNA of the HBV genome. The shaded lines represent the four overlapping open reading frames (ORF) S, C, P, and X and their corresponding encoded proteins and detailed structure of the main HBV antigens encoded (HBcAg, HBeAg, and HBsAg). The outermost thin black lines represent the 3.5, 2.4, 2.1, and 0.7 kb mRNA transcripts, which are all terminated near the poly-A signal. Details of the HBcAg dimmers that assemble into the inner core and the structure of the HBsAg domain, common to all HBV surface proteins, containing the main “a” are also shown (see also text).

X 145/154 aa

0,8 kb RNA

[ie, during antiviral treatment or hepatitis B immune globulin (HBIG) prophylaxis]. Most common and clinically relevant HBV mutants have been well characterized and affect the precore/core promoter, preS/S, and polymerase genes.40

Precore and Core Promoter Variants HBeAg, which is not required for virus replication, seems to act as a decoy for the immune system, inducing tolerance. Viral clearance is usually associated with decreased HBeAg production followed by seroconversion to anti-HBe. However, documented severe liver disease in anti-HBe-positive patients with chronic hepatitis B (CHB), (HBeAg-negative CHB), led to the recognition of mutations in the precore region that prevent HBeAg synthesis. By far the most common of these mutations is a transition (G1896A), that creates a stop codon blocking translation of the

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precore protein and HBeAg production. Less frequently, point mutations in the start codon can also abrogate HBeAg production. In the United States, precore variants have been found in 27% of patients with chronic HBV infection.39 The most common core promoter variant involves a dual mutation A1762T, G1764A (TA change). In the United States, these variants have been found in 44% of patients with chronic HBV infection, and may also be associated with HBeAg-negative CHB.39 Most patients with HBeAg-negative CHB harbor HBV variants in the precore or core promoter regions, have lower serum HBV DNA levels, and are more likely to run a fluctuating course.43,44 HBeAg-negative CHB has been estimated to account for 7% to 30% of patients with CHB worldwide, being predominant in Mediterranean countries and Asia.45,46 The most common HBV variant associated with HBeAg-negative CHB, G1896A, usually occurs in HBV genotype D infection (more prevalent in the Mediterranean basin) and is rare in genotype A infections (more common in the United States and northwestern Europe). Although increasing data support an association between core promoter variants and more severe liver disease, hepatic decompensation and hepatocellular carcinoma (HCC), large-scale longitudinal studies are needed to clarify this association. Precore and core promoter variants have also been described in association with fulminant acute hepatitis B, and have been associated with higher resistance to interferon and nucleoside analog therapy.

Pre-s/s Variants The major B-cell neutralizing epitope of HBsAg resides in the “a” determinant region located at amino acid positions 124 to 149. Antibodies to the “a” determinant confer protection against all HBV serotypes. HBV S gene mutants have been described in infants infected with HBV despite an adequate anti-HBs response to HBV vaccination (vaccine escape mutants). The most common mutation involves an amino acid substitution at codon 145 (G145R) in the “a” determinant of HBsAg. Recurrent hepatitis B after liver transplantation despite HBIG prophylaxis has also been associated with this mutation. Finally, in rare instances, mutations in the HBV genome downregulate S gene expression or produce aberrant surface proteins undetectable by conventional HBsAg assays, despite ongoing HBV replication. Such “occult” HBV infections may have an impact on blood transfusion, organ transplantation, and diagnosis of cryptogenic liver disease. Polymerase Gene Mutations The RT/DNA polymerase region has five conserved domains: A, B, C, D, E, with the putative catalytic domain believed to reside in the YMDD locus in domain C. Naturally occurring polymerase gene mutations are rare, but many mutants are selected in patients receiving treatment with oral nuclos(t)ide analogs.

Profile of Serologic and Molecular Markers During Hepatitis B Virus Infection Infection with HBV is associated with characteristic changes in the serum levels of hepatitis B antigens and antibodies and HBV

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DNA, which define different clinical states.47 Acute infection is characterized by the appearance of HBsAg, HBeAg, and HBV DNA 1 to 10 weeks after exposure, followed soon thereafter by anti-HBc (initially of the IgM and then the IgG class). After exposure, there is a phase, lasting approximately 3 weeks (1 to 6 weeks), in which serum HBV DNA is intermittently detectable at low levels, followed by a “ramp-up” phase during which HBV DNA increases rapidly with an estimated doubling time of 2.56 days.48 The period between exposure and first appearance of detectable HBsAg is referred to as the window period (WP). During this WP, the duration of which depends on the sensitivity of the serologic assay, HBV DNA is the only marker of HBV infection. Hepatitis is defined by the observed onset of increased alanine aminotransferase (ALT) activity, 25% or fewer of the cases being associated with jaundice. Usually, HBeAg and HBV DNA, detectable by hybridization assays with limits of 105-106 copies/mL, disappear early, and HBsAg titers decline as the ALT peaks. Soon thereafter, the enzyme values begin to decline. What remains is IgM antiHBc, which, after 6 months or longer, is replaced by IgG antiHBc, the latter persisting for life in decreasing titers. During the interval between loss of HBsAg and development of anti-HBe and anti-HBs, which may extend for weeks or months, IgM anti-HBc may be the sole marker of acute HBV infection. The usual recovery sequence begins with the loss of replicative markers (HBeAg and HBV DNA), seroconversion to anti-HBe and disappearance of HBsAg, return of ALT levels to normal, and, finally, appearance of anti-HBs, which conveys immunity to reinfection [Fig 46-2(A)]. Although recovery is associated with disappearance of serum HBV DNA as determined by insensitive assays; when very sensitive assays [polymerase chain reaction (PCR) or transcription-mediated amplification (TMA)] are used, HBV DNA may remain detectable (sometimes intermittently) for many years.49 Persistence of HBsAg and usually of HBeAg beyond 6 months defines chronic infection (which occurs with variable frequency depending on the age at infection and immune status of the host). HBV DNA is usually detectable with insensitive assays (levels
105-106 copies/mL) as well as anti-HBc. HBeAg may persist for years or decades during chronic infection and its presence is usually associated with high levels of serum HBV DNA and active liver disease. Over time during chronic infection, spontaneous or treatment-induced seroconversion to anti-HBe occurs, usually associated with a marked decrease in serum HBV DNA and remission of liver disease. Some patients with mutations that prevent HBeAg production, however, continue to have active liver disease despite anti-HBe seroconversion (HBeAgnegative CHB) [Fig 46-2(B)]. There exists a subset of patients with “occult HBV infection” defined as being HBsAg negative with persistent HBV DNA detectable by PCR, in the presence (80%) or absence (20%) of anti-HBc with or without anti-HBs. Most of these patients have very low or only intermittently detectable serum HBV DNA levels; although HBV DNA is usually present in the liver. Infection with preS/S HBV variants accounts for a small number of these cases. HBV transmission from recently infected blood donors

Chapter 46: Transfusion-Transmitted Hepatitis

(A) HBV DNA

Anti-HBe

HBeAg

ALT

HBsAg

IgG anti-HBc Anti-HBs IgM anti-HBc

Figure 46-2. Clinical, serologic, and virologic course of acute and chronic HBV infection. (A) Schematic profile of serologic and virologic markers of a typical acute resolving hepatitis with the average timing of detection of serum HBV DNA, HBsAg, and HBeAg and their corresponding antibodies, with respect to the ALT elevation reflecting immune-mediated liver cell destruction after HBV replication (as reflected by serum HBV DNA levels) has been mostly controlled by noncytolytic mechanisms. (B) Course of chronic hepatitis B after perinatal or early-childhood-acquired infection. The four distinct phases shown are: immune tolerance (high-level HBV replication with normal ALT levels), immune clearance (high ALT level, lower HBV DNA levels and frequent hepatitis flares, depicted with dashed lines for both HBV DNA and ALT, which may lead to loss of HBsAg and anti-HBe seroconversion), inactive carrier state (normal ALT, presence of antiHBe and low or even undetectable HBV DNA), and reactivation phase after many years of inactive carrier state (lower and fluctuating HBV DNA and ALT levels in the presence of anti-HBe), commonly associated with selection of core or precore HBV variant (see text for further details).

0

4

8

12

16

20

24

28

32

36

Weeks after exposure (B) Anti-HBc HBsAg HBeAg Anti-HBe HBV DNA

ALT

Immune tolerance

with precore mutants, who never developed detectable HBsAg, has been described in Japan.50 Most cases of occult HBV infection represent late stages of persistent HBV infection associated with a strong immune response from the host, and/or viral interference because of coinfection with HCV.51 Among blood donors and the general population in developed countries cryptic HBV infection is rare,52 but it is frequently detected in countries that have a high prevalence of HBV (⬃10%-15%).53

Epidemiology and Prevention According to WHO estimates, 2 billion people have been infected with HBV, of whom 350 million are chronic carriers,

Immune clearance

Inactive carrier state

Reactivation

and 4 million new infections occur annually. HBV is readily transmitted by percutaneous or transmucosal transfer of blood or body fluids (eg, semen, cervicovaginal fluid, saliva). Three patterns of infection exist. In developing countries of Asia and sub-Saharan Africa, the disease is transmitted predominantly during the perinatal period by carrier mothers or, less commonly, through horizontal household transmission during infancy or early childhood. In developed countries, infection occurs mainly in adolescents and young adults through intimate (usually sexual) contact with chronic carriers or by percutaneous exposure (injection drug use, tattoos, multiple transfusions, accidental needlestick exposure, etc). There is a striking inverse

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relationship between age at infection and the risk of chronicity54,55; the chronicity rate is ~90% for perinatal infection, 20% to 50% for infection acquired before 5 years of age and 1% to 5% for adult acquired infections. Hence, the prevalence of HBV carriers varies from 0.1% to 2% in low-prevalence areas (United States, Canada, Western Europe, Australia), to 2% to 5% in intermediate-prevalence regions (Mediterranean countries, Japan, Central Asia, Eastern Europe, Middle East, and Latin America), to 7% to 20% in high-prevalence areas (Southeast Asia, China, sub-Saharan Africa). Several studies undertaken soon after the implementation of HBsAg screening showed that it was more common in males, in African and Asian Americans, and in paid donors. In addition, the HBsAg prevalence was higher among certain population groups (injection drug users and promiscuous men who have sex with other men).56 A study of the risk of blood-borne viral infections at five blood donor centers from 1991 to 1996, involving 1.9 million donors showed no reduction in the frequency of HBV infection (0.2% HBsAg-positive first-time donors during this 6-year period).57 A more recent study has shown a progressive decline from 1995 to 2002 to a prevalence of 0.07% among first-time donors.58 However, changing blood donor demographics as a result of immigration of people from areas of moderate to high endemicity may increase HBV prevalence again. Among the preventive measures for HBV infection, adequate information to HBV carriers about transmission mechanisms and counseling to avoid spread should be provided (use of condoms during sexual intercourse with occasional partners; not sharing toothbrushes and razors; covering open cuts and scratches; cleaning blood spills with bleach; not donating blood, sperm, or organs; and vaccinating regular sexual partners and household members). A highly effective HBV vaccine (recombinant HBsAg), confers protection to all HBV genotypes and subtypes in 95% of immunocompetent individuals. In highor intermediate-prevalence areas vaccination campaigns of all newborns (along with HBIG in children born to HBV carrier mothers) and children under 12 years of age, have been widely implemented during the last 10 to 20 years so that, in many countries most individuals 25 years old are protected from HBV infection. Recommendations for vaccination have been established in recent Centers for Disease Control and Prevention (CDC) and Advisory Committee on Immunization Practices (ACIP) guidelines.59,60

Screening Tests and Detection Systems Serologic Assays After the donor selection process, testing for HBsAg remains the first-line screen for HBV. Since its introduction in 1970, HBsAg assays have increased their sensitivity by more than 2 log10. During the 1970s, extensive prospective studies (undertaken to evaluate the frequency of hepatitis and the efficacy of immune globulin prophylaxis) that coincided with the appearance of serologic tests for HBsAg, documented the impact of screening tests with greater assay sensitivity. For example, in a study

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performed at the blood bank of the NIH, the frequency of transfusion-associated hepatitis B was 4.8% with the gel diffusion test, 3.7% with counterelectrophoresis (CEP) screening, and 0.6% after screening by radioimmunoassay (RIA) became available.3 Currently licensed tests are third-generation EIA or chemiluminescent immunoassays (ChLIA), with analytical sensitivities varying from 0.13-0.62 to 0.07-0.12 ng/mL, respectively, as assessed in seroconversion panels. 48,61 The infectious WP for licensed EIA tests has been estimated at 37 to 50 days, and 33 to 44 days for the recently licensed ChLIA tests.62 Aside from the WP, HBsAg immunoassays may fail to detect HBV DNA positive donors infected with some preS/S mutants, acutely infected donors with transient antigenemia and chronic carriers with occult HBV infection.50, 51 Testing for anti-HBc, originally introduced in 1986 as a surrogate marker for NANB infection, was retained because of its potential value for the detection of HBV-infected donors. The effectiveness of screening for anti-HBc as a means to reduce HBV transmission may be very low, because of its low specificity, and the deferral of a large proportion of otherwise eligible donors; the vast majority of repeatedly reactive anti-HBc-positive units also carry high-titer anti-HBs and are consistently negative for HBV DNA. Although cases of hepatitis B infection have been described in recipients of HBsAg-negative, anti-HBc-positiveonly blood, their prevalence is quite low (and often represent false-positive test results) thus limiting the value of anti-HBc screening in low-prevalence populations.63 Furthermore, in contrast to studies from high-prevalence regions, where HBV DNA has been identified by PCR in 5% to 25% of anti-HBconly donors,64,65 in low-prevalence areas (United States, Canada, Western Europe), HBV DNA has been detected in only 0.5% to 3.5% of anti-HBc-only blood donors66-68 and infectivity of these low-level HBV DNA-containing units has not been well established. Nonetheless, anti-HBc screening has been implemented in several low-prevalence countries (United States, Germany, United Kingdom, France, Canada, New Zealand), often along with HBV DNA testing, because HBV infection continues to pose the greatest residual risk of transmission of all major blood-borne viruses.

Molecular Assays for HBV DNA Qualitative and quantitative tests for HBV DNA in serum (and liver tissue) have been developed to assess HBV replication. The sensitivity limit and dynamic range of these assays depends upon the techniques used. These vary from several hybridization assays (detection limit 103-106 copies/mL) to the most sensitive amplification procedures such as real-time PCR and TMA assays. Commercial assays based on the latter procedures are available and have excellent performance with low-end detection limits of 5 to 50 IU/mL (25-250 copies/mL).48,69,70 Although the main clinical utility of quantitative tests for HBV DNA is the selection and monitoring of patients for antiviral treatment, sensitive (quantitative or qualitative) tests have also been developed in semiautomated or automated platforms for HBV NAT of large numbers of blood donors, often in multiplexing formats for

Chapter 46: Transfusion-Transmitted Hepatitis

simultaneous detection of HCV RNA, HIV RNA, and HBV DNA in MP-NAT (minipool) or ID-NAT (individual donor).71 The use of NAT testing of blood donors for HBV DNA is intended to shorten the infectious WP and to detect occasional cases of occult HBV infection. Transmission from occult late-stage chronic carriers may contribute little to HBV transfusion risk in countries performing routine anti-HBc screening, but might be more relevant in areas with intermediate prevalence where anti-HBc screening is not performed. Nonetheless, the infectivity of such donors (3% in a look-back study conducted in Japan),50 and the clinical significance for the donors remain largely unknown and are being actively investigated.72 Most of the residual risk (RR) of transfusion-transmitted HBV comes from recently infected individuals who donate during the infectious WP and this risk, in the absence of NAT, has been estimated to range between 0.75 and 200 per million donations from repeat donors in areas where anti-HBc screening has not been implemented and between 0.91 and 8.5 per million donations in areas screening for antiHBc (3.6 per 106 donations in the United States).73 However, implementation of NAT for HBV remains controversial because of uncertainties in the length of the infectious WP on the incidence of infection among first and repeat donors and on the minimum inoculum size required for transmission. In addition, the new HBsAg tests may be 10-fold more sensitive than current EIA tests and may have already shortened the WP significantly. A mathematical model to estimate the potential impact of HBV NAT testing in reducing WP and relative risk according to the analytical sensitivity of NAT assays, pool size, doubling time of the virus, the incidence rate and back-extrapolation to a putative infectious dose of one DNA copy per 20 mL (the plasma volume of an additive solution red cell concentrate) has been proposed.62 According to this model, ID-NAT for HBV would reduce the WP to 20 days (a 50% reduction from current estimates).

Pathogenesis and Mechanisms of Viral Persistence HBV is not directly cytopathic and both liver injury and control of HBV replication appear to involve HBV-antigen-specific cytolytic T cells (CTL) and CD4 T cells, as well as cytokines released by both virus-specific T cells and nonspecific lymphocytes.74,75 The early immune response to HBV does not appear to involve a significant direct innate immune response during the first weeks of infection.76 Nonetheless, HBV DNA can be cleared from serum and liver before detectable T-cell infiltration and liver injury. Downregulation of HBV replication, with removal of both single-stranded intermediates and cccDNA from the cytoplasm and nucleus, is mediated primarily by interferon-γ (IFN-γ), presumably released by HBV-specific CD8 T cells as well as well as other activated cells.77,78 Clearance of most HBV DNA from blood and liver is followed by development of multispecific CD4 and CD8 T cells, cytolysis of HBV-infected hepatocytes, and ALT elevation. A broad and vigorous multispecific CD4 and CD8 T-cell response is essential for recovery as evidenced by in-vivo depletion studies in the chimpanzee model.79

Viral persistence occurs mainly in vertically transmitted HBV infection (90% of newborns to HBeAg-positive carrier mothers with high HBV DNA levels) presumably because the immune system of neonates is not fully developed, allowing development of immune tolerance to nucleocapsid proteins induced by HBeAg. The mechanisms of chronicity in settings other than perinatal transmission may involve qualitative and quantitative differences in both innate and adaptive immune response.80 The T-cell immune response not only plays a role in viral control and recovery, but also contributes to liver damage in acute and chronic hepatitis. Flares of disease activity during the immune clearance phase of CHB coincide with increased serum interleukin (IL)-12 levels, increased HBV-specific CD4 and CD8 T-cell responses, and recruitment of large numbers of neutrophils, natural killer (NK) cells and activated bystander lymphocytes. These nonspecific immune cells are believed to mediate most of the liver damage.

Clinical Spectrum and Natural History of HBV Infection Acute Hepatitis B The spectrum of clinical manifestations of acute HBV infection varies, depending on host factors (age, immune status, and underlying liver disease), ranging from subclinical or anicteric hepatitis to icteric hepatitis and, in some cases, fulminant hepatic failure. After an incubation period of 1 to 3 months, approximately 70% of patients with acute HBV infection have subclinical hepatitis. In those with symptomatic disease, the clinical picture is indistinguishable from that of other viral hepatitis with symptoms and jaundice lasting 1 to 3 months. The disease may be more severe in patients coinfected with other hepatitis viruses or with underlying liver disease. Fulminant hepatitis is unusual (0.1 to 0.5%). Laboratory testing during the acute phase reveals elevation of aminotransferases (values up to 1000-2000 IU/L), and bilirubin, which usually return to normal within 1 to 4 months in patients who recover. Despite clinical, analytic, and serologic recovery from acute infection, traces of HBV DNA remain detectable in blood and/or liver tissue by PCR for many years, suggesting that eradication of HBV rarely occurs and that latent infection is kept under control by the immune system. Data from several prospective studies conducted during the early 1970s indicate that transfusion-transmitted hepatitis B begins after an average incubation period of 11 to 12 weeks.81 In general, the clinical symptoms of the different types of viral hepatitis are indistinguishable from one another, although symptoms are more frequently absent among persons with HCV than among those with HBV infections. The biochemical dysfunction also tends to be more severe with HBV disease than with transfusion-transmitted HCV, with higher mean ALT values (705 vs 471 U/liter) and a higher frequency of jaundice (60% vs 25%).81 It is difficult to assess the disease course after transfusion because of the scarcity of well-defined cases studied prospectively. Although non-transfusion-related acute hepatitis B in immunocompetent adults leads to chronicity in 1% to 5%

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of cases, too few large-scale, prospective studies have been conducted to establish whether the frequency of chronicity in transfusion-associated cases is similar. The frequency of chronic HBV infection is clearly influenced by the age at onset (neonates and infants vs adults or elderly people) and the immune status of the patient; it is debated as to whether the size of the inoculum (transfusion vs needlestick or sexual) also affects the likelihood of evolution to chronicity.

Chronic Hepatitis B Persistence of HBsAg beyond 6 months after acute infection is the hallmark of CHB. The overall view of the natural course of chronic HBV infection has changed with the recognition that HBV replicates throughout the course of CHB, that the immune response plays a major role in liver damage, and that the balance between immune response and viral replication is a dynamic process.82,83 The natural course of CHB consists of four phases [Fig 46-2(B)]. The first phase of immune tolerance is classically seen in patients infected at birth by vertical transmission and is characterized by the presence of HBeAg and high-level HBV DNA in serum but with normal ALT levels and minimal or absent liver inflammation. It may last 1 to 4 decades, a period during which spontaneous HBeAg seroconversion is infrequent. In childhood or adult-acquired HBV infection this phase is short-lived or absent. The second phase (immune clearance/ HBeAg-positive CHB) is characterized by HBeAg persistence, high or fluctuating HBV DNA levels, persistent or intermittent ALT elevation, and active hepatic necro-inflammation. A hallmark of this phase is the occurrence of hepatitis flares, often accompanied by transient increases in serum HBV DNA, that are sometimes severe enough to lead to hepatic decompensation. The duration of the “immune clearance” phase, and the frequency and severity of the flares, correlate with the risk of cirrhosis and HCC.84 An important outcome of this phase is spontaneous HBeAg clearance accompanied by anti-HBe seroconversion; clearance of HBeAg correlates with, but is not limited to, older age, higher ALT levels, and infection with HBV genotype B. The third phase (inactive HBsAg carrier state) is characterized by the presence of anti-HBe, normal ALT levels, low serum HBV DNA (105 IU/mL), and mild hepatitis with variable fibrosis, depending on the duration and severity of the prior immune clearance phase. The inactive carrier state may persist indefinitely, in which case the prognosis is generally favorable. This is supported by a long-term follow-up study of 296 HBsAg-positive, healthy blood donors in Italy,85 whose survival was similar to that of 157 uninfected controls over a 30-year period, and in whom no episodes of hepatic decompensation occurred. However, 4% to 20% of inactive carriers have episodes of reversion to HBeAg positivity and 10% to 30% of inactive carriers have spontaneous reactivation of HBV replication and liver disease activity after years of quiescence. The fourth phase (HBV reactivation of inactive carrier state/HBeAg-negative CHB) is characterized by positive anti-HBe, detectable HBV DNA, elevated ALT, and active liver

726

disease.86 Most cases represent reactivation after a period of the inactive carrier state of variable duration, but some progress directly from HBeAg-positive to HBeAg-negative CHB. Because this is a later phase in the course of CHB, patients are usually older and have more advanced liver disease. Most patients with HBeAg-negative CHB harbor precore or core promoter HBV variants and tend to have lower serum HBV DNA levels than during the immune clearance phase. A clinical course characterized by fluctuating ALT and HBV DNA levels. HBeAg-negative CHB, originally reported in Mediterranean countries,86,87 is now being recognized with increasing frequency in all parts of the world.88,89 Prevalence varies according to the predominant HBV genotype in different geographic regions. Spontaneous clearance of HBsAg occurs in 0.5% to 1% of patients with CHB per year, usually in association with undetectable serum HBV DNA, normalization of ALT, and absence of liver disease activity. In the absence of cirrhosis or HCV coinfection, loss of HBsAg conveys a good prognosis. CHB is usually asymptomatic until the evolution to cirrhosis or HCC and the ensuing complications. Collectively, among patients with CHB, 40% develop cirrhosis, liver failure, and/or HCC. Cirrhosis develops during CHB with an annual incidence of 2% to 6% in HBeAg-positive and 8% to 10% in HBeAg-negative patients. Certain cofactors increase the risk, such as alcohol, HCV or HIV coinfection, high HBV DNA levels, and HBV genotype C.45,89,90 Among patients with cirrhosis, persistent, high-level HBV DNA is associated with increased risk of hepatic decompensation and mortality. The annual incidence of HCC has been estimated to be 1% for noncirrhotic carriers and 2% to 3% for patients with cirrhosis. Factors associated with increased risk of HCC include male gender, older age, a family history of HCC, core promoter mutation, HBV genotype C, HCV coinfection, and the presence of cirrhosis. Although preexisting cirrhosis is a strong risk factor, 30% to 50% of HBV-associated HCC occurs in the absence of cirrhosis.91

Treatment The aims of treatment of CHB are induction of sustained suppression of HBV replication and remission of liver disease activity, with the goal to prevent cirrhosis, hepatic failure, and HCC. Currently, six therapeutic agents have been approved for the treatment of adults with CHB in the US (IFN-α, and nucleoside or nucleotide analogs: lamivudine; adefovir; entecavir; and telbivudine), while other analogs are still undergoing trials (emtricitabine, tenofovir, and clevudine). Responses to treatment are categorized as biochemical (normalization of ALT levels), virologic (loss of HBV DNA and anti-HBe seroconversion), and histologic (reduction in activity and halting of fibrosis progression). Standard and pegylated IFN-α (which are given for finite periods, by subcutaneous injection), are associated with substantial side effects and have limited efficacy in inducing virologic response (35% in HBeAg-positive and 70% in anti-HBe-positive CHB). Despite its low initial efficacy, the advantage of pegylated IFN-α is that the virologic response in HBeAg-positive patients

Chapter 46: Transfusion-Transmitted Hepatitis

who achieve anti-HBe seroconversion is sustained after discontinuation of therapy in up to 90% of cases and hence long-term therapy is not required. However, a sustained virologic response occurs in only 20% of HBeAg-negative CHB patients treated with pegylated IFN-α. Drug resistance does not occur in patients treated with IFN. In contrast to IFN, nucleos(t)ide analogs are given orally, have minimal side effects, and are more potent inhibitors of viral replication. However, they usually require long-term or indefinite administration (because of the low rate of sustained remission after withdrawal) and 30% to 70% develop resistant mutants after 2 to 5 years of treatment (1% after 2 years with entecavir monotherapy). Although there is no doubt that antiviral therapy modifies the natural history of chronic HBV infection—reducing liver-related morbidity, mortality, and HCC incidence—the current complexity of treating CHB (criteria for selection of candidates, choice of antiviral agent, monitoring of response, diagnosis and management of resistant mutants) has necessitated the development of evidencebased treatment guidelines for the management of CHB.91-93 These guidelines will continuously evolve as new medications are added and as new drug interactions and resistance patterns emerge.

HBV Summary Hepatitis B virus, a hepadnavirus, causes acute and chronic infection, the latter evolving to cirrhosis, end-stage liver disease and HCC in 40% of cases, unless properly treated. It is transmitted perinatally and by blood and secretions and was a major cause of posttransfusion hepatitis until the risk was reduced by elimination of paid donors and by screening for HBsAg. A highly effective vaccine is available and vaccination campaigns in high and intermediate prevalence areas have been widely implemented in the last two decades. The near elimination of the risk of HBV transmission by transfusion has been a major part of the success of contemporary transfusion medicine. The small residual risk of HBV infection by blood transfusion will be further reduced with more sensitive serologic and molecular assays with the ultimate goal of eradicating this transmission mechanism from the epidemiology of HBV infection.

Hepatitis D Hepatitis D virus (HDV), also known as hepatitis delta virus, is a 35- to 37-nm defective RNA virus that contains a 1700-nucleotide RNA genome similar to that of plant satellite viruses and viroids. Hepatitis D virus is a hybrid virus that relies on HBV for its surface coat (HBsAg), which surrounds a nucleocapsid core expressing HDV antigen. HDV requires concurrent infection with HBV to support its replication and clinical expression.94 Hepatitis B virus and HDV infections can occur simultaneously, or a patient with chronic HBV infection can become superinfected with HDV. Ultimately, the duration of HDV infection is determined by the duration of HBV infection because HDV invariably relies on HBV for its growth and expression. A diagnosis of HDV infection can be made by demonstrating antibody

(IgM or IgG) to HDV (anti-HDV), identifying HDV antigen (HDAg) in liver nuclei by immunohistochemical staining, or by detecting HDV RNA in serum by PCR or in liver by cDNA hybridization. The detection of HDAg in serum is difficult and therefore not a practical diagnostic marker. Hepatitis D tends to segregate into two different epidemiologic patterns. In endemic areas, such as Mediterranean countries, HDV infection appears to be transmitted from person to person. In contrast, in nonendemic areas, such as northern Europe and North America, transmission of HDV infection appears to be confined to specific percutaneously exposed populations, such as drug users and hemophiliacs. Occasionally, even in nonendemic areas, protracted outbreaks of hepatitis D occur either by HBV-HDV coinfection or HDV superinfection. In nonendemic areas, such outbreaks have a tendency to amplify HDV infection in the community and to blur the epidemiologic distinction between endemic and nonendemic areas. The frequency of HDV infection, even in endemic areas of the world, has been declining. The clinical features of hepatitis D are similar to those of hepatitis B, and most of the more severe outcomes of hepatitis B are caused by hepatitis D coinfection. In particular, superinfection of chronic hepatitis B with HDV often leads to more serious and rapidly progressive liver disease and can transform asymptomatic or mild chronic hepatitis B into severe chronic hepatitis and cirrhosis. In addition, fulminant hepatitis can follow HBVHDV coinfection or HDV superinfection of an HBV carrier. 95 Because HDV infection cannot occur in a patient who is immune to HBV, prevention of HDV infection is accomplished readily by HBV vaccination in persons susceptible to hepatitis B. For those already infected by HBV, prevention of superinfection is limited to mechanical efforts to avoid intimate contact with HDV-infected persons or HDV-contaminated needles or blood components.

Hepatitis D and Blood Transfusion Because, under ordinary circumstances, HDV infection cannot occur in the absence of HBV infection, the chance that a blood donor screened and found to be negative for HBsAg and antiHBc could harbor HDV is exceedingly small. Before the advent of current screening techniques, transmission of HDV undoubtedly occurred and contributed to the previously identified high frequency of HDV infection in multitransfused persons, including those with hemophilia and thalassemia.96 In a study of American patients with hemophilia A, 75% had a serologic marker of previous HBV infection, and 13% of them had antibodies to HDV.97 Although HDV is a blood-borne viral agent, contemporary, highly sensitive screening methods used in blood banks should be effective in reducing the frequency of transfusion-associated HDV hepatitis to a negligible level, at least in HBsAg-negative blood recipients. There is a very small residual risk for HBsAgpositive blood recipients because rare donors have occult HBV infection in which HBsAg is nondetectable despite the presence of HBV DNA. Theoretically, such donors could carry low levels

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of HDV, though some level of HBsAg production would have to exist in order to support replication of HDV. Even this remote possibility of a donor with coexistent occult HBV infection and HDV infection would be interdicted in regions that screen donors for anti-HBc antibody.

hydrophobicity patterns are similar. The pestiviruses, like HCV, are characterized by blood-borne transmission and by lifelong viremia. Unlike dengue and yellow fever viruses, they do not have a mosquito vector.

Cloning and Molecular Characterization

Hepatitis C Virus Historical Perspective Before hepatitis C virus (HCV) was cloned, considerable knowledge of this agent was garnered from experiments in chimpanzees. In 1978, it was found that serum from patients with acute or chronic NANB hepatitis and from asymptomatic carriers could transmit infection to chimpanzees.98,99 Although chimpanzees did not develop clinical illness, they had transaminase elevations and liver biopsy evidence of hepatitis after an appropriate incubation period. Animals inoculated with serum from patients with NANB hepatitis did not develop HBV markers, emphasizing the clear distinction between HBV and the agent of NANB hepatitis. In addition to elevations in transaminase levels, electron microscopy revealed that infected chimpanzees generally had characteristic tubular ultrastructures in hepatocyte cytoplasm.100 These tubular structures, consisting of an electrondense center and a double-unit membrane, represent proliferation of the rough endoplasmic reticulum, a change now shown to be induced by interferon. The tubules are an epiphenomenon of NANB/HCV infection and are useful indicators of such infection in chimpanzees because of their relative specificity. They are not induced during the course of HAV or HBV infection in chimpanzees and are not seen in human NANB/HCV infection. The findings of elevated transaminase levels, light microscopic changes of hepatocellular damage, and electron microscopic evidence of cytoplasmic tubules made it possible to define NANB hepatitis in chimpanzees, even in the absence of a specific serologic marker. Using the chimpanzee as an infectivity model, it was shown that the cytoplasmic tubule-inducing NANB agent was sensitive to chloroform and other lipid solvents and thus that it contained essential lipid, presumably in its surface membrane.101,102 Filtration studies subsequently showed that the NANB agent was 30 to 60 nm in diameter.103 Among known viruses, only the flaviviruses and the hepadnaviruses (HBV, woodchuck hepatitis virus, duck hepatitis virus) are lipid encapsulated and fall into this size range. It was predicted that the NANB agent was a flavivirus and the subsequent cloning and characterization of HCV6 proved this prediction correct. Flaviviruses are a large and diverse family, formerly known as arboviruses (arthropod-borne viruses) and togaviruses. They include the dengue and yellow fever viruses, the rubivirus (rubella), and the pestiviruses (bovine diarrheal virus, hog cholera virus, and other mucosal disease viruses of animals). HCV represents a new genus in the family Flaviviridae and is most closely related to pestiviruses. Although there is only limited homology with the pestiviruses, the genomic structure and

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In the mid-1980s, a long series of experiments conducted by Houghton et al at the Chiron Corporation, in collaboration with Bradley at the CDC, culminated in the cloning and serologic characterization of HCV.6 Using large volumes of plasma derived from a chimpanzee with chronic NANB hepatitis, these investigators extracted whole nucleic acid and made complementary DNA (cDNA) from RNA and single-stranded DNA templates. The cDNA was inserted into a gt11-phage expression vector and used to infect Escherichia coli. Expressed proteins were blotted and overlaid with serum from patients with NANB hepatitis. A single clone was identified that expressed an antigen reactive with chronic- and convalescent-phase serum from persons with NANB hepatitis, but not with serum from control subjects without infection or from patients with type A or type B hepatitis.6 Cloned nucleic acid was used to generate probes that were shown to hybridize with RNA derived from the starting plasma, thus closing the loop between the cloned agent and the infectious plasma. Once the cloned agent of NANB hepatitis was molecularly characterized, it was designated HCV. Amplification of the reactive clones and expression of the genome in large cultures of yeast produced sufficient antigen to formulate radioimmune and enzyme-linked assays for the detection of antibody to HCV. Characterization of HCV shows that the viral genome consists of single-stranded, linear RNA of 9.6 kb that is positively stranded and hence can serve directly as message for the synthesis of viral proteins.6,104 The viral proteins are encoded from a single, large open reading frame that produces a polyprotein that is posttranslationally cleaved.104 The genome (Fig 46-3) is characterized by a highly conserved, untranslated region at the 5 terminus (5UTR) that is now used as the primary target for molecular amplification assays to detect HCV RNA and for viral genotyping. The 5UTR contains an internal ribosomal entry site that is essential for viral protein synthesis and that initiates translation at the start codon of the large open reading frame.104 Downstream of the 5UTR are the coding regions for envelope proteins designated E1 and E2, followed by the coding region for the viral core (nucleocapsid). In the terminal portion of the E1 region is a hypervariable segment (HVR1), which is the most highly mutable region of the genome, presumably because its position in the viral envelope places it under a high degree of immune pressure. It is presumed that this region is the target for neutralizing antibody responses. Downstream from the structural genomic regions are a series of nonstructural coding regions designated NS2, NS3, NS4, and NS5. The full function of these regions is not known, but they have been shown to code for critical enzymes, including an NS3 protease, an NS3 helicase, and an NS5 polymerase.

Chapter 46: Transfusion-Transmitted Hepatitis

Proposed genome of HCV: Single-stranded RNA virus in Flaviviridae Family Non coding Core envelope 5‘

Helicase Membrane protease binding

C E1 E2/NS1 NS 2

Conserved

NS 3

NS 4

RNA polymerase NS 5

3‘

Initial clone

5-1-1

HV

First-generation EIA

c100-3

c22

c33c

c100-3

Second-generation EIA c22

c200

c22

c200

AA No. 1

500

1000

1500

Third-generation EIA

NS 5 2000

2500

3000

Figure 46-3. Proposed genome of hepatitis C virus (HCV). Functional equivalents and major antigens used in antibody detection assays. The structural organization of the HCV genome has been deduced from genomic sequencing and from parallels with other flaviviruses. The entire genome contains approximately 9500 bases coding for approximately 3000 amino acids. The 5 end has a highly conserved noncoding region, the one generally used for polymerase chain reaction amplification and presumed to have important regulatory functions. Downstream of the noncoding region are the regions coding for the structural elements, including the core or nucleocapsid (C) and the envelope (E1, E2/NS1). It is unclear whether NS1 is part of the envelope region or the first nonstructural gene. The 5 end of E2/NS1 contains a hypervariable (HV) region that mutates rapidly

and probably plays a key role in the ability of the virus to escape neutralization. There follow a series of nonstructural genes (NS2-NS5) with enzymatic or membrane-binding functions. The initial clone discovered was from the NS4 region; the derived protein was designated 5-1-1. This was expanded to form the c100-3 antigen, the basis of the first-generation anti-HCV assay. Secondgeneration assays added the c22 core antigen and the c33c antigen from the NS3 region. These antigens increased the sensitivity of the second-generation assay approximately 20% over the first-generation test. The third-generation assay (pending licensure in the United States) adds an NS5 protein and reconfigures some of the earlier antigens. (Used with permission from Alter.105)

There are six major genotypes of HCV that are designated in Arabic numerals 1 through 6 and each has been subtyped as designated by lowercase lettering. The genotypes differ from each other in nucleotide sequence by at least 15%. Genotypes 1a and 1b are the most prevalent forms in the United States, Europe, and Japan. In the United States, genotypes 1a and 1b are found in almost equal frequency, whereas in Japan genotype 1b predominates. Genotypes 2 and 3 are found as minor populations in the United States (10% genotype 2 and 6% genotype 3) and Europe. Genotype 3 is the predominant genotype in Southeast Asia. Genotype 4 appears isolated to Northern Africa (especially Egypt), and the Middle East, whereas genotype 5 has been localized to South Africa. Genotype 6 is rare and found predominantly in Japan. Although useful as epidemiologic tools and predictors of treatment response, the clinical significance of HCV genotypes is uncertain. Although many studies have claimed that patients with genotype 1 have more severe histologic outcomes and clinical disease, an equal number of studies have failed to show this association. The primary use of HCV genotypes is in the prediction of response to interferon-based therapies. In general, patients with genotypes 1 and 4 have an approximate 45% to 50% sustained virologic response to interferon plus ribavirin, whereas patients with genotype 2 or 3 infection have an

approximate 80% sustained virologic response rate that is akin to “cure” (see below).

Detection Systems Surrogate Tests Mounting evidence for the role of blood transfusion in the induction of chronic liver disease and continuing frustration in the development of a specific assay for the agent of NANB hepatitis prompted the adoption of surrogate (nonspecific) tests for the detection of NANB hepatitis virus carriers. These surrogate tests, ALT and anti-HBc, were implemented for routine donor screening early in 1987. Use of these tests evolved from retrospective analyses of prospective studies of TAH conducted by the Transfusion Transmitted Virus Study Group106,107 and by the department of transfusion medicine at the NIH.108,109 These studies found a threefold to fourfold increase in hepatitis risk among recipients of ALT-elevated or anti-HBc-positive blood compared to the rate among recipients of blood negative for these two indices. It was predicted from these studies that the adoption of ALT and/or anti-HBc tests for donor screening might result in a 30% to 40% reduction in TAH. The value of surrogate assays as an interim measure was validated when it was later shown that approximately one-third of donors with

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confirmed anti-HCV were also positive for one or both of these surrogate markers.110 A controlled prospective study conducted in Canada further validated the use of surrogate assays for the prevention of TAH.111

Serologic Assays Humoral responses to HCV are vigorous and are directed to a multiplicity of antigenic sites.104,112 As shown in Fig 46-3, the original clone produced a protein designated 5-1-1, which was then expanded and fused to form the c100-3 antigen that served as the basis for first-generation anti-HCV assays. This early assay was highly effective in interdicting HCV carrier blood donors, but anti-c100-3 did not appear for a protracted period after exposure, creating a window of infectivity that ranged from 12 to more than 26 weeks. Some patients with HCV infection never developed antibody that was reactive in the first-generation assay. The second-generation assays added two critical epitopes to both the screening EIA and the confirmatory recombinant immunoblot assay (RIBA). These were a core protein, designated c22-3, and an NS3 protein, designated c33c. Antibodies to these new epitopes generally appear much earlier than anti-c100-3, and the window period in which a donor might be seronegative has considerably narrowed. In an NIH prospective study, 41% of persons with HCV infection had specific antibody detected with second-generation assays within 10 weeks of exposure, 80% had antibody within 15 weeks, and all patients had antibody within 6 months.113 Third-generation EIA tests added an HCV antigen from the NS-5 region and have shown a marginal increase in sensitivity with a further reduction of the window period of approximately 1 week compared with second-generation assays. Because this increment in sensitivity would have minimal benefit in disease prevention, the use of third-generation antiHCV assays has not been mandated by the US Food and Drug Administration (FDA). Antibodies, particularly to 5-1-1, c100-3, and E1, may disappear spontaneously, during immunosuppression, or after successful antiviral therapy. Antibodies to c22-3 and c33c rarely disappear from persons with chronic infection and only infrequently disappear even from persons with apparent recovery. However, a minority of patients who clear HCV RNA also lose anti-HCV over time and are termed seroreverters; in most persons, some HCV antibodies persist for long periods, perhaps for a lifetime. The prevalence of HCV antibody, found with second-generation EIAs and confirmed with RIBA, among US blood donors ranges from 0.2% to 0.4%. Similar rates have been observed in Europe and higher rates are seen in Japan and other Eastern nations. These rates among highly selected blood donors are underestimates of the prevalence in the general population, which is estimated to be around 1%. Because of nonspecific reactivity, it is important to confirm EIA reactivity with a supplemental assay. The only licensed supplemental test is a recombinant immunoblot strip assay that displays the key epitopes in a linear format on a nitrocellulose strip. With second-generation assays, approximately 40% of

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EIA-reactive samples are confirmed by RIBA in a low-risk blood donor setting. Among high-risk populations or EIA-reactive donors who have elevated ALT levels, the confirmation rate is much higher (60% to 80%). In the initially licensed supplemental assay, approximately 30% of EIA-positive samples were classified as indeterminate because only one band appeared on the strip. A third-generation RIBA replaces the recombinant proteins c22-3 and c100-3 with synthetic peptides representing only a portion of the respective coding regions and replaces 5-1-1 antigen with recombinant NS5. This format resolves many RIBA-2-indeterminate patterns. Approximately 60% of RIBA-2 indeterminates are negative with RIBA-3, whereas 20% are RIBA-3 positive and 20% remain indeterminate. Thus RIBA-3 resolves approximately 80% of RIBA-2 indeterminate results. There is good, but not absolute, correlation between a positive RIBA result and NAT documentation of HCV RNA. Patients who have resolved HCV infection are RIBA positive, but repeatedly NAT negative. In contrast, almost all anti-HCV-positive persons who are HCV RNA positive also are RIBA positive. An alternative to RIBA testing is to go directly from a positive EIA screening assay to amplification of the viral genome by NAT or other technique. If HCV RNA is detected, there is no need to perform RIBA. However, if HCV RNA is negative, the RIBA assay should be performed to distinguish HCVrecovered subjects from those with false-positive EIA reactivity.

Molecular Assays for Detection of HCV RNA The most sensitive way to detect HCV is measurement of HCV RNA by means of NAT or another gene amplification technique. The NAT has shown that HCV RNA is almost universally detected in the early phase of HCV infection, generally within 1 to 3 weeks of exposure. Thus, HCV RNA detection precedes the increase in serum ALT level, sometimes by as much as 10 to 12 weeks. It also has been shown by means of quantitative NAT or branched DNA assay that the highest levels of HCV RNA appear early in the course of infection, generally preceding or coinciding with the first significant elevation in ALT level.105 In persons who appear to recover from HCV infection, HCV RNA generally declines or disappears near the time of the peak in ALT elevation. However, in at least 75% of persons, HCV RNA persists, generally with associated fluctuating ALT elevations, but up to 30% of infected individuals have persistently normal serum ALT. These relationships imply that infectivity is probably greatest before signs or symptoms of acute hepatitis develop, that chronic HCV infection is more frequent than is indicated by ALT elevations alone, and that a proportion of persons with HCV infection may be true asymptomatic carriers with normal serum ALT levels and minimal histologic abnormalities in the liver. The extreme sensitivity of NAT techniques and the long seronegative window between the first detection of virus and the first detection of anti-HCV has prompted the introduction of NAT into routine donor screening. NAT testing has been universally implemented in US blood establishments and in most blood centers in developed nations. Nucleic acid testing is being performed by two primary methods—standard

Chapter 46: Transfusion-Transmitted Hepatitis

reverse-transcription PCR and TMA. Transcription-mediated amplification offers two advantages in the blood bank setting because extraction and amplification take place in the same tube under isothermal conditions and because the test lends itself to a duplex configuration, whereby HIV and HCV RNA can be measured simultaneously. A triplex TMA assay that also detects HBV DNA has been developed, but testing for HBV DNA is not mandated by the FDA at this time. As indicated earlier, the routine use of NAT has generated a better understanding of the dynamics of HCV and HBV viremia. With serial stored collections from commercial plasma donors who subsequently are found to be HCV-infected, it has been shown that HCV RNA usually is undetectable until the level increases precipitously to reach peak levels over a brief interval. This phase of rapid acceleration has been termed the ramp-up phase, and it usually occurs 2 to 4 weeks after exposure. During ramp-up, the doubling time of the virus has been calculated to be 13 to 17 hours.114 The pattern of HBV viremia differs in that the ramp-up is more delayed and not as steep and in that the ramp-up is preceded by a persistent low-level of viremia. During the period of low-level HBV viremia before the ramp-up phase, possibly infectious donations can be missed at MP-NAT testing, but detected with ID-NAT testing. The doubling time for HBV during the ramp-up phase is calculated to be 2.8 days. Another distinction between HBV and HCV testing is that MP-NAT dramatically decreases the infectious window for HCV (60 days, an 85% reduction) compared with detection of antibody to HCV. In contrast, MP-NAT (minipool) testing for HBV DNA reduces the window by only 6 days (13%) compared to detection of HBsAg; ID-NAT (individual donor) for HBV would decrease the window by to 25 days (55%) compared to current HBsAg detection,114 but newer, more sensitive HBsAg assays will narrow the window between detection of HBV RNA and detection of HBsAg to as little as 1 week. In essence, to have a significant effect on HBV transmission compared to current practice, one would have to implement ID-NAT testing. For HCV, MP-NAT testing seems sufficient in most instances. A serologic test for HCV core antigen has been developed115 and has shown sensitivity similar to that of HCV MP-NAT testing. This test may be an option for blood establishments that have not committed to NAT technology.

HCV Cell Culture The holy grail of HCV research has been to develop an in-vitro culture system. After 15 years of failure, this crusade now has come to fruition, spearheaded by the groundbreaking work of Wakita and collaborators in Japan116 and subsequently expanded by several investigative groups.117,118 The groundbreaking event was the isolation of a clone, designated JFH-1, derived from a single patient in Japan with fulminant hepatitis caused by HCV genotype 2a infection.116 This full-length genome replicated without modification in a hepatoma cell line designated Huh-7. Replication was indicated by the finding of HCV RNA and HCV antigens within cultured liver cells, the secretion of intact viral particles, and the ability to passage the virus in culture and to

chimpanzees. Thus far, only this single JFH-1 isolate or its chimeras have been shown to grow in Huh-7 and other hepatoma cell lines. No genomic or structural difference has been identified to account for JFH-1’s unique growth characteristics, nor is it known whether its high replicative capacity in vitro relates to the development of fulminant hepatitis in the infected patient. In regard to the latter, clones from other patients with fulminant hepatitis have not grown in tissue culture, nor did JFH-1 cause fulminant hepatitis in the chimpanzee. Building on the discovery by Wakita et al, Rice and coworkers117 constructed chimeras comprising the core to NS-2 region of the related genotype 2a clone, J6, and the nonstructural regions from JFH-1. This chimera grew very well in Huh-7.5 cells, spreading more rapidly and reaching higher infectivity titers (105) than JFH-1. Serial passage was achieved without the loss of infectivity. Further, it was shown that virus replication was inhibited by interferon and by protease and polymerase inhibitors, demonstrating the value of this culture system for drug testing. Zhong,118 in the Chisari lab, took this a step further by deriving an “interferon-cured” cell line designated Huh 7.5.1 that allowed very efficient release of virions with sustained infectivity titers of 104 to 105 IU/mL, about 50-fold higher than in the original Wakita experiments. It is postulated that a defect in the RIG-1 pathway of innate immunity may be the reason these cells are so permissive. In sum, these three landmark studies published in 2005 demonstrated that 1) HCV virions could replicate in human hepatoma cell lines, could be serially and efficiently passaged, and that particles produced in vitro could transmit infection to chimpanzees at reasonably high titer; 2) the HCV virion is 5060 nm in diameter, as previously predicted, but now directly visualized; 3) infectivity could be blocked by monoclonal antibodies to E2 and by patient sera, confirming that neutralizing antibodies to HCV exist and that E2 contains a key neutralizing epitope; 4) CD81, a membrane tetraspanin, is a critical component of the HCV entry pathway; and 5) infectivity could be blocked by interferon and protease/helicase inhibitors, demonstrating the suitability of this system for evaluation of candidate antiviral agents. Because most HCV infections are genotype 1, it was critical to establish a culture system for this dominant genotype. In 2006, Yi and Lemon119 produced adaptive mutations in the nonstructural regions of the prototype genotype 1 strain, H77, and transfected this adapted genome (H77-S) into Huh 7.5 cells. Although infectivity titers with H77-S were two logs lower than with JFH-1, the system is viable and now allows study of genotypic differences in an in-vitro system. Sera from genotype-1a-infected individuals neutralized H-77S virus, but could not neutralize genotype 2a virus, a finding that has implications for the development of therapeutic hyperimmune globulins and HCV vaccines.

Mechanisms of Viral Persistence Viral Quasispecies The most striking feature of HCV is its ability to persist in the host. Measurements of HCV RNA in serum and liver suggest that the prevalence of persistent infection is higher than the

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prevalence of chronic hepatitis and may be in the range of 75% to 85%. The mechanism of persistence appears to involve the ability of the virus to mutate rapidly under immune pressure and to exist simultaneously as a series of related but immunologically distinct variants, any one of which can become the predominant strain when a coexistent strain comes under immune pressure. This coexistence of multiple genetic variants has been termed “quasispecies.” It provides an efficient and rapid mechanism for the virus to escape the host immune response. Although mutations occur throughout the HCV genome, most occur in a relatively short hypervariable segment of the envelope (E2) domain. This hypervariability suggests that this region contains important target epitopes that are under intense immune attack. Among HCV isolates, the average mutation rate is 103 to 104 base substitutions per genome site per year.120 This high rate of mutation, reflected in the quasispecies nature of the virus and in the ability of HCV to rapidly evolve escape mutants, appears to be the primary mechanism underlying the absence of effective neutralization and the development of persistent infection. A study by Farci et al121 showed that early in HCV infection, patients who ultimately recover have a decreased degree of viral diversity after the appearance of anti-HCV. In these persons, antibody or coexistent cell-mediated immune responses seem to contain viral replication and quasispecies formation such that the virus becomes increasingly homogeneous and is eventually cleared. In contrast, in most patients, the development of antibody appears to drive viral diversity until the population becomes so heterogeneous that it cannot be fully contained by the immune system. Hepatitis C virus has evolved to become an efficient machine for survival. In this survival scheme, a master strain is accompanied by a series of subservient strains that under immune pressure can themselves become the master. Defective particles are formed that can protect replicative particles, and a low level of replication can allow the virus to both hide from the host and protect its environment by inducing disease so indolent that the cells that nurture the virus are well maintained or only incrementally destroyed. These efficient mechanisms for self-survival have allowed HCV to chronically infect approximately 1% of the world’s population.

Neutralizing Antibody and Cell-Mediated Immunity There is little evidence that HCV is directly cytopathic. It is generally believed that liver damage is the result of the host’s immune response, including CTLs, cytokines, and apoptotic factors. The immune response to HCV infection is brisk and yet generally ineffective in viral clearance. High titers of antibody coexist with high levels of virus. Antibodies broadly directed across the entire genome rarely have neutralizing potential. Those directed at the critical neutralizing epitopes in the viral envelope more often tend to drive viral diversity than to eradicate the agent. Nonetheless, neutralizing antibodies to HCV have been found in mixing experiments in which chimpanzee infectivity was used as the endpoint. These neutralizing responses, however, have been shown to be transient and highly

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strain-specific.122 Recently, pseudoviruses, consisting of a retroviral core, HCV envelope proteins and a green fluorescence protein marker, have been developed and allow for in-vitro measurement of neutralizing antibodies to HCV.123 In this system, pseudoviruses infect an HCV susceptible hepatoma cell line (Huh-7) and infected cells fluoresce green. By premixing the pseudoviruses with serum suspected to contain antibody, neutralization can be measured by diminution of green fluorescence compared to controls. This system has shown that neutralizing antibodies are formed in HCV infection, but that they appear late in the acute infection and probably play a minimal role in viral clearance.124 Paradoxically, neutralizing antibodies increase over time during chronic infection and also become more broadly reactive against multiple genotypes125; this broad, cross-reactive neutralization has now been demonstrated in virus culture assays as well as in the pseudovirus system.126 It is presently unclear whether these neutralizing antibodies play a role in containing chronic infection even though they are incapable of clearing the virus. It is also unknown whether these late-developing neutralizing antibodies can be harvested to produce a clinically effective HCVspecific hyperimmune globulin. A protective HCV immune globulin would be a major weapon in preventing infection of transplanted HCV-naïve livers; reinfection of transplanted livers, presumably from extrahepatic HCV reservoirs, is now universal. An expanding array of assays to measure in-vitro lymphoproliferative and cytotoxic (CTL) responses, including enzymelinked immunospot (ELISPOT) assays, and tetramer assays, have elucidated the cellular immune responses to HCV infection.127 It has been found that cell-mediated immune responses occur early (1 to 2 weeks) after the onset of infection. These initial responses involve innate immunity, primarily effected by CD56 NK cells. Although the liver is particularly rich in NK cells, HCV has evolved mechanisms to bypass innate immunity. It is now clear that specific HCV proteins, particularly core and NS3, directly inhibit critical steps in the transcription cascade of the innate immune response, blocking upregulation of interferon regulatory factors and the generation of natural interferons.128 This impaired innate immune response allows the virus to gain an early foothold such that HCV reaches very high levels of replication before generation of the adaptive immune response, forcing the cellular immune system to combat a massively expanding and rapidly evolving viral antagonist. Results suggest that the Class II major histocompatibility complex restricted helper T-cell (TH) response is particularly important in determining the outcome of acute HCV infection.129 Patients with acute, self-limited hepatitis C have been shown to mount an early, vigorous, and multispecific TH response that can be measured in the peripheral blood. In contrast, if this TH response is weak or poorly sustained, persistent infection and chronic hepatitis ensue. It appears that in most patients with HCV infection, the TH response is blunted. In addition to the early stimulation of TH responses, there is a corresponding HCV-specific CD8 CTL response that is probably driven by cytokines (IFN, IL-2) released by TH1 lymphocytes.

Chapter 46: Transfusion-Transmitted Hepatitis

Early, vigorous, and multispecific CTL responses have been associated with viral clearance and recovery from HCV infection. Conversely, in patients with persistent HCV infection, CTL responses are weak and targeted against fewer HCV epitopes.129 Although patients with chronic HCV infection have blunted CTL responses to HCV, they have normal CTL responses to other viruses such as influenza and Epstein-Barr virus (EBV). Hence, there is no generalized immunodeficiency state, but rather a specific tolerance to HCV. The net effect is that the adaptive cellmediated immune response appears to be blunted or exhausted in those destined to become chronic carriers. It has been debated whether the exhaustion of T cells is the result of, or the cause of, viral persistence, but recent data130 have demonstrated that diminished HCV-specific T-cell responses occur early in HCV infection, suggesting that the failure of this critical immune response is central and causal to viral persistence. Thus, several mechanisms are at play in the development of persistent HCV infection including the high mutability of the virus, inhibition of innate immunity induced by viral proteins, and the blunting or exhaustion of HCV-specific adaptive T-cell responses. Further, although the total number of virions produced is large, the number of viruses per cell is small. This small intracellular viral burden may decrease the display of viral antigens on the cell surface, blunt the lymphoid immune response, and protect the cell from immune destruction. There is little evidence that HCV is directly cytopathic, so it is probable that CTL and other immune responses while attempting to contain the virus also cause liver cell damage through a variety of mechanisms, including Fas-ligand apoptosis, cytokine-induced injury (including that from tumor necrosis factor α), and perforin-induced injury.127 It has been shown that Fas is upregulated on HCV-infected hepatocytes and on uninfected bystander cells and that Fas ligand is expressed on activated liver-infiltrating T cells.127 Such apoptotic cell death may account for liver cell destruction that is sometimes out of proportion to the number of cells actually infected. In contrast, IFN-γ produced by CTLs can result in intrahepatic viral clearance without accompanying liver cell injury. The T-cell-mediated attack on the HCV-infected liver may lead to sufficient inflammation and fibrotic repair that cirrhosis develops. However, in most patients with HCV infection, an equilibrium develops in which ongoing viral replication and T-cell- and cytokine-mediated cell destruction are balanced by controlled hepatic regeneration such that chronic hepatitis develops without progressive fibrosis and without impairment of liver function.

Epidemiology Although HCV was discovered through investigations of transfusion-transmitted hepatitis, transfusion was never the primary source of transmission and currently is only a minor mechanism for dissemination of this agent. Nonetheless, therapeutic blood transfusion is the prototype for parenteral (percutaneous) transmission of viral agents. Such percutaneous spread is defined by the intravenous infusion of contaminated blood

components or drugs or by the shared use of contaminated needles or other sharp instruments. Studies of HCV-infected blood donors showed that 27% were infected as the result of blood transfusion in the pretesting era before 1990, whereas 42% were infected by intravenous drug use (IVDU) with shared needles.131 It is noteworthy that none of the blood donors in this study were drug addicts, but rather persons who experimented with drugs in their youth and generally for intervals of less than 5 years. Sharing of needles was almost universal. It is also noteworthy that there was an independent cohort who snorted cocaine and repeatedly denied IVDU. While the veracity of this denial cannot be proved, the statistical association of cocaine snorting with HCV infection is exceedingly high and substantiated in a multivariate analysis.131 Further, approximately 25% of cocaine snorters provided a history of experiencing nosebleeds while snorting or observing nosebleeds in those with whom they shared snorting devices, thus providing a rationale for blood transmission by this covert route. Other routes of overt or covert blood transmission include improperly sterilized, reusable needles and dental instruments, particularly in developing nations, but also in the developed world in the decades before 1970. Although it is now difficult to prove, tattooing with shared needles or inks undoubtedly contributed to viral spread as did ritual scarification practices in native populations. Although rare transmissions have been traced to chronically infected health-care workers, the main health-care burden has been the reutilization of unsterilized needles and contaminated instruments, including early dialysis machines and inadequately sterilized diagnostic instruments such as colonoscopes and bronchoscopes. It is important to keep in mind that HCV infection requires only one exposure to result in a lifelong infection and that most of those now found to be HCV-infected were actually exposed 20 to 50 years ago when health care and transfusion practice were markedly different than they are today. Thus, our current HCV-related disease burden is primarily the residual of past parenteral spread that, except for IVDU, essentially has been interdicted in the developed world. IVDU, however, remains unchecked and has been the primary source of HCV infection since proliferation of the drug culture in the 1960s. It is conceivable that if the practice of shared-needle IVDU was eradicated, HCV infection would ultimately disappear among populations in developed countries. Although parenteral routes are the predominant modes of HCV transmission, a parenteral source cannot be identified in up to one-third of infected individuals. This does not rule out covert parenteral exposures, but has raised the possibility of nonparenteral modes of acquisition. The CDC has conducted comprehensive epidemiologic studies of community-acquired HCV infection in four selected (sentinel) counties in the United States.132 The sentinel counties studies initially revealed that among patients with clinically overt NANB hepatitis, only 15% gave a history of blood transfusion, 25% used intravenous drugs, fewer than 10% were in health-related fields or had a presumed sexual or household contact, and more than 50% had no known route of exposure to this agent. More recent evaluations133 have

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shown that fewer than 1% of cases of community-acquired hepatitis are related to transfusion, further demonstrating the effectiveness of donor screening measures implemented since 1990. The precise routes of nonparenteral transmission of hepatitis C remain an enigma even in the face of sensitive serologic and molecular assays for HCV. There is evidence that HCV, like HBV and HIV, has a sexual and perinatal, as well as parenteral, route of transmission. However, HCV is much less efficiently spread by these nonparenteral routes than are HBV and HIV. The CDC has implicated sexual transmission in up to 10% of acute, clinically apparent hepatitis C cases based on the absence of an identified parenteral source and on a history of having had sex with more than two sexual partners in the preceding 6 months or having had sex with a prostitute or having a recent history of a sexually transmitted disease. In contrast, the sexual partners of patients with chronic HCV infection are very rarely infected despite years of unprotected sex.131 This paradox might be explained by the fact that HCV may be more transmissible early in HCV infection when viral titers are higher and when the virus is more likely to exist as free virions rather than complexed to antibody. In the final analysis, it is probable that HCV can be spread sexually, but that such spread is very inefficient and more likely to occur early in the course of infection. Because sexual transmission is rare, particularly during chronic infection, there is no mandate that committed, monogamous partners should routinely use condoms or other protective devices; rather, the probabilities and potential risks should be presented to affected couples and the decision for condom use left to their discretion. Perinatal transmission of HCV is also inefficient, but has been documented to occur in about 5% of cases where the mother is HCV RNA positive at the time of delivery.

Clinical Spectrum of Hepatitis C Virus Infection Acute Manifestations Only 25% of acute hepatitis C cases present with jaundice or substantial clinical symptoms; subclinical cases generally remain undetected until uncovered by the incidental finding of elevated ALT levels or anti-HCV during the course of blood donation or general medical/surgical evaluation. Long-term observation in prospective studies has revealed that asymptomatic acute hepatitis has the same propensity to evolve into chronic liver disease, including cirrhosis, as do the more overt cases. Thus, silent TAH may have major clinical significance. Although patients with HCV rarely have sufficient symptoms to seek medical care, individual cases can be acutely severe and even fulminant. However, fulminant hepatitis is an exceedingly rare complication of hepatitis C. Three major patterns of elevation of ALT level have been observed in hepatitis C—monophasic, polyphasic (fluctuating), and plateau.134 The monophasic pattern involves a single acute phase peak followed by complete normalization of ALT level. The polyphasic pattern is the most characteristic and includes dramatic fluctuations of ALT level over brief intervals. Over time, the amplitude of the fluctuation diminishes, but the periodicity persists into chronicity. In the plateau pattern, a relatively constant and generally low-level ALT abnormality is observed in the

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acute and chronic phases of the disease. Such patients often have biopsy evidence of chronic liver disease early in the posttransfusion course. These patterns have prognostic implications. In one study,134 42% of patients with a monophasic peak progressed to chronic hepatitis, compared with 87% of patients with the fluctuating pattern and 95% of patients with the plateau pattern. Levels of ALT occasionally normalize for prolonged intervals that suggest recovery, but are followed months to years later by secondary elevations that indicate the presence of chronic liver disease. This prolonged normalization of ALT level followed by recrudescence makes it difficult to ascertain when, or if, biochemical recovery from HCV infection has occurred. The best current assessment of recovery from HCV infection is serial measurement of HCV RNA. Three negative determinations of HCV RNA over the course of 3 to 6 months generally connote sustained clearance of the virus and full recovery from the infection.

Chronic Sequelae The most important component of HCV infection is its ability to evolve into chronic hepatitis, cirrhosis, and more rarely, HCC. Long-term follow-up evaluation of transfusion-associated cases indicates that at least 50% of patients contract chronic hepatitis as evidenced by elevations in ALT level that persist longer than one year. More recent data indicate that chronic hepatitis may occur in nearly 70% of cases. Similar frequencies of chronic hepatitis have been documented among community-acquired cases of hepatitis C. In eight early studies, 339 patients with transfusion-associated NANB hepatitis underwent prospective observation; 47% had biochemical evidence of chronic hepatitis.81 Of 102 patients who underwent biopsy, 41% had chronic active hepatitis and 20% had cirrhosis; five of the 20 patients with cirrhosis died of liver disease. Subsequent studies in which serial liver biopsies were performed provided unequivocal documentation of the progression of histologic abnormalities from acute to chronic hepatitis to cirrhosis.135-138 In summary, numerous studies of transfusion recipients have revealed the following: 1) at least 50% of patients with acute hepatitis C eventually have biochemical evidence of chronic hepatitis; 2) approximately 20% of patients with chronic hepatitis C have biopsy evidence of cirrhosis; 3) progression from acute hepatitis to cirrhosis can be documented histologically and generally evolves slowly over the course of decades but sometimes can occur more rapidly, the mean time to the development of cirrhosis being approximately 20 years; 4) the progression to cirrhosis is enhanced in patients with HCV infection who abuse alcohol—the cumulative effect of alcohol and HCV infection is more than additive, and the risk of cirrhosis may be 15-fold higher among persons who abuse alcohol than among persons with HCV infection alone139; and 5) when HCC occurs, it is almost always in patients with established cirrhosis—the risk of development of HCC among patients with cirrhosis has been estimated to be 1.7% per year.140 The foregoing considerations allow a schematic depicting the typical course of acute resolving and chronic hepatitis C (Fig 46-4). The essential features of viral replication, humoral immune responses, hepatocellular inflammation, and clinical

Chapter 46: Transfusion-Transmitted Hepatitis

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Figure 46-4. Biochemical, serologic, and molecular biologic profile of acute and chronic transfusion-associated hepatitis C virus (HCV) infection. Acute, resolving hepatitis C is shown in (A) and chronic hepatitis C in (B). Resolving disease cannot be differentiated from progressive disease by the time of onset of detectable HCV RNA by means of polymerase chain reaction (PCR), the magnitude of HCV RNA elevation as measured by means of branched DNA assay, the interval to the first elevation in the level of alanine aminotransferase (ALT), the magnitude of ALT elevation in the acute phase, or the interval between exposure and the first appearance of antibody. Progression to chronic disease cannot be predicted in the acute phase, and the only distinguishing features in these patterns are the persistence of ALT elevation and the persistence of HCV RNA in persons with chronic hepatitis C. The acute, resolving pattern (A) occurs in 10% to 15% of patients with transfusion-associated hepatitis C and the chronic pattern (B) in 85% to 90%. Note: 1) HCV RNA is detectable soon after exposure. Here the PCR results was positive 2 weeks after exposure, but it can become positive even sooner. 2) HCV RNA may be detected by means of branched DNA assay coincident with PCR reactivity, but the reaction may be delayed, as shown here. 3) The major

peak of viral replication (assessed with HCV RNA level) occurs before the first increase in ALT level and before any clinical or biochemical evidence of hepatitis; it is presumed that persons might be most infectious in this interval before the acute phase. 4) In acute resolving infection, HCV RNA levels increase rapidly before the decline in serum ALT level. 5) In chronic infection, HCV RNA level diminishes and can remain low, fluctuate, or become undetectable; HCV RNA levels sometimes show a periodicity that parallels the fluctuations in ALT level; in (B) the level of HCV RNA increases a short time before ALT level does and decreases before the decline in ALT. 6) Second-generation anti-HCV assays shorten the seronegative window in HCV infection much more than do first-generation assays; nonetheless, anti-HCV was not detectable for 12 to 15 weeks after exposure and for 6 to 7 weeks after the first significant rise in ALT level. Antibody to HCV (detected with second-generation assays) almost always persists in chronic cases and generally persists in acute resolving cases. Antibodies detected with the first-generation assay (anti-C100, anti-5-1-1) generally disappear in resolving cases. (Used with permission from Alter.105)

outcome are described earlier and in the legend to Fig 46-4. These are representative diagrams of classic cases—there is considerable variability in these patterns from case to case. The most consistent features are that viral replication can be detected soon after exposure, that peak viremia occurs in the acute or preacute phase in patients who recover and in those who have chronic hepatitis, and that progression to chronic hepatitis cannot be predicted by the time of onset or the peak level of viremia. Untoward occurrences among recipients of standard blood transfusion are exaggerated in recipients of clotting factor

concentrates. Before recent inactivation procedures were developed, such concentrates, derived from massive pools of paid blood donors, were invariably contaminated with HBV and HCV. Overall, more than 50% of patients with hemophilia have ALT elevations and serologic results that suggest chronic hepatitis C.141 That proportion increases to 80% among patients with severe hemophilia who received clotting factor concentrates before virus inactivation.142 In a landmark multicenter study,142 liver biopsy specimens from 155 patients with hemophilia and chronic elevations in ALT level revealed cirrhosis in 15% and

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severe chronic active hepatitis, likely to progress to cirrhosis, in an additional 7%. Similar severe outcomes were found in a subsequent prospective study in England143 wherein 71% of 79 patients with hemophilia had persistent abnormalities in ALT level, 25% of the 34 patients who underwent biopsy had cirrhosis, and 12% had radiographic evidence of esophageal varices. One patient died of complications of portal hypertension. Before AIDS, chronic liver disease accounted for 5% to 11% of deaths in patients with hemophilia. The advent of AIDS has further complicated the course of hepatitis C in this population. It is now evident that HIV infection accelerates the course and increases the severity of coexistent HCV infection.144 Now that patients with HIV infection have long survival because of highly active antiretroviral therapy, severe liver disease is emerging as a major cause of morbidity and mortality among patients with HCV and HIV coinfection. In Japan, where hepatitis C is common and where a similar progression from acute to chronic hepatitis has been well documented, it has been possible to examine the role of blood transfusion in the development of chronic liver disease. Kiyosawa et al145 found that among patients with chronic hepatitis and cirrhosis, presumably related to hepatitis C (then designated NANB hepatitis), 44% and 38% of patients, respectively, had a history of previous transfusion. In HCV-related chronic liver disease, a history of transfusion was 6 to 12 times more frequent than in HBV-related disease. This finding suggested a causal relation in the former, but less so in the latter, where the virus tends to be transmitted more readily by sexual and maternal-fetal routes. Another important observation in the study of Kiyosawa et al was the long interval between transfusion and the first clinical manifestations of chronic liver disease. The mean interval from transfusion to the clinical recognition of chronic hepatitis was 11.3 years; to the development of overt cirrhosis, 21.2 years; and to the development of clinically apparent HCC, 29.0 years.145 These long intervals may account for the apparent disparity between the number of expected cases of transfusion-associated cirrhosis and the number of cases actually detected; because patients who undergo transfusion tend to be elderly, many die of an underlying illness before chronic liver disease becomes clinically evident. Such intercurrent deaths obscure the course of this slowly evolving disease and mask the possible effect of HCV-related chronic hepatitis and cirrhosis on younger blood recipients. Infection with HCV can unequivocally lead to HCC.146 In a composite of prospective studies, it appears that the overall incidence of HCV-associated HCC is 1% to 5%. However, once cirrhosis develops in chronic hepatitis C, the risk of developing HCC is about 4% per year. Currently, the rate of HCV-related HCC is relatively low in Western nations and relatively high in Japan. Okuda et al147 conducted a study of 113 patients with nonalcoholic HCC and found that 69% of the cases of HCC were HBsAg negative and presumably related to HCV infection, as subsequently documented by serologic testing.148 The disparity between the frequency of HCV-related HCC in the United States and Japan probably reflects the duration of infection in the

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respective populations. Calculations using the molecular clock methodology suggest that the major spread of HCV in Japan preceded that in the United States by 30 to 40 years.149 Hence, the infected population in Japan is decades older and their duration of infection places them at increased risk of HCC evolution. It is anticipated that as the duration of infection in Western populations increases, the rates of HCC will increase concomitantly and hence that many more cases will be observed in the next 2 to 3 decades unless chronic HCV carriers can be successfully treated in the interim. The long-term consequences of TAH were comprehensively studied by Seeff et al.150 Patients who had TAH in five major prospective studies conducted in the 1970s were traced after a mean interval of 18 years and prospectively evaluated for evidence of chronic hepatitis. Over this interval, there was no increase in overall mortality among patients who sustained an episode of TAH compared with controls who underwent transfusion and did not have an episode of TAH. Although the mortality was not different, 35% of patients with previous episodes of hepatitis had biochemical and histologic evidence of chronic hepatitis compared to 1% of controls. The results of this study were subsequently updated to a mean follow-up period of 23 years.151 Overall mortality was similar among patients with hepatitis and control subjects without hepatitis, but patients with hepatitis had a small, but significant increase in liver-related mortality (4.1% vs 1.3%; p  0.05). Among 103 patients with positive results for anti-HCV in 1974, 76% were still anti-HCV positive and HCV RNA-positive approximately 20 years later. An additional 17% had specific anti-HCV but no longer had viremia, suggesting they had spontaneously cleared the infection. A surprising finding was that 7% of patients had no residual markers of HCV infection, suggesting they not only had cleared the virus, but also had lost anti-HCV. Hence, prevalence estimations based on serologic status may underestimate the extent of HCV infection in the population. More important, this study showed that the spontaneous clearance rate of HCV infection may exceed the commonly accepted estimate of 15% and actually be between 20% and 25%. In a separate study of longer duration, Seeff et al152 found that only 1 of 17 persons with chronic HCV infection had died of liver disease in the nearly 50 years after their HCV infection was first documented. Milder outcomes of HCV infection have been observed in studies of asymptomatic blood donors found to be anti-HCV positive at the time of donation. In an NIH study,131 15% of donors appeared to have spontaneously cleared HCV infection on the basis of the presence of RIBA-confirmed anti-HCV and repeatedly negative determinations for HCV RNA. Among those with persistent HCV infection, the peak ALT level exceeded two times the upper limit of normal in only 16%, and clinical symptoms were minimal. Liver biopsy was performed on 94 patients. No patients had severe inflammatory changes, 13% had stage 3 or 4 fibrosis, and only 2% had cirrhosis after an average duration of infection of 19 years on the basis of a defined parenteral exposure. Repeated liver biopsies were performed 5 years later on

Chapter 46: Transfusion-Transmitted Hepatitis

60 of these patients. Fibrosis progression over that interval was minimal; no new cases of cirrhosis were found even when the follow-up was extended to a mean of 24 years.153 Three additional long-term outcome studies also suggest relatively mild outcomes in chronic HCV infection. Kenny-Walsh154 performed a 17 year follow-up study of 376 women with HCV infection from a contaminated lot of Rh Immune Globulin (RhIG). Liver biopsies were performed on 363 patients a mean of 17 years after exposure; only 4% had moderate to severe grades of inflammation. Periportal fibrosis (stage 1) was found in 34% of patients; bridging fibrosis (stage 3) in 15% and cirrhosis in only 2%; 49% had no fibrosis. Two of seven patients with cirrhosis also had a history of alcohol abuse. A similar study in Germany155 enrolled 152 women with HCV infection traced to contaminated lots of RhIG and found that none had cirrhosis or severe fibrosis after a mean follow-up period of 15 years. Further supporting mild outcomes of HCV infection, a 20-year followup study156 of children who were infected at the time of heart surgery revealed that 45% had spontaneously recovered based on the findings of RIBA-confirmed anti-HCV and persistently negative assays for HCV RNA. Among the children with viremia who underwent biopsy, only two of 17 had portal fibrosis and only one had cirrhosis 20 years after exposure. The one patient with cirrhosis also had chronic HBV infection. There is a paradox in the course of HCV infection. If one studies patients who already have evidence of severe liver disease, it is evident that HCV is responsible for the majority of cases. From this perspective, HCV projects as a frequent cause of cirrhosis, end-stage liver disease, and HCC. On the other hand, if one prospectively evaluates cohorts of persons with HCV infection, a more balanced picture emerges. First, as indicated above, a minimum of 15%, and possibly as high as 25% of infected adults spontaneously recover from HCV infection, usually within the first year after onset. The rate of recovery among children with HCV infection is even higher, reaching 45% in a retrospective-prospective study conducted in Germany.156 It is now also apparent that in the absence of alcohol abuse or coexistent HIV infection, chronic hepatitis C is generally an indolent process in which severe outcomes may never occur or may take decades to evolve. Most adults who undergo transfusion die of the disease for which they were transfused or of an intercurrent illness before the potentially fatal complications of chronic hepatitis C become manifest. Nonetheless, over the span of 20 or more years, 20% to 30% of patients with HCV infection may have histologic evidence of cirrhosis that can ultimately lead to liver failure and the need for liver transplantation. That the majority of patients with HCV infection spontaneously recover or have a nonprogressive form of chronic liver disease gives hope to the individual patient; however, this does not diminish the global impact of HCV infection engendered by the sheer magnitude of the population that is infected. On a global scale, the absolute number that face life-threatening events related to HCV is staggering even if the proportion that encounter these events is encouragingly small.

Treatment The last decade has seen marked advances in the treatment of hepatitis C and new classes of drugs are in the pipeline. The foundation of therapy since the inception of treatment has been IFN-α, but the introduction of combination therapy with ribavirin and subsequently, the use of pegylated interferon have been the innovations that have increased sustained efficacy from approximately 10% in the early 1990s to near 50% at present.157 The efficacy of treatment is assessed by the sustained loss of HCV RNA. During treatment, four patterns of HCV RNA response are observed: 1) Non-response wherein HCV RNA levels do not fall or are only minimally reduced during therapy; 2) Breakthrough wherein a good initial response is followed by a return of HCV RNA to original levels despite continued therapy; 3) Relapse wherein HCV RNA becomes undetectable during therapy, but then rebounds to initial levels soon after therapy is discontinued; the main benefit of ribavirin is its ability to prevent this virologic relapse; 4) Sustained virologic response (SVR) wherein HCV RNA becomes undetectable during therapy and remains undetectable at least 6 months after the cessation of therapy. Considerable evidence now shows that a sustained virologic response is tantamount to “cure” because long-term follow-up studies have not shown recrudescence of infection beyond 6 months after therapy is terminated. Serial measurements can be used early after initiation of therapy to assess the likelihood of response. Those who have a more rapid early response are more likely to have a sustained response.157 There are several parameters that predict treatment outcome, but none are absolute. In general, higher response rates are seen in females, in those less than age 40, in those of European ancestry compared to those of African ancestry, in the nonobese and in those with viral loads less than 2 million copies/mL. In addition, persons with severe liver disease, particularly cirrhosis, and those with immunodeficiency, particularly HIV infection with CD4 counts 200 mm3, are less likely to respond. However, none of these parameters are a contraindication to treatment and their value lies in predicting the probability of response. The main determinant of virologic response is the viral genotype; SVR in persons who test positive for genotype 1 or 4 approximates 45% to 50% with combined IFN-α and ribavirin, whereas patients with genotype 2 or 3 infection have an approximate 80% sustained virologic response. In addition, patients with genotype 2 or 3 infection have to be treated for only 6 months compared to a 1-year course for those with genotypes 1 and 4.157 Hence, it is essential to obtain a viral genotype before initiating therapy. Another important treatment guideline is that treatment can be stopped after 3 months in persons whose viral load has failed to become undetectable by that time; the continued presence of virus in the peripheral blood 3 months after initiation of therapy is highly predictive of treatment failure. In summary, the combined use of pegylated IFN-α (180 µg/ wk, subcutaneously) and weight-based dosing of ribavirin (8001200 mg/day, orally) will result in an approximate 50% SVR for genotypes 1 and 4 and an approximate 80% SVR for genotypes 2 and 3. These treatments have multiple side effects that are

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often poorly tolerated. Interferon can cause flu-like symptoms, marked fatigue, mood changes (particularly depression and irritability), hair loss, anorexia, nausea and vomiting, thyroid dysfunction (generally hypothyroidism) and cytopenias, particularly thrombocytopenia and leukopenia. All these side effects, except those involving the thyroid, are reversible upon cessation of therapy. Cytopenias may necessitate dose reduction or cessation of interferon therapy. The primary serious side effect of ribavirin is hemolytic anemia. This can be offset by administration of erythropoietin, but often requires dose reduction or discontinuation of this arm of the therapy. The mechanisms of interferon’s and ribavirin’s antiviral effects are diverse and not completely understood, but they are general antiviral agents and not HCVspecific therapies. Under development are several drugs that are specifically directed to critical steps in the HCV life cycle, including inhibitors of HCV protease and helicase enzymes. Early evidence indicates that this class of virus-specific enzyme inhibitors are very potent and can decrease viral loads rapidly.158 However, as with similar inhibitors for HBV and HIV, resistance develops rapidly and hence these drugs will not be given singly, but rather in combination with interferon, interferon plus ribavirin, or with each other. It is probable that interferon will remain the backbone of HCV therapy and that these new designer drugs will be additive rather than replacements. It is anticipated that under these new combination regimens, treatment efficacy will increase significantly, but the substantial side effects of interferon will remain and side effects of the new agents will be added. It is hoped that the anticipated additive efficacy of these new therapies will offset their side effects and resistance patterns, especially because the drug cocktails may need to be given for only 6 months. Many other therapeutic approaches, including small interfering RNAs, are under investigation and the ability to study new agents has been enhanced, not only by increased knowledge of the structure of the virus, but also by the recent availability of in-vitro culture systems116-119 and the chimeric severe combined immunodeficiency (SCID) mouse model that harbors human hepatocytes.159 Last, the potential for liver transplantation is increasingly available for those who fail antiviral therapy and progress to end-stage liver disease. Although the transplanted liver is universally reinfected, the 5-year survival for liver transplantation now approximates 70%.160 Nonetheless, in a subset of transplant recipients, recurrent hepatitis C is severe and cirrhosis may develop in as little as 3 to 5 years.161,162 The main impediment to liver transplantation is not graft or patient survival, but rather cost and the inability to procure a sufficient number of viable livers for the very large population that has developed, or subsequently will develop, HCV-related end-stage liver disease that is not amenable to current drug therapies.

Non-A, Non-B, Non-C Hepatitis Within the spectrum of TAH, cases were identified that met the biochemical and exclusionary definition of NANB hepatitis,

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but were subsequently shown to be negative for serologic and molecular markers of HAV, HBV, and HCV infection. Such cases have been termed non-A, non-B, non-C hepatitis and have been assumed to be the consequence of another, as yet undiscovered, human hepatitis virus. However, such cases could also be the result of a known hepatitis agent present at levels below the detection limit of current assays, or be of nonviral origin. After more than a decade of intensive investigation, and despite many published and unpublished claims of discovery, a non-A, non-B, non-C human hepatitis agent has yet to be conclusively identified. Evidence for the existence of more than one NANB hepatitis agent initially derived from the clinical observation of more than one episode of HBV- and HAV-negative hepatitis occurring in the same patient163 and the observation that some episodes of NANB hepatitis among persons with hemophilia occurred after an unusually short incubation period.164 Results of early chimpanzee cross-challenge studies further supported the existence of two NANB agents.165 One of these agents proved to be HCV, and the other is still undefined. Confounding the issue of whether there are additional NANB agents is the observation that both humans and chimpanzees can sustain more than one episode of HCV infection.166 Thus, what appeared to be two episodes of NANB hepatitis may instead have been two distinct episodes of hepatitis C. Nonetheless, 10% to 30% of cases of chronic NANB hepatitis cannot be accounted for by HCV infection, and most cases of fulminant hepatitis and hepatitis-associated aplastic anemia appear to be unrelated to hepatitis viruses A, B, or C, suggesting the existence of an additional agent of hepatitis in humans. Encouraged by the blind cloning of HCV, investigators have used sophisticated molecular approaches to search for the agent or agents of non-A, non-B, non-C hepatitis. This has led to the sequential discovery of the GB virus167 the hepatitis G virus,168 the TT virus,169 and the SEN virus.170 None of these agents has yet been proven to be a hepatitis virus in humans, but each has been shown to be transmitted by transfusion and to result in persistent infection in some blood recipients. The GB agent derives from the blood of a surgeon (G.B.) who in the 1950s contracted acute icteric hepatitis. Inoculation of GB serum into marmosets reproducibly caused hepatitis, but controversy arose as to whether this represented transmission of a human hepatitis agent or reactivation of a latent marmoset hepatitis virus. The controversy was never resolved. Decades later, investigators at Abbott Laboratories analyzed frozen samples that had been collected during serial passages of the GB agent in the marmoset model. Using representational difference analysis to compare preinoculation and postinoculation marmoset serum,167 the investigators identified unique nucleic acid sequences in the postinoculation specimens.171 Sequencing of the full genome identified three distinct GB agents that were designated GBV-A, GBV-B, and GBV-C.171 The genomic structure identified these agents as flaviviruses, but the agents showed little homology to HCV or other members of that family. Extensive population studies involving

Chapter 46: Transfusion-Transmitted Hepatitis

patients with and without liver disease and studies of marmosets established that GBV-A was a primary marmoset agent, that GBV-C was a human virus, and that GBV-B might infect both species. In independent investigations, Gene Labs (in collaboration with NIH) and the CDC, used sequence-independent single primer amplification to identify what was considered a novel hepatitis agent and called the hepatitis G virus.168 Subsequent sequence comparisons revealed that GBV-C and HGV were essentially the same agent and represented strain variants of this new member of the Flaviviridae family. Although GBV-C/HGV was unequivocally found to be transmitted by transfusion, extensive clinical studies did not show a causal relation between the presence of the virus and the development of hepatitis.172 The designation hepatitis G virus now appears to be a misnomer; it would be better to refer to this agent as the GB virus because that designation does not infer causality. In 1997, Nishizawa et al169 in Japan used representational difference analysis to compare pre- and posthepatitis sera from five patients with TAH. A unique viral clone was found in three of the five cases of TAH studied, and viral titers generally correlated with the ALT level. The presumed virus was designated TT virus after the initials of the index case. Biophysical characterization173 suggested that TT virus was a novel, 3700 bp, nonenveloped, circular, single-stranded human DNA virus most closely related to the Circoviridae, a family of plant and animal viruses not previously associated with human disease.174 The TT virus family is highly divergent and at least 16 genotypes have been identified. The TT virus has been reported to replicate in the liver based on PCR detection and in-situ hybridization.175 However, there were no morphologic changes in the liver cells that showed hybridization signals, and there was no correlation between the percentage of TT-virus-infected hepatocytes and the histologic activity index or its composite scores. Although the TT virus can infect liver cells, it may not cause hepatitis. Epidemiologic studies have shown that TT virus is transmitted by both parenteral and enteral routes. The original studies of TT virus suggested a relation to acute and chronic liver disease, but subsequent studies with inclusive primers that targeted conserved sequences showed an exceedingly high background prevalence (93%) and thus established that most persons with TT virus infection did not have hepatitis.176 Matsumoto et al177 studied TT virus infection in the transfusion setting using the more restrictive primers used in the original publications. The background prevalence in the volunteer US donor population was 7.5%. The key finding of this NIH study was that the frequency of transfusion-associated TT virus infection was identical among patients with non-A–E hepatitis (see later) (23%) and control patients who did not have hepatitis (22%). Overall, fewer than 4% of TT virus infections were associated with hepatitis, and there was poor correlation between TT virus DNA levels and ALT levels. Hence, as had occurred in investigations of HGV, the NIH prospective series did not support a causal association between TT virus infection and

posttransfusion hepatitis. However, there are caveats to the interpretation that the TT virus lacks pathogenicity in that the agent may cause disease in only a small number of infected persons who have particular host susceptibility factors or there may be particular TT virus variants that are pathogenic while the wildtype virus is not. Many other isolates subsequently were identified in the TT virus family, including agents designated SANBAN, YONBAN, and TLMV.178 Each of these agents is phylogenetically distant from the prototype TT virus agent and shows only 50% to 60% sequence homology. Unlike HCV variants that primarily represent strain differences of 1% to 2% (quasispecies) or genotypes diverging by approximately 15%, in the TT virus family, sequence differences between variants frequently exceed 30% and sometimes exceed 50%. These differences are so great that the agents, although linked by common biophysical characteristics, may have totally different clinical spectrums and disease associations. A virus detection program conducted by the Diasorin Corporation (Stillwater, MN) uncovered what was at the time considered a novel infectious agent and designated SEN virus for the initials of the patient in whom it was initially found. Like TT virus, SEN virus proved to be a small, nonenveloped, circular, single-stranded DNA virus.179 The SEN virus represented a subfamily of very heterogeneous agents differing in nucleotide sequences by 15% to 50% from each other and by 40% to 60% from the prototype TT virus sequence. The SEN virus is now considered a member of the TT virus family in the genus Circoviridae. The clinical significance of SEN virus is uncertain. Two SEN virus variants, designated SENV-D and SENV-H, have been extensively studied.179 SENV-D and -H were found in 1.5% to 2% of volunteer US donors and were shown to be transfusion transmitted. They were present in 40% of 155 subjects who underwent transfusion but in only 3% of 97 control subjects who did not undergo transfusion (p  0.0001).179 A significant relationship of infection to transfusion volume was observed, and donor-recipient linkage was established by means of sequencing. It is noteworthy that new SEN virus infections occurred in 3% of patients who did not receive transfusions. This finding suggested that, as with TT virus, there was nosocomial transmission or reactivation of latent virus. In the NIH prospective study of TAH,179 13 cases of nonA–E hepatitis were found. One of these patients was infected with SEN virus before transfusion. Among the remaining 12 susceptible recipients, 11 (92%) became SENV-D or SENV-H positive after transfusion. There was a good, although imperfect, temporal association between the level of virus and the level of elevation of ALT value. In contrast, acute SEN virus infection was found in 24% of patients who underwent transfusion but did not have hepatitis (p  0.0001). Despite the strong statistical link to cases of transfusion-associated non-A-E hepatitis, it was projected that no more than 5% of cases of SEN virus infection were accompanied by biochemical evidence of hepatitis.

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Further, there has been no significant association between SEN virus infection and acute liver failure or cryptogenic cases of chronic hepatitis, cirrhosis, or HCC. If SEN virus is a hepatitis agent, it causes disease in only a small number of infected individuals, the difference perhaps depending on host susceptibility, viral load, or immune responsiveness. A parallel might be drawn to viruses such as cytomegalovirus or EBV that also cause disease in only a small number of infected persons. Nonetheless, it is important to emphasize that the statistical association between SEN virus infection and TAH does not establish causality. There is currently no proof that either GB virus, TT virus, or SEN virus represents the elusive agent of non-A, non-B, non-C hepatitis. An epidemic form of NANB hepatitis has resulted in massive outbreaks, most prominently in India, other parts of the Asian subcontinent, and South America.180,181 These explosive outbreaks are waterborne and epidemiologically resemble HAV infection, but they are serologically and to some degree clinically distinct. Particles ranging from 27 to 38 nm have been associated with the epidemic form of NANB hepatitis and a specific serologic assay has been developed.182 This form of hepatitis is now known to be caused by a distinct RNA virus designated hepatitis E virus (HEV) and to be classified in the family of Caliciviruses. Although HEV was first recognized in its epidemic form, it is endemic in many areas of the world and is probably maintained in animal hosts, particularly swine. An HEV vaccine has been developed, but not yet widely applied. Thus, the characterized human hepatitis viruses range from A to E, including HAV (infectious), HBV (serum), HCV (former NANB), HDV (δ agent), and HEV (enterically transmitted nonA, non-B). It is unclear at present whether a non-A, non-B, nonC transfusion-transmitted hepatitis agent exists or whether these biochemically defined cases represent cryptic (sero-silent) forms of hepatitis B or C or are of nonviral etiology. Viral discovery programs are ongoing, but their likelihood of success diminishes as the incidence of non-A, non-B, non-C hepatitis declines in the wake of enhanced blood donor screening. Clearly, the discovery of HCV and the introduction of molecular assays for HCV RNA coupled with increasingly sensitive assays for HBsAg has diminished TAH incidence to minuscule levels and proportionately reduced the clinical relevance of non-A, non-B, non-C hepatitis in this setting.

Summary A marked decline in the incidence of TAH was observed in the early 1970s as commercial blood supplies were phased out and the United States adopted an all-volunteer donor system. No subsequent interdictive measure has had such dramatic effect, although additional contributions were made by the development of sensitive assays for HBsAg and by the introduction of surrogate tests for the NANB hepatitis agent. Ironically, AIDS had the next most profound effect on the incidence of hepatitis.

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The development of a highly sensitive test for antibody to HIV and the intensified donor screening initiated to exclude highrisk AIDS groups served to exclude the primary source of donors transmitting viral hepatitis. The overlap between groups at risk of AIDS and groups at risk of viral hepatitis is so marked that a single exclusionary process diminishes both occurrences. As the result of these indirect measures, the incidence of TAH had declined significantly even before the introduction of anti-HCV testing. In sequential prospective studies at the NIH, the incidence of TAH had decreased from 8% to 10% to approximately 4% just before the introduction of routine anti-HCV donor screening. Implementation of anti-HCV testing resulted in an approximate 80% reduction in TAH incidence from pretesting levels, bringing the frequency down to less than 1%. It can be calculated from the prevalence of the HCV carrier state (0.3%), the number of units of blood components transfused (15 million annually), and the infectivity of anti-HCV-positive, RIBA-positive blood (90%) that the introduction of first-generation assays initially prevented 111 transfusion-associated HCV infections per day in the Untied States alone, or approximately 40,000 infections per year. A subsequent analysis showed that second-generation assays would find one additional true positive donor in every 1000 tested and thus could have prevented an additional 15,000 HCV transmissions per year. A prospective study at the NIH in which the second-generation anti-HCV assay was used for donor screening showed that the risk of transfusion-transmitted hepatitis C is now exceedingly low and, indeed, approaches zero—a remarkable achievement given that the incidence of hepatitis was 33% in the 1960s and 10% to 12% in the early 1980s. Mathematical modeling after the introduction of HCV-NAT places the risk of transfusion-associated hepatitis C at one case per 1.5 to 2.0 million transfusions. Although anti-HCV screening has been a critical determinant in risk reduction, it has been only one of many interventions since 1985 that have increased blood safety. Other measures include the more judicious use of blood components and derivatives by physicians and a concerned recipient population; the increasing use of autologous blood and blood management techniques; intensive donor questioning and interdiction based on high-risk behavior; the shift to apheresis platelets; the introduction of additional infectious disease tests; bacterial testing of platelet products; and virus inactivation of clotting factor concentrates. Under development are chemical pathogen reduction procedures for plasma, platelets, and red cells. These combined sociologic, serologic, molecular, and virus inactivation measures raise the realistic potential that transfusion-associated viral hepatitis will be totally eradicated and become a historical footnote in the practice of transfusion medicine.

Disclaimer The authors have disclosed no conflicts of interest.

Chapter 46: Transfusion-Transmitted Hepatitis

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Chapter 46: Transfusion-Transmitted Hepatitis

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105. Alter HJ. To C or not to C: These are the questions. Blood 1995; 85:1681-95. 106. Aach RD, Szmuness W, Mosley JW, et al. Serum alanine aminotransferase of donors in relation to the risk of non-A, non-B hepatitis in recipients: The transfusion-transmitted viruses study. N Engl J Med 1981;304:989-94. 107. Stevens CE, Aach RD, Hollinger FB, et al. Hepatitis B virus antibody in blood donors and the occurrence of non-A, non-B hepatitis in transfusion recipients: An analysis of the transfusion-transmitted viruses study. Ann Intern Med 1984;101:733-8. 108. Alter HJ, Purcell RH, Holland PV, et al. The relationship of donor transaminase (ALT) to recipient hepatitis: Impact on blood transfusion services. JAMA 1981;246:630-4. 109. Koziol DE, Holland PV, Alling DW, et al. Antibody to hepatitis B core antigen as a paradoxical marker for non-A, non-B hepatitis agents in donated blood. Ann Intern Med 1986;104:488-95. 110. Kleinman S, Alter HJ, Busch M, et al. Increased detection of hepatitis C virus (HCV)-infected blood donors by multiple-antigen HCV enzyme immunoassay. Transfusion 1992;32:805-13. 111. Blajchman MA, Bull SB, Feinman SV. Post-transfusion hepatitis: Impact of non-A, non-B hepatitis surrogate tests. Canadian Post-Transfusion Hepatitis Prevention Study Group. Lancet 1995;345:21-5. 112. Kuo G, Choo OL, Alter HJ, et al. An assay for circulating antibodies to a major etiologic virus of non-A, non-B hepatitis. Science 1989;244:362-4. 113. Alter HJ. New kit on the block: Evaluation of second-generation assays for detection of antibody to the hepatitis C virus. Hepatology 1992;15:350-3. 114. Busch MP. Closing the windows on viral transmission by blood transfusion. In: Stramer SL, ed. Blood safety in the new millennium. Bethesda, MD: AABB, 2001:33-54. 115. Tanaka E, Ohue C, Aoyagi K, et al. Evaluation of a new enzyme immunoassay for hepatitis C virus (HCV) core antigen with clinical sensitivity approximating that of genomic amplification of HCV RNA. Hepatology 2000;32:388-93. 116. Wakita T, Pietschmann T, Kato T, et al. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 2005;11:791-6. 117. Lindenbach BD, Evans MJ, Syder AJ, et al. Complete replication of hepatitis C virus in cell culture. Science 2005;309:623-6. 118. Zhong J, Gastaminza P, Cheng G, et al. Robust hepatitis C virus infection in vitro. Proc Natl Acad Sci U S A 2005;102:9294-9. 119. Yi M, Villanueva RA, Thomas DL, et al. Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells. Proc Natl Acad Sci U S A 2006;103:2310-15. 120. Ogata N, Alter HJ, Miller RH, et al. Nucleotide sequence and mutation rate of the H strain of hepatitis C virus. Proc Natl Acad Sci U S A 1991;88:3392-6. 121. Farci P, Shimoda A, Coiana A, et al. The outcome of acute hepatitis C predicted by the evolution of the viral quasispecies. Science 2000;288:339-44. 122. Farci P, Alter HJ, Govindarajan S, et al. Lack of protective immunity against reinfection with hepatitis C virus. Science 1992;258:135-40. 123. Bartosch B, Bukh J, Meunier JC, et al. In vitro assay for neutralizing antibody to hepatitis C virus: evidence for broadly conserved neutralization epitopes. Proc Natl Acad Sci U S A 2003;100:14199-204. 124. Meunier JC, Engle RE, Faulk K, et al. Evidence for crossgenotype neutralization of hepatitis C virus pseudoparticles and

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125.

126.

127. 128.

129.

130.

131.

132.

133.

134.

135.

136.

137.

138.

139.

140.

141.

142.

143.

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enhancement of infectivity by apolipoprotein C1. Proc Natl Acad Sci U S A 2005;102:4560-5. von Hahn T, Yoon JC, Alter H, et al. Hepatitis C virus continuously escapes from neutralizing antibody and T-cell responses during chronic infection in-vivo. Gastroenterology 2007;132:667-78. Scheel TKH, Gottwein JM, Jensen TB, et al. Development of JFH1-based cell culture systems for hepatitis C virus genotype 4a and evidence for cross-genotype neutralization. Proc Natl Acad Sci U S A 2008;105:997-1002. Rehermann B. Interaction between the hepatitis C virus and the immune system. Semin Liver Dis 2000;20:127-41. Sumpter R Jr, Loo YM, Foy E, et al Regulating intracellular antiviral defense and permissiveness to hepatitis C virus RNA replication through a cellular RNA helicase, RIG-I. J Virol 2005;79:2689-99. Ferrari C, Valli A, Galati L, et al. T-cell response to structural and non-structural hepatitis C virus antigens in persistent and self-limited hepatitis C virus infections. Hepatology 1994;19:286-95. Folgori A, Spada E, Pezzanera M, et al. Early impairment of hepatitis C virus specific T cell proliferation during acute infection leads to failure of viral clearance. Gut 2006;55:1012-19. Conry-Cantilena C, VanRaden M, Gibble J, et al. Routes of infection, viremia, and liver disease in blood donors found to have hepatitis C virus infection. N Engl J Med 1996;334:1691-6. Alter MJ, Gerety RJ, Smallwood LA, et al. Sporadic non-A, non-B hepatitis: Frequency and epidemiology in an urban U.S. population. J Infect Dis 1982;145:886-93. Alter MJ, Kruszon-Moran D, Nainan OV, et al. The prevalence of hepatitis C virus infection in the United States, 1988 through 1994. N Engl J Med 1999;341:556-62. Pastore G, Monno L, Santantonio T, et al. Monophasic and polyphasic pattern of alanine aminotransferase in acute non-A, non-B hepatitis: Clinical and prognostic implications. Hepatogastroenterology 1985;32:155-8. Alter HJ, Hoofnagle JH. Non-A, non-B: Observations on the first decade. In: Vyas GN, Dienstag JL, Hoofnagle JH, eds. Viral hepatitis and liver disease. Orlando, FL: Grune & Stratton, 1984:345-54. Realdi G, Alberti A, Rugge M, et al. Long-term follow-up of acute and chronic non-A, non-B post-transfusion hepatitis: Evidence of progression to liver cirrhosis. Gut 1982;23:270-5. Omata M, Iwama S, Sumida M, et al. Clinico-pathological study of acute non-A, non-B post-transfusion hepatitis: Histological features of liver biopsies in acute phase. Liver 1981;1:201-8. Iwarson S, Lindberg J, Lundin P. Progression of hepatitis non-A, non-B to chronic active hepatitis: A histologic follow-up of two cases. J Clin Pathol 1979;32:351-5. Corrao G, Arico S. Independent and combined action of hepatitis C virus infection and alcohol consumption on the risk of symptomatic cirrhosis. Hepatology 1998;27:914-19. Kato Y, Nakata K, Omagari K, et al. Risk of hepatocellular carcinoma in patients with cirrhosis in Japan: Analysis of infectious hepatitis viruses. Cancer 1994;74:2234-8. Seeff LB. Hepatitis in hemophilia: A brief review. In: Burk PB, Chalmers C, eds. Frontiers in liver disease. New York: ThiemeStratton, 1981:231-41. Aledort LM, Levine PH, Hilgartner M, et al. A study of liver biopsies and liver disease among hemophiliacs. Blood 1985;66:367-72. Hay CRM, Preston FE, Triger DR, et al. Progressive liver disease in hemophilia: An understated problem? Lancet 1985;i:1495-7.

144. Tefler P, Sabin C, Devereux H, et al. The progression of HCCassociated liver disease in a cohort of hemophiliac patients. Br J Haemotol 1994;87:555-61. 145. Kiyosawa K, Akahane Y, Nagata A, et al. Significance of blood transfusion in non-A, non-B chronic liver disease in Japan. Vox Sang 1982;43:45-52. 146. Kiyosawa K, Akahane Y, Nagata A, et al. Hepatocellular carcinoma after non-A, non-B posttransfusion hepatitis. Am J Gastroenterol 1984;79:777-81. 147. Okuda H, Obata H, Motoike Y, et al. Clinicopathological features of hepatocellular carcinoma: Comparison of hepatitis B seropositive and seronegative patients. Hepatogastroenterology 1984;31:64-8. 148. Bruix J, Calvet X, Costa J, et al. Prevalence of antibodies to hepatitis C virus in Spanish patients with hepatocellular carcinoma. Lancet 1989;ii:1004-6. 149. Tanaka Y, Kurbanov F, Vargas V, et al. Molecular tracing of the global hepatitis C virus epidemic predicts regional patterns of hepatocellular carcinoma mortality. Gastroenterology 2006;130:703-14. 150. Seeff LB, Buskell-Bales Z, Wright EC, et al. Long-term mortality after transfusion-associated non-A, non-B hepatitis. N Engl J Med 1992;327:1906-11. 151. Seeff LB, Hollinger FB, Alter HJ, et al. Long-term mortality and morbidity of transfusion-associated non-A, non-B and type C hepatitis—a National Heart, Lung and Blood Institute collaborative study. Hepatology 2001;33:455-63. 152. Seeff LB, Miller RN, Rabkin CS, et al. 45-Year follow-up of hepatitis C virus infection in healthy young adults. Ann Intern Med 2000;132:105-11. 153. Shakil AO, Conry-Cantilena C, Alter HJ, et al. Volunteer blood donors with antibody to hepatitis C virus: Clinical, biochemical, virologic and histologic features. Ann Intern Med 1995;123:330-7. 154. Kenny-Walsh E. Irish Hepatology Research Group. Clinical outcomes after hepatitis infection from contaminated anti-globulin. N Engl J Med 1999;340:1228-33. 155. Muller R. The natural history of hepatitis C: Clinical experiences. J Hepatol 1996;24(Suppl):52-4. 156. Vogt M, Lang T, Frosner G, et al. Prevalence and clinical outcome of hepatitis C infection in children who underwent cardiac surgery before the implementation of donor screening. N Engl J Med 1999;341:866-70. 157. Hoofnagle JH, Seeff LB. Peginterferon and ribavirin for chronic hepatitis C. N Engl J Med 2006;355:2444-51. 158. Reesink HW, Zeuzam S, Weegink CJ, et al. Rapid decline of viral RNA in hepatitis C patients treated with VX-950: A phase 1b, placebocontrolled, randomized study. Gastroenterology 2006;131:997-1002. 159. Mercer DF, Schiller DE, Elliott JF, et al. Hepatitis C virus replication in mice with chimeric human livers. Nat Med 2001;7:890-91. 160. Davis GL. Chronic hepatitis C and liver transplantation. Rev Gastroenterol Disord 2004;4:7-17. 161. Berenguer M, Ferrell L, Watson J, et al. HCV-related fibrosis progression following liver transplantation: Increase in recent years. J Hepatol 2000;32:673-84. 162. McCaughan GW, Zekry A. Pathogenesis of hepatitis C virus recurrence in the liver allograft. Liver Transpl 2002;8(10 Suppl 1):S7-13. 163. Mosley JW, Redeker AG, Feinstone SM, et al. Multiple hepatitis viruses in multiple attacks of acute viral hepatitis. N Engl J Med 1977;296:75-8. 164. Craske J, Dilling N, Stern D. An outbreak of hepatitis associated with intravenous injection of factor VIII concentrate. Lancet 1975;ii:221-3.

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165. Yoshizawa H, Itoh Y, Iwakiri S, et al. Demonstration of two different types of non-A, non-B hepatitis by reinjection and cross-challenge studies in chimpanzees. Gastroenterology 1981;81:107-13. 166. Lai ME, Mazzoleni AP, Argiolu F, et al. Hepatitis C virus in multiple episodes of acute hepatitis in polytransfused thalassaemic children. Lancet 1994;343:388-90. 167. Simons JN, Leary TP, Dawson GJ, et al. Isolation of novel viruslike sequences associated with human hepatitis. Nat Med 1995;6:564-9. 168. Linnen J, Wages J Jr, Zhang-Keck ZY, et al. Molecular cloning and disease association of Hepatitis G virus: A transfusion-transmissible agent. Science 1996;271:505-8. 169. Nishizawa T, Okamoto H, Konisi K, et al. A novel DNA virus (TTV) associated with elevated transaminase levels in posttransfusion hepatitis of unknown etiology. Biochem Biophys Res Commun 1997;241:92-7. 170. Tanaka Y, Primi D, Wang RY, et al. Genomic and molecular evolutionary analysis of a newly identified infectious agent (SEN virus) and its relationship to the TT virus family. J Infect Dis 2001;183:359-67. 171. Leary TP, Muerhoff AS, Simons JM, et al. Sequence and genomic organization of GBV-C: A novel member of the Flaviviridae associated with human non-A-E hepatitis. J Med Virol 1996;48:60-7. 172. Alter HJ, Nakatsuji Y, Melpolder J, et al. The incidence of transfusion-associated HGV infection and its relation to liver disease. N Engl J Med 1997;336:747-54. 173. Okamoto H, Nishizawa T, Kato N, et al. Molecular cloning and characterization of a novel DNA virus (TTV) associated with posttransfusion hepatitis of unknown etiology. Hepatol Res 1998;10:1-16.

174. Mushahwar IK, Erker JC, Muerhoff AS, et al. Molecular and biophysical characterization of TT virus: Evidence for a new virus family infecting humans. Proc Natl Acad Sci U S A 1999;96:3177-82. 175. Rodriguez-Inigo E, Casqueiro M, Bartolome J, et al. Detection of TT virus DNA liver biopsies by in situ hybridization. Am J Pathol 2000;156:1227-34. 176. Okamato H, Takahashi M, Nishizawa T, et al. Marked genomic heterogeneity and frequent mixed infection of TT virus demonstrated by PCR with primers from coding and noncoding regions. Virology 1999;259:428-36. 177. Matsumoto A, Yeo AE, Shih JW, et al. Transfusion-associated TT virus infection and its relationship to liver disease. Hepatology 1999;30:283-8. 178. Hijikata M, Takahashi K, Mishiro S. Complete circular DNA genome of a TT virus variant (isolate name SANBAN) and 44 partial ORF2 sequences implicating a great degree of diversity beyond genotypes. Virology 1999;260:17-22. 179. Umemura T, Yeo AET, Sottini A, et al. SEN virus infection and its relationship to transfusion-associated hepatitis. Hepatology 2001;33:1303-11. 180. Khuroo MS. Study of an epidemic of non-A, non-B hepatitis: Possibility of another human hepatitis virus distinct from posttransfusion non-A, non-B type. Am J Med 1980;68:818-824. 181. Kane MA, Bradley DW, Shrestha JM, et al. Epidemic non-A, non-B in Nepal: Recovery of a possible etiologic agent and transmission studies in marmosets. JAMA 1984;252:3140-5. 182. Tsarev SA, Tsareva TS, Emerson SU, et al. ELISA for antibody to hepatitis E virus (HEV) based on complete open-reading-frame 2 protein expressed in insect cells: Identification of HEV infection on primates. J Infect Dis 1993;168:369-78.

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47

Retroviruses and Other Viruses John A.J. Barbara1 & Brian C. Dow2 1

Emeritus Microbiology Consultant to National Blood Service, London, and Visiting Professor, University of West of England, Bristol, United Kingdom 2 Consultant Clinical Microbiologist and Head of National Microbiology Reference Unit, Scottish National Blood Transfusion Service, Glasgow, United Kingdom

Ever since the advent of the global human immunodeficiency virus (HIV) epidemic, transfusion medicine has maintained a heightened awareness of the potential risk of transmitting infection through blood transfusions. This awareness has paradoxically increased despite the actual infectious risks of blood transfusion being lower than ever in developed countries. The enhanced level of safety has resulted from a series of stepwise interventions and policies in blood services throughout the world. Microbial risks were once mainly related to persistent infections such as HIV, hepatitis B virus (HBV), and hepatitis C virus (HCV), where viral carriage or latency were the key factors predisposing an agent to transmission by transfusion. More recently, several acute infections have posed a threat to blood safety when they have occurred at high incidence in certain populations. West Nile virus (WNV) in North America is a good example of this but other infections such as chikungunya and the specter of respiratory viruses such as pandemic influenza have made transfusion microbiologists widen their areas of vigilance. These issues are reflected in this chapter.

Retroviruses Overview Retroviruses are widely distributed in nature, with examples in insects, reptiles, and almost all mammals. There are four main pathogenic human retroviruses—namely, HIV-1 and HIV-2, belonging to the lentivirus group of the retrovirus family, and human T-cell lymphotropic viruses (HTLV-I and HTLV-II), belonging to the oncorna group. It is generally accepted that HTLV-I and HTLV-II evolved from simian T-lymphotropic retroviruses that were transmitted to humans centuries ago. HIV1 is also thought to have derived from a simian ancestor, as simian immunodeficiency viruses are endemic in chimpanzees Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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in Central Africa.1 HIV-2 originated from sooty mangabey monkeys in West Africa. It is thought that transmission to African natives may have occurred only over the last century.2,3 There are three groups of HIV-1, the main group (M) containing several genotypes A through K, new group (N), and the outlier group (O). Group M is widely distributed throughout the world and is the predominant cause of the AIDS pandemic. Many of the group M subtypes and most of the circulating recombinant forms have a limited geographic spread (Table 47-1). Genotype B is by far the most widespread HIV genotype in the developed world, whereas genotype C is the most common overall, accounting for 50% of HIV infections. The Democratic Republic of Congo has the largest degree of HIV-1 genetic diversity, suggesting that this geographic area is the source of the virus.4,5 Groups O and N are confined to Cameroon in Central Africa. HIV-2 was originally thought to be confined to West Africa, but international travel has resulted in a wider distribution that represents 1% or less of all HIV infections. Retroviruses are membrane-coated, single-stranded RNA viruses that require the essential enzyme, reverse transcriptase, to transcribe their viral RNA to complementary doublestranded DNA that will integrate into the host cell chromosome (Fig 47-1). The host cell enzymes aid the virus to complete its life cycle by synthesising virions that bud from the plasma membrane to infect other cells or organisms. More virologic and clinical details on retroviral infections can be accessed in the scientific literature.7-12

Human Immunodeficiency Virus Originally described in the early 1980s by both Montagnier and Gallo, respectively, as lymphadenopathy virus (LAV) and human T-cell lymphotropic virus type III (HTLV-III), HIV-1 (as it subsequently became known) was established as the cause of AIDS in 1984 (Fig 47-2). Shortly after infection, the virus integrates into the host cell DNA using reverse transcription. The infected individual may suffer a “glandular fever”-like “seroconversion” illness within 2 to 3 weeks of infection, then develops antibodies, and usually remains asymptomatic for many

Chapter 47: Retroviruses and Other Viruses

Table 47-1. Human Immunodeficiency Virus Strains Found Worldwide* HIV Strain HIV-1 Group M

HIV-1 CRF

HIV-1 URF

Subclassification

Main Geographic Concentration

Worldwide Prevalence

A

Africa and former USSR

High

B

America, Western Europe, Australia

High

C

South and East Africa, India

High

D

East and West Africa

High

F

West Central Africa

Very low

G

West Central Africa

Very low

H

West Central Africa

Very low

J

West Central Africa

Very low

K

West Central Africa

Very low

CRF01_AE

South East Asia

High

CRF02_AG

West Africa

High

CRF03 to CRF16

Africa, China, Thailand, Argentina, Western Europe

Very low

Several

Africa, South America, South East Asia

Moderate

West Central Africa

Very low

West Central Africa

Very low

West Africa

Moderate

HIV-1 Group O HIV-1 Group N HIV-2

Sub types A and B

CRF ⫽ circulating recombinant forms (component subtypes after the underscore); URF ⫽ unique recombinant forms *Adapted from McCutchan.3

becomes increasingly prone to opportunistic infections, indicating the start of clinically apparent AIDS. In the developed world the use of combination drug therapy can prolong life expectancy, but the majority of those denied such medication will inevitably reach the terminal stage of the disease.

RNA DNA

RNA AAA

Figure 47-1. Simplified replication cycle of a retrovirus. The virion containing two RNA genome copies enters the cell via a specific cell surface receptor. The single-stranded RNA genome is converted into a double-stranded RNA provirus by the virion enzyme reverse transcriptase. The provirus inserts into host chromosomal DNA in the same orientation as the original virion RNA. Transcription of RNA from the integrated DNA provirus is mediated by cellular RNA polymerases, and this RNA serves both as messenger RNA for the synthesis of viral antigens and as genomic RNA, which becomes packaged into progeny virion budding from the cell surface. Used with permission from Weiss et al.6

years. However, during this asymptomatic period, the latent virus commonly reactivates and actively replicates in lymphoid tissues, macrophages, and other tissues. The number of CD4⫹ lymphocytes gradually decreases until the infected individual

Incidence and Prevalence Among Blood Donors Since 1983 men who have had sex with other men (MSM) have been permanently excluded from donation in most countries because the HIV prevalence was high in this population. With improvements in HIV serologic tests [including the introduction of HIV nucleic acid amplification testing (NAT)], the risk reduction measures have been subject to review following pressure from this population group. Although it is expected that potential donors will answer donor questionnaires honestly, it has been obvious from counseling HIV-positive donors that a significant proportion admit to a risk factor that would have resulted in deferral.14 The number of new cases of HIV infection (incidence) among blood donors should reflect the situation within the general heterosexual population. In the United Kingdom (UK) new cases of HIV have doubled in the general population during the past 5 years. This is a cause for concern and donor statistics have also shown a general increased trend to over twice the usual number of HIV-positive donors being detected in 2002 to 2003 compared with 2000 to 2001. The rate in new donors peaked in 2005 with nine cases per 100,000; the rate in repeat donors peaked in 2002 to 2003 with 0.8 case per 100,000.15 The HIV-positive donor rate

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Section IV: Part II

gp120env(SU) gp41env(TM)

Reverse transcriptase (RT)

p6gag(NC) Vpr

Integrase (IN) Protease (PR)

Lipid bilayer

Singlestranded HIV-1 RNA Host proteins

p7gag(NC)

p24gag(CA)

p17gag(MA)

Figure 47-2. Schematic of the human immunodeficiency virus type 1 (HIV-1) virion. Each of the virion proteins making up the envelope (gp120env and gp41env) and inner core (p24gag, p17gag, p7gag, and p6gag) is identified. The diploid RNA genome is shown associated with reverse transcriptase (RT), an RNA- and DNA-dependent DNA polymerase. Integrase (IN) and protease (PR) also are present in the mature HIV-1 virion. The auxiliary protein Vpr is incorporated into the HIV-1 virion through interaction with the P6gag protein, which composes the carboxyl terminus of the p55gag precursor protein. CA ⫽ capsid protein; MA ⫽ matrix protein; NC ⫽ nucleocapsid protein; SU ⫽ surface protein; TM ⫽ transmembrane protein. Used with permission from Geleziunas and Greene.13

1st gen Ab Detection

2nd gen Ab 3rd gen Ab 4th gen Ag/Ab p24 Antigen HIV NAT Infectivity

⫺56 ⫺48

⫺40

⫺32 ⫺24 ⫺16 Days

⫺8

0

8

has not increased further, although more HIV transmissions are being detected in the general population. In the United States the incidence is reported to be 7.2 cases per 100,000 in new donors (1993 to 1996) and 2.9 cases per 100,000 in repeat donors.16 In Western Europe 1.7 donations per 100,000 were HIV positive; in Eastern Europe (former USSR) this rose to 36.7 per 100,000 for the period 2000 to 2004. Ukraine had the highest prevalence with 128.4 HIV-positive donors per 100,000. The dramatic increase in the prevalence of HIV infection in Eastern European blood donors parallels the high ongoing transmission among the general populations, particularly in injecting drug users. This causes considerable concern over the safety of the blood supply in Eastern Europe.

Donor Testing and Counseling Shortly after the discovery of HIV as the cause of AIDS, serologic enzyme-linked immunosorbent assay (ELISA) tests for anti-HIV became commercially available during 1985 and most blood services in developed countries began screening every donation. HIV screening tests have gradually been improved over the years by replacing viral lysates with recom-

748

Figure 47-3. HIV window period detection by various HIV assays (generalized). Assuming that the first-generation HIV antibody test could detect early HIV seroconversion at Day 0, second-generation tests were able to detect seroconversion up to 23 days before, third-generation tests 34 days before, fourth-generation Ag/Ab assays 39 days before, HIV p24 antigen assays 40 days before, and HIV NAT 45 days before. This still leaves a period of around 11 days when a donor could be infective but is nonreactive in current tests.

binant antigens, including HIV-2 proteins and HIV-1 group O components; using sandwich technology; and, more recently, incorporating simultaneous detection of both HIV antibody and HIV p24 antigen in the fourth-generation HIV antigen/ antibody tests.17 These improvements have led to a considerable shortening (almost fourfold) of the seronegative gap between potential infectivity following infection and reactivity in serologic tests (Fig 47-3). Numerous commercial HIV serologic assays exist; transfusion services select a suitable assay on the basis of sensitivity, specificity, and other criteria. Normally, the selected assay should be capable of detecting early seroconversion samples without any associated specificity problems.18 External quality control samples19 should be used to monitor the performance of routine screening kits and batches of test kits should also be checked to ensure sensitivity is maintained before general use.20 The advent of HIV NAT-based systems for screening blood donors (introduced in some countries in 1995) is thought to reduce the time by detection by only a few days compared with the latest fourth-generation HIV antigen/antibody test systems. Current commercial HIV NAT assays can detect 14 copies/mL

Chapter 47: Retroviruses and Other Viruses

Blot

30 31 32 Figure 47-4. HIV Western blot (Genelabs HIV 2.2). Strip 30 shows a confirmed HIV-1 positive sample; strip 31 is a negative sample; strip 32 is an indeterminate (p24).

Control band

HIV-1 RNA (50% detection limit). The blood services in the United States, Canada, Germany, and France perform HIV NAT in minipools (MPs) of six to 24 donations and detect around 1 in 3 million as HIV-positive (by NAT only). Facilities in Italy observe around 1 in 450,000 and those in South Africa detect 1 in 25,00014 as HIV-positive (by NAT only, serology negative). Look-back investigations on HIV MP-NAT-negative donations from donors who later were found to be confirmed HIV positive, have revealed a number of HIV transfusion transmissions,21 leading to consideration of reducing the number of donations in minipools to the ultimate individual donation (ID)-NAT assay. HIV NAT should not replace serologic HIV donor screening assays, because there are occasional examples of HIV-seropositive donors with HIV viral loads below the threshold of HIV MP-NAT, being barely detectable even by HIV ID-NAT.22 Western blot testing of repeatedly reactive samples is often considered the “gold standard” for confirmation of reactivity, although reactivity in two or three alternative ELISAs of equal sensitivity is also a recognized confirmatory strategy in highprevalence populations. Western blot displays a wide range of antibodies against various components of the virus with the identification of antibodies to p24 (core) and gp120 and gp160 (envelope) regions being essential to conclude HIV infection (Fig 47-4). However, Western blot testing has several drawbacks. It is incapable of confirming HIV infection in those who are recently infected and who may be HIV NAT-reactive or HIV p24 antigen-reactive. However, the assay invariably will produce a clear positive result on the follow-up sample from such a donor. It should be pointed out that many weak bands can appear in individuals who have never been infected. With the use of widely accepted criteria to interpret results, such “indeterminate” reactions can be considered as uninfected if no change in blot pattern is seen on follow-up samples. This position has been verified by extensive analysis of many such samples worldwide.23-25 The finding of sole HIV NAT reactivity in the absence of serologic activity requires confirmation by either an alternative HIV NAT assay or a sensitive HIV p24 antigen test (generally up to four times more sensitive than any of the combined assays).

p17

p24

gp41

gp120

p55 p51

p66

gp160

In the absence of any confirmatory procedure, preliminary counseling of donors should be considered that includes the acquisition of another blood sample that would reveal any further development of serologic markers. If HIV infection is confirmed, the donor should be invited to attend a clinic with a donor consultant who will advise the donor of his or her HIV status and ask for a follow-up sample for repeat testing to confirm identity. The donor would be probed regarding any potential risk situations that may have led to the infection. Use of a detuned assay on confirmed HIV-positive donors can aid in the determination whether new donors were recently infected.26,27 Often donors will admit to a risk situation that they had denied when they gave their index donation. The infected donor would then be referred to the appropriate followup health department for clinical management.

Window Period and Risk of Transmission Transfusion transmission has infected approximately 2% to 5% of those with HIV, with the vast majority of these transmissions occurring before the advent of HIV donor testing.28 Around 90% of recipients of seropositive units became infected with HIV.29 Since HIV screening commenced, HIV transfusion transmission has become a relatively rare event, usually associated with a blood donor in the early course of HIV seroconversion, ie, in the “window period.” This window period is defined as the time between exposure to the virus (or onset of infectiousness in the host) and the appearance of a diagnostic marker of the infection in the blood. In the late 1980s some well-documented cases of HIV-1 transmission from screening test-negative blood were reported.30,31 This led to an estimate that five cases of transfusion-transmitted AIDS had occurred per year in the United States from anti-HIV-negative, early seroconversion donations. The residual risk has been estimated between 1 in 450,000 and 1 in 3 million before NAT; NAT screening has halved this risk.32 The risk of transmission of HIV-2 has been estimated at less than 1 in 15 million and other subtypes are of even lesser concern. It is notable that the risk of HIV infection from blood has been reduced by almost four orders of magnitude in comparison with the transfusion AIDS epidemic of 1982 to 1984, which was responsible for around 2% to 5% of those infected

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with HIV. Furthermore it has been suggested that the infectivity of a transfusion in the early 11-day window period (before HIV NAT detection) may be lower than later stages of primary infection because of the low level of virus in this eclipse phase.12 It has been estimated that the doubling time of HIV is 20.5 hours in this period.33 Nevertheless, despite the improvements in HIV testing, there have been reports of HIV-NAT-negative blood involved in transfusion-transmitted HIV.22,34 This demonstrates that despite the employment of the most sensitive type of assay to screen blood donations, zero risk is presently still unattainable. The residual risk of HIV transfusion-transmitted infection (TTI) has been estimated to be 0.14 per million donations for the UK blood supply during the period 1996 to 2003.15 Despite improvements in testing (HIV antigen/antibody assays and HIV NAT in some centers) the residual risk has risen to 0.22 per million because the improvements in tests have been overshadowed by the gradual doubling of new infections in the general population. These new infections have been associated with heterosexual spread of genotype C and other non-B genotypes, whereas MSM spread is associated with genotype B. A similar increase in non-B HIV genotypes has been found in other European countries and, to a lesser extent, Asia and the Americas.35-38 In the United States the proportion of non-B HIV-positive blood donors rose from 0.8% to 3.1% between 1993 and 2000.39 Worldwide it has been estimated that up to 13 million units are not tested for HIV, HBV, or HCV. The World Health Organization (WHO) has estimated that this would result in 160,000 TTI cases per year. More recently, the Global Database on Blood Safety stated that HIV testing is being conducted in all 152 surveyed countries, but poor quality test performance together with sporadic supply problems have contributed to HIV transmission.40

Clinical Course Although HIV can be inactivated by heat treatment of lyophilized products at 68ºC for 72 hours, this became apparent only after a considerable number of hemophilia patients had already been infected through HIV-contaminated Factor VIII products. Indeed, around 50% of those treated with the non-heat-treated product experienced HIV seroconversion.41 Initially it was hoped that those with anti-HIV were merely immunized rather than infected but studies proved that those with anti-HIV were also HIV-positive by polymerase chain reaction (PCR), indicating that they were indeed infected. This has been amply borne out by the clinical outcomes in such patients. Because HIV can withstand the storage temperatures of blood components, it is to be expected that recipients of blood would be infected. The Transfusion Safety Study (TSS) in the United States was unique in actively tracing and enrolling recipients of known HIV-positive units. In this study 111 (89.5%) of 124 recipients of HIV-infected components experienced seroconversion to anti-HIV positivity.42-44 It was noted that washed Red Blood Cell (RBC) units and RBC units stored longer than 26

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days had lower transmission rates than other components. This would also suggest that leukocyte reduction might have a beneficial effect in reducing infectivity.45 Following infection with HIV, there is usually an asymptomatic period of around 2 to 3 weeks that progresses to an acute flu-like (seroconversion) illness in 40% to 90% of patients. Fever, enlarged lymph nodes, sore throat, rash, joint and muscle pains, headache, diarrhea, and vomiting are some of the signs and symptoms that can be experienced for a period of up to 2 weeks or longer. Correct diagnosis at this stage is usually unlikely unless symptoms are protracted. The infection will then become clinically latent but with active viral replication. The virus can be transmitted via blood and genital secretions during the asymptomatic and clinically latent period. Persistent asymptomatic HIV infection lasts on average 10 years in the absence of treatment. Eventually there is a sharp decrease in the number of CD4 T lymphocytes, leading to immunosuppression and opportunistic infections that may prove fatal when not treated. When an HIV diagnosis has been made and treatment is undertaken, the many advances in HIV management can extend survival and delay the onset of AIDS. However, worldwide the majority of those infected live in developing countries where effective therapy is usually not available. The 112 recipients enrolled in the TSS study provided valuable data as to the progression of the disease in those individuals infected through transfusion. Seven years following their transfusion, 37 (33%) had developed AIDS.46 The transfusion recipients tended to have a faster rate of disease progression compared with both infected donors and infected hemophilia patients who were followed in parallel. This higher rate was mainly because the patients were older and with underlying clinical conditions.47 Overall this study led to suggestions that the route of infection, size of inoculum, and other viral co-infections do not significantly alter the course of the disease. However, the importance of the viral virulence gene, nef, was demonstrated in an Australian cohort of a blood donor and eight transfusion recipients infected with an HIV strain lacking a functioning nef gene. The cohort members had a much milder illness with longer disease-free survival in the absence of therapy.48 Whether transfusion of AIDS patients is beneficial is debatable.49,50 The results of the Viral Activation by Transfusion Study (VATS) showed no evidence that leukocyte reduction benefits this patient group.51

Treatment Antiviral treatment has evolved from monotherapy to cocktails of highly active antiretroviral therapy (HAART) that has achieved remarkable success in decreasing the mortality from AIDS and in prolonging useful active life in those with HIV infection. Three classes of antiretroviral drugs are available—nucleoside and nucleotide analogs (NRTIs); reverse transcriptase inhibitors (RTIs); and protease inhibitors (PIs). There is no cure for HIV or AIDS. It should be realized that where viral load is measured routinely, any increase may be associated with the development

Chapter 47: Retroviruses and Other Viruses

of drug-resistant strains, necessitating a drug substitution in the patient’s therapy.

1

Human T-Cell Lymphotropic Viruses Human T-cell lymphotropic virus (HTLV-I) was identified in 1978 when it was isolated from a Japanese patient. The virus was found to cause adult T-cell leukemia and lymphoma (ATL)52 and tropical spastic paraparesis (TSP), otherwise known as HTLV-I-associated myelopathy or HAM.53 The virus is now found globally but is endemic in Japan, the Caribbean, South America, and West and Central Africa, where infection rates are often above 1%. HTLV-II was originally identified in 1982 in a patient with hairy-cell leukemia. HTLV-I and HTLV-II show 65% homology. HTLV-II is found in American Indian populations and in some populations of intravenous drug users.

Incidence and Prevalence Among Blood Donors The overall incidence in first-time donors in the United States is around 40 per 100,000. In repeat donors a rate of approximately 1.6 per 100,000 person years was identified from 1991 to 1996. HTLV-II is found more often than HTLV-I among US first-time donors, reflecting the epidemic of HTLV-II among intravenous drug users. In the UK, routine screening of blood donations commenced in 2002 and has resulted in over 100 HTLV-positive donors being detected. However, in the UK there is a remarkable geographic variation in distribution, with southern England having an incidence as much as 10 times higher than northern regions.54,55 Donor Testing and Counseling Current HTLV donor screening assays are based solely on antibody detection by ELISA or chemiluminescent (ChLIA) format. The more sensitive assays have used recombinant HTLV proteins on both solid phase and conjugate in a sandwich assay format. Assays detect both HTLV-I and HTLV-II. In the UK a lengthy pilot study was performed using antiHTLV to test the residues of the pools of samples used for HCV NAT assays (pools of up to 95 donations).56 It was estimated that this procedure was around 95% sensitive compared with individual donation testing. However, adjustment of the cut-off to enhance the test sensitivity together with reduction in the minipool size to no more than 48 donations led to an HTLV screening strategy that was comparatively inexpensive, around 98% sensitive, and with remarkable specificity (the almost ideal situation). Furthermore, the knowledge that all UK blood components were subjected to leukocyte reduction meant that this strategy carried even less residual risk of viral transmission by transfusion. Confirmatory testing of reactive HTLV donations is more complex than confirmatory testing for HIV, partly because of the relatively poor Western blots that were commercially available. Modifications to those Western blots to include recombinant materials in addition to viral lysate and also the development of recombinant immunoblots (Fig 47-5) (similar to HCV immunoblot formats) have led to more accurate interpretations. Indeed, some of the immunoblots were capable of discriminating the

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Streptavidin 3⫹ 1⫹ ⫹/⫺ gap p 19 I/II gap p 24 I/II env gp46 I/II env gp21 I/II gag p 19-I env gp46-I env gp46-II

Figure 47-5. HTLV Immunoblot (Innolia). Strips 09 and 10 show confirmed HTLV-I specimens; strip 11 shows confirmed HTLV-II; strip 12 is a negative control; strip 13 is a positive control.

pooled donation samples (up to 95 donations) as either HTLV-I or HTLV-II positive. Nucleic acid testing as part of a confirmatory algorithm is still under development.57 Most UK HTLV-positive blood donors were shown to be of Afro-Caribbean ancestry or had been sexual partners of someone of Afro-Caribbean ethnicity. Therefore, it is important to ensure that sexual partners and other family members are also tested for HTLV in any clinical follow-up.

Window Period and Risk of Transmission A window period of 51 days has been calculated.58 A study of US transfusion recipients estimated the risk of HTLV infection at 12 per 100,000 units before screening and 1.4 per 100,000 following the implementation of blood donor HTLV screening. Over time, this risk has changed to 0.16 per 100,000 screened donations. Generally, transfusion-transmitted HTLV has been associated with (fresher) cellular components so that plasma transfusions are thought to be of little risk. Furthermore, the use of leukocyte reduction procedures has lessened the risk considerably such that in combination with HTLV donor screening, virtual elimination of HTLV transfusion transmission has been claimed.59 Clinical Course Although the majority of HTLV-positive donors exhibit no clinical symptoms of disease, they should be made aware that a small percentage (2% to 4%) may develop ATL up to 40 years after infection. HAM/TSP is characterized by spinal cord degeneration and has a

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Table 47-2. Human Herpesviruses Herpes Subfamily

New Nomenclature

Common Name

Alphaherpesvirinae Alphaherpesvirinae Alphaherpesvirinae Gammaherpesvirinae Betaherpesvirinae Betaherpesvirinae Betaherpesvirinae Gammaherpesvirinae

HHV-1 HHV-2 HHV-3 HHV-4 HHV-5 HHV-6 HHV-7 HHV-8

Herpes simplex virus-1 (HSV-1) Herpes simplex virus-2 (HSV-2) Varicella zoster virus (VZV) Epstein-Barr virus (EBV) Cytomegalovirus (CMV) HHV-6 HHV-7 Kaposi’s sarcoma-associated virus

much shorter incubation period, developing in a few (1.5% to 3%) patients infected with HTLV-I and HTLV-II. HTLV-I has also been associated with uveitis and may lead to eventual blindness.

Treatment The management of asymptomatic HTLV infection generally should not involve drug therapy because of the low chance of clinical disease development. Chemotherapy for the treatment of ATL is often less effective than for other forms of leukemia or lymphoma. Therefore, there is a high mortality associated with this disease. HAM/TSP can be treated with systemic steroids or azathioprine immunosuppression with variable success.

Other Retroviruses The finding of a high proportion of individuals with antibodies to the p24 core antigen on HIV Western blot is highly suggestive that these individuals may have been infected with other retroviruses. Testing sequential donations or samples from such individuals over many years has shown that their “indeterminate” banding patterns usually do not alter for a particular individual. In addition, PCR testing of such individuals has not identified any reactivity. Also, it is known that the human genome, like that of other species, harbors some ancient endogenous retroviral sequences. These observations have not resulted in any real proof of disease association. However, there is some concern over the possibility that the use of animal-derived blood constituents could harbor such retroviruses that may re-activate following transfusion. Indeed, the latest fad of “bush meat cuisine” may also result in cross-species transmission of retroviruses.60,61 Blood donor-recipient repositories in developed and developing countries will provide reassurance against such potential risks to blood safety.62

Herpesviruses Overview Herpesviruses are ubiquitous in nature, with practically every living creature known to be susceptible to some associated herpesvirus. Humans are known to have several herpesviruses (Table 47-2). All human herpesviruses (HHVs) have the

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capability to lie dormant in tissues after an acute infection. With reference to transfusion, cytomegalovirus (CMV or HHV-5) is by far the most important herpesvirus infection. Herpes simplex (HHV-1 and HHV-2) and herpes varicella-zoster (HHV-3) have restricted tissue involvement, but CMV can manifest itself in many organs. Therefore, CMV is a major problem in immunosuppressed patients requiring transfusion therapy. Patients who should receive components that are selected or processed to minimize the risk of CMV infectivity include the following: ● Transplant recipients ● Patients with severe immunodeficiency ● Fetus (intrauterine transfusion) ● CMV-negative pregnant females ● Low birthweight premature infants and neonates It is important to be able to distinguish between CMV infection, identified by means of serologic tests or viral isolation, and CMV disease identified by laboratory evidence of infection combined with specific symptoms associated with the virus. Epstein-Barr virus (EBV or HHV-4) is responsible for infectious mononucleosis, is associated with Burkitt lymphoma and nasopharyngeal cancer, and can also be transfusion transmitted. Human herpesvirus 6 (HHV-6) causes “roseola infantum” or “sixth disease”—a childhood rash. Primary infection of adults is rare. Human herpesvirus 8 (HHV-8) has been found in Kaposi’s sarcoma and other tumors such as Castleman disease and bodycavity-based lymphoma.63,64 It has also been associated with polyneuropathy, organomegaly, and endocrinopathy. As with CMV and EBV, HHV-8 appears to cause a persistent infection with the viral genome, lying latent in host immune cells.

Incidence and Prevalence Among Blood Donors Figure 47-6 shows the prevalence of CMV infection in a Scottish blood donor population with an apparent 2% CMV seroconversion rate per year. The prevalence figures for a similar population 20 years earlier showed figures that were considerably higher. Thus, it would appear that few donors had been infected in recent times (possibly because of improved hygiene and living conditions). Support for this hypothesis comes from a German study65 in which an overall CMV seroconversion rate of 0.55% per year was observed.

Chapter 47: Retroviruses and Other Viruses

80%

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CMV antibody positive (2004)

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CMV antibody positive (1984)

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Figure 47-6. Comparison of cytomegalovirus antibody status of West of Scotland blood donors according to age and samples 20 years apart.

Figure 47-7. Western blot for cytomegalovirus antibodies showing the various antibodies that are elicited.

Antigen bands

EBV and HHV-6 have a very high prevalence in the general population (⬎80%). HHV-8 has a seroprevalence in blood donors between 0 and 20%. African populations have been reported to have a prevalence of 35% to 75%, with 10% to 50% showing evidence of HHV-8 DNA. In the UK, HHV-8 has been reported more frequently in MSMs attending sexually transmitted disease clinics and this was a contributory factor in the decision to maintain permanent deferral of this population group as blood donors.66

Donor Testing Most transfusion testing sites use ELISA-based assays to test blood donations for the presence of anti-CMV. Unlike mandatory marker assays, anti-CMV assays are not as strictly controlled by regulatory agencies. Validation and concordance of assays is hampered by the number of antibodies that CMV can elicit.67 Most transfusion testing sites tend to use “total” antiCMV assays that can detect both IgG and IgM class antibodies. Hemagglutination assays are inherently capable of detecting both classes of antibodies and some commercial assays have now become available that can be performed on automated blood group analyzers. In contrast, diagnostic virology laboratories use both IgG and IgM antibody tests to differentiate acute from

IE1 p150 CM2

p65 gB1

gB2

recrudescent CMV infections. Although it is accepted that those donors with IgM reactivity are most likely to have CMV viremia, the capacity of CMV to lie dormant in white cells means that any antibody-reactive donation has the capability of infecting the recipient with CMV. Donors with reactivity in anti-CMV screening assays usually are flagged as “CMV positive” on donor databases so that they will not be retested and their blood will not be included in the preparation of CMV-negative components. Problems associated with specificity of some ELISA tests can lead to some donors being labeled falsely as CMV positive; transfusion services are hesitant to retest these donors because of operational constraints. Recently, a CMV Western blot assay (Fig 47-7) has become available, allowing a check to be made on samples found to show discrepant reactivity with different manufacturers’ assays. Because CMV is a common infection and not clinically significant in immunocompetent individuals, there is no need to counsel donors. No routine tests are performed for EBV, HHV-6, or HHV-8 infection. Indeed, tests for HHV-8 frequently show discrepant results.68

Window Period and Risk of Transmission Although CMV is transmissible by transfusion, this is now relatively uncommon in developed countries, particularly those where

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all blood components are leukocyte reduced. Despite the average 3 log10 reduction in leukocyte concentration achieved by leukocyte reduction,69 there has been no abandonment of CMV-seronegative blood bank inventories, particularly for the patient populations at high risk of CMV disease.70 It is thought that only 1% to 5% of blood units found to be CMV seropositive will actually lead to infection of CMV-seronegative recipients. The incubation period of primary CMV infection varies between 4 and 8 weeks. The residual risk of transfusion-transmitted CMV infection from anti-CMV screened blood components (not leukocyte reduced) is around 1.2% to 1.6% per donation, whereas the risk is between 2.3% and 3.0% for leukocyte-reduced blood components.71 The present use of CMV screened leukocyte-reduced blood would be expected to be associated with a risk of less than 1%. Before leukocyte reduction, EBV infection occurred between 3 and 7 weeks following transfusion in around 33% to 46% of recipients lacking EBV antibodies. Such recipients are a small minority, especially in older individuals. The transmissibility of HHV-8 by transfusion is still debatable. Studies in the United States provided evidence of seroconversion in two of 284 cardiac surgery patients from nonleukocyte-reduced blood between 1986 and 1990.72 A Ugandan study showed a 6% frequency of seroconversion with non-leukocyte-reduced blood compared with 3.5% among recipients of HHV-8 seronegative blood.73

Clinical Course In marrow recipients, CMV is one of the most significant infections, responsible for 15% of mortality before the advent of good antiviral drugs. CMV matching of kidney recipients has a beneficial effect on clinical outcome more so than matching patients for HLA Class I status. The use of seropositive blood for seropositive patients can lead to reinfection of some CMV seropositive recipients; therefore, CMV-seronegative components are preferred (but not always available) for all immunocompromised patients. The leukocyte reduction of blood components has lessened the demand on CMV-seronegative components. Transfusion-transmitted EBV infections usually are asymptomatic, but some appear as an infectious mononucleosis with mild fever, anorexia, and malaise. Most HHV-8 infection is benign with a low frequency of disease development.

Treatment Antiviral drugs that are effective against CMV (and also HHV-6) include ganciclovir, foscarnet, cidofovir, and valaciclovir. Drugs can be prescribed either for prophylaxis, suppression, or treatment.

Other Viruses Several viruses have had a considerable impact on blood services in certain parts of the world. These “other” viruses include parvovirus B19 (human erythrovirus), West Nile virus, chikungunya, and pandemic influenza.

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Parvovirus B19 Parvovirus B19 was so named because it was the 19th sample on a hepatitis B counter electrophoresis assessment panel that reacted; subsequently it was shown to contain parvovirus. Parvovirus is a single-stranded nonenveloped DNA virus of 5500 nucleotides that code for two capsid proteins (VP1 and VP2) and other proteins.74 Parvovirus is the agent that causes “fifth disease” or “slap-cheeked sydrome” in young children. However, parvovirus can also cause serious, even life-threatening, disease in certain immunosuppressed individuals and women in the second trimester of pregnancy, where hydrops fetalis may result. Individuals lacking the P blood group globoside are naturally immune to parvovirus because the virus requires the globoside for binding to the cell. Various cell types contain this surface antigen and are susceptible to infection. The clinical manifestations of parvovirus are, therefore, widespread, involving fetal myocardium, placenta, liver, kidney, thyroid, megalokaryocytes, and erythroid progenitor cells. Compared to some blood derivatives, blood components are only rarely associated with parvovirus B19 transmission. This may be because of a lack of recognition of an association of parvovirus infection with transfusion or, more likely, asymptomatic infection in the recipient. Nevertheless, recent studies suggest between 0.5% and 0.9% of individual blood donations are contaminated with parvovirus B19 by evidence of B19 DNA. It could be argued that other components containing antibodies may prevent infection in some recipients, but ongoing studies with an archive repository in the United States may reveal the true transmission rate. Various parvovirus serologic assays are now commercially available.75 The Netherlands have reacted to the risk of parvovirus infection in selected patient groups by providing a “parvovirussafe” panel of donors deemed to be safe from transmitting parvovirus by evidence of parvovirus antibodies for a period of at least 6 months. This panel was created by large-scale ELISA screening of the donor panel for parvovirus antibodies (against VP2) over a period of over a year. The demand for “parvovirus-safe” blood components has been limited. Recent studies76,77 raise doubt over the strategy, as they describe low-level parvovirus B19 carriage in individuals with cocirculating antibodies. In the past, parvovirus B19 transmissions have occurred through plasma-derived Factor VIII or IX blood derivatives. Although parvoviruses are generally stable and resist most physiochemical inactivation procedures, parvovirus B19 is more readily inactivated than the other parvoviruses using either dry or wet heat, extreme pH, ultraviolet C irradiation, photochemical reaction, or even nanofiltration procedures in blood derivative manufacture. Despite the incorporation of some of these inactivation procedures and the use of recombinant-derived clotting factor concentrates, reports of potential parvovirus B19 transmission with high-purity clotting factor concentrates, immunoglobulin preparations, and solvent/detergent-treated plasma still occurred. This led plasma fractionators worldwide to screen their source plasma for parvovirus DNA and use only pools with less than 10,000 copies/ mL. This level of reactivity was ascertained by considering the level

Chapter 47: Retroviruses and Other Viruses

of parvovirus antibodies that were also contained within these pools and its ability to neutralize small concentrations of the virus. A pilot study on parvovirus NAT testing was able to demonstrate some exceedingly high viral concentrations in maxipools of donations. The level of parvovirus mirrored the level of infection in the general population where epidemics appear every 3 to 4 years, usually in late spring.78 The prevalence of parvovirus is age related, with most infections occurring in young children, who often then infect their (non-immune) siblings, parents, or grandparents. By the age of 15 years, 60% of the population will possess antibodies to parvovirus.79 Most parvovirus assays detect three genotypes, Parvovirus 1, 2, and 3. Two new parvoviruses, Parvovirus 4 and Parvovirus 5, have been described with no known pathogenicity.80

West Nile Virus West Nile virus, a flavivirus, was first recognized in 1937 and now has a wide distribution in Africa, Europe, Asia, Australia, and, in the past decade, in Northern America. WNV mainly infects birds and is normally transmitted by mosquito. However, horses and also humans can be bitten by infected mosquitoes and subsequently can develop West Nile fever.81 In the United States, WNV was first identified in 1999 and over the next few years spread from the eastern seaboard to the Pacific states. Most human cases occur between July and November with a peak period in August and September, but viremic blood donors have been found throughout the year. Cases in the United States peaked in 2003 with 9862 cases and 264 deaths, but WNV is likely to continue to cause around 2000 cases and 100 deaths per year. The incubation period is between 3 and 15 days in humans with over 80% of infections being asymptomatic or with a mild flu-like illness. In rare cases (fewer than 1% of those infected), a more severe disease results and fatal encephalitis can occur. Asking donors about predonation headache and fever was found to have no effect on blood safety.82 The risk of transfusion transmission is associated with a period of viremia occurring around 3 days after infection and lasting around a week.32 Since July 2003, the US and Canadian blood supply has been screened using WNV NAT assays that were developed quickly by two major diagnostic companies, Roche and Chiron.83 Both assays can be used on minipools of six or 16 donations. However, following reported WNV transmissions from MP-NAT-negative donations, an algorithm was devised to switch to ID-NAT when there was demonstrable WNV activity in a region.84 This strategy seems sensible, particularly because 294 (22%) of 1332 viremic blood donors tested during the period 2003 to 2005 were detected solely by ID-NAT.85 Following an outbreak in horses in southern France, a survey of 2157 blood donors in that region showed that 0.8% had evidence of antibodies to WNV.86 Various outbreaks in Europe have not equalled the WNV epidemic in the United States. Perhaps the American WNV is more pathogenic, or alternatively, perhaps Europeans are less susceptible. European blood services have found that deferring donors from North America for a period of 28 days offers sufficient protection to their blood supply. Fresh frozen

plasma imported from the United States is currently pathogen inactivated (eg, with methylene blue) to combat any WNV risk.

Chikungunya Chikungunya virus (CHIKV) is an alphavirus of the Togaviridae family with a single-stranded RNA genome. The virus is transmitted by mosquitoes (Aedes albopictus) to humans, where after 1 to 10 days symptoms of chikungunya fever develop. Derived from the Swahili name for “that which bends up,” Chikungunya disease symptoms include arthralgia, myalgia, rash, headache, and fever. Three main genotypes exist associated with either West Africa, Central and East Africa, or Asia. More recently, there has been an epidemic associated with the Central and East African genotype that was first reported in 2005 in French Indian Ocean islands, but subsequently spread to cover most Indian Ocean islands and the Indian subcontinent. It has been estimated that at the height of the epidemic over 250,000 (35% of the population) had been infected on Reunion Island, with 250 deaths. French blood service authorities originally managed the problem by importing blood to the French territories as a short-term measure, with the implementation of chikungunya NAT testing and pathogen inactivation as longer-term measures.14 Blood services outside the affected areas were able to defer (for a period of 28 days) those donors who had visited areas where chikungunya was known to be epidemic. During 2007, European blood services realized that foreign travel can aid the spread of chikungunya not only as isolated cases in returning visitors but also as an outbreak (eg, chikungunya fever in an Italian province). The latter event occurred after a visit to the region from a foreigner incubating the disease, together with the spread of the disease by the Aedes albopictus mosquito within that region. This mosquito species is also capable of transmitting dengue virus and is found elsewhere in Italy, France, and Albania. This highlights the need for continued vigilance of emerging infections elsewhere in the world, particularly because world travel can quickly transfer disease from one area to another. In addition, the effects of global warming allow insect vectors to survive in what once were less temperate regions.

Pandemic Influenza Pandemic influenza has been a continuing threat arising shortly after the severe acute respiratory syndrome (SARS) outbreak. The lethal influenza viruses that have infected humans have been adequately managed so far by existing public health measures.87 This has been helped by the relative inability of “bird flu” viruses to infect humans. However, concern exists that eventually a mutation or a mixed infection will occur that will allow one of these lethal viruses to spread among humans. Because there have been a number of bird flu cases in humans involving the influenza subtype H5N1, it is felt that any new emerging influenza threat is likely to be closely related. Vaccines have been prepared, but as with normal human influenza vaccines, they have good efficacy in immunocompetent individuals but are of limited use among those who are at greatest risk from such a pandemic (ie, the old and the very young). It is believed that transfusion-transmitted influenza is relatively rare,88 but theoretically

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possible. Thus, the deferral of donors from areas at the height of an epidemic may be the safest option, along with the backfilling of blood stocks from areas with few or no cases. At the height of any epidemic it is likely that the use of blood will be severely curtailed— partly because of cancellation of elective surgery, but also because of a lack of health-care personnel and, indeed, blood donors.

Lymphocytic Choriomeningitis Virus Lymphocytic choriomeningitis virus (LCMV) was first described in 1931 as one of the main causes of meningitis. Eight solid-organ transplant recipients have been infected with LCMV from two organ donors,89 raising the concern that LCMV may also be transfusiontransmitted despite a lack of reported cases in the literature. A French study90 ascertained a low (0.33%) prevalence in the blood donor population, suggesting that the risk of transmission is low. However, the researchers did suggest that history of recent rodent contact should lead to a temporary deferral of potential donors.

Simian Foamy Virus Strategies for limiting the spread of simian foamy virus are similar to those used to manage LCMV. Donors who have occupational exposure to primates have been deferred in Canada as a measure to prevent potential transmission.

Emerging Nonpathogenic Viruses The use of new powerful molecular genetic techniques to identify previously undescribed viruses has led to a plethora of “new” viruses that have yet to be shown to have any human pathogenicity.91 Two such viruses have instigated much research and literature in the past 20 years—GB virus-C (GBV-C, once known as hepatitis G virus) and TT virus (TTV).

GBV-C GBV-C is an enveloped RNA virus and a member of the Flaviviridae family (which also includes HCV). The viral genome is structured similarly to HCV but has only 25% homology with it. Several genotypes exist, mainly confined to various continents. Transfusion transmission of GBV-C has been demonstrated. The prevalence of GBV-C RNA in blood donors ranges from 1% to 5% dependent on geographic location. Antibody as measured by anti-E2 is usually found three to four times more frequently than is viral RNA.64 GBV-C, and independently “hepatitis G virus (HGV),” was discovered in patients with sporadic “non-A, non-B” hepatitis, inferring that it was a causal agent of hepatitis. However, subsequent investigations have concluded that GBV-C was not the cause of viral hepatitis, nor had any hepatotropic or other pathologic role. It was merely a “passenger” virus, isolated by chance. Blood screening is therefore not necessary.

TT Virus As with GBV-C, TT virus was discovered during the search for the elusive cause of the minor (non-C) component of “non-A through

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E” posttransfusion hepatitis. The virus was discovered in a posttransfusion hepatitis patient with the initials TT. TTV is a small (18-50 nm) DNA nonenveloped virus. TTV can be transmitted by transfusion and has a TTV RNA prevalence of between 5% and 80%; there is again a marked geographic variation in infection rates with higher prevalence in Africa (up to 80%).64 It is thought that 50% of African children will have been infected by TTV by 1 year of age. More sensitive NAT assays detect higher rates of viral infection. The relevance of TTV in transfusion is limited because of the lack of any clinical symptoms. However, the fact that some current fractionated plasma product inactivation procedures are unable to prevent infection of recipients is of some concern.

Conclusion The pathogenicity of any virus is extremely important when considering the value of introducing specific blood safety interventions. Another issue is the difficulties of testing and excluding infected donations when the prevalence of an agent in the donor population is high. Nevertheless, there are pragmatic actions that can be taken to reduce the likelihood of “at-risk” recipients being given infected blood. The blood supply will never be deemed to have “zero risk,” but there is an expectation by the public for blood services to provide blood that is as safe as possible. This will include steps such as excluding “high-risk” donors temporarily or permanently, using serologic or NAT-based assays to identify donors infected with pathogenic blood-borne agents, or using pathogen inactivation/reduction techniques on particular blood components to ensure that there are considerable reductions in microbial infectivity (see Chapter 51). The latter intervention is likely to be a potential basis for blood safety strategies to avert the emerging diseases of the future. Effective microbial inactivation of the full inventory of blood components would be one prerequisite of such a strategy. The case to discontinue the use of any of the present arsenal of tests/procedures will be unlikely to succeed unless the inactivation procedure is extremely reliable and robust. Commercial companies promoting pathogen inactivation systems suggest that the cost savings realized with discontinuing CMV and bacterial testing will more than adequately compensate for the costs of introducing their systems, claimed to be effective against nearly all transfusion-transmissible agents.

Disclaimer The authors have disclosed no conflicts of interest.

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origin and evolution of the human immunodeficiency virus (HIV) and significance of HIV-1 genetic subtypes. Philos Trans R Soc Lond B Biol Sci 2001;356:867-76. McCutchan FE. Global epidemiology of HIV. J Med Virol 2006;78(Suppl 1): S7-12. Janssens W, Buve A, Nkengasong JN. The puzzle of HIV-1 subtypes in Africa. AIDS 1997;11:705-12. Vidal N, Peeters M, Mulanga-Kabeya C, et al. Unprecedented degree of human immunodeficiency virus type 1 (HIV-1) group M genetic diversity in the Democratic Republic of Congo suggests that the HIV-1 pandemic originated in central Africa. J Virol 2000;74:10498-507. Weiss RA, Dagleish AG, Loveday, Human immunodeficiency viruses. In: Zuckerman AJ, Banatvala JE, Pattison, eds. Principles and practice of clinical virology, 4th ed. Chichester, UK: Wiley and Sons, 2000:659-94. Lee CA. The natural history of HIV disease in haemophilia. Blood Rev 1998;12:135-44. Schochetman G, Kuhns MC. HIV variants and hepatitis B surface antigen mutants: Diagnostic challenges for immunoassays. J Med Virol 2006;78(Suppl 1):S3-6. Fiebig EW, Murphy EL, Busch MP. Human immunodeficiency virus, human T-cell lymphotrophic viruses, and other retroviruses. In: Hillyer CD, Silberstein LE, Ness PM, et al, eds. Blood banking and transfusion medicine: Basic principles and practice. Philadelphia, PA: Churchill Livingstone, 2003:447-64. Hammer SM. Management of newly diagnosed HIV infection. N Engl J Med 2005;353:1702-10. Sepkowitz KA. One disease, two epidemics—AIDS at 25. N Engl J Med 2006;354:2411-14. Merson MH. The HIV-AIDS pandemic at 25—the global response. N Engl J Med 2006;354: 2414-17. Geleziunas R, Greene WC, Molecular insights into HIV-1 infection and pathogenesis. In: Sande MA, Volberding PA, eds. The medical management of AIDS, 6th ed. Philadelphia, PA: WB Saunders, 1999:23-40. Laperche S, Pillonel J. Influence of epidemiological factors on blood transfusion. ISBT Science Series 2007;2:78-84. Soldan K, Davison K, Dow B. Estimates of the frequency of HBV, HCV, and HIV infectious donations entering the blood supply in the United Kingdom, 1996 to 2003. Eurosurveillance 2005;10:17-19. Glynn SA, Kleinman SH, Schreiber GB, et al. Trends in incidence and prevalence of major transfusion-transmissible viral infections in US blood donors, 1991 to 1996. Retrovirus Epidemiological Donor Study (REDS). JAMA 2000;284:229-35. Dow BC. Microbiology confirmatory tests for blood donors. Blood Rev 1999;13:91-104. Barbara J, Ramskill S, Perry K, et al. The National Blood Service (England) approach to evaluation of test kits for detecting infection. Transfus Med Rev 2007;21:147-58. Ferguson M, Holmes H, Sands D. NIBSC/UK Blood Transfusion Service working standards for HBsAg, anti-HCV and anti-HIV 1 (‘go/no-go’ controls). Vox Sang 2001;80:205-10. Nightingale MJ, Ramskill S, Newham J, et al. The systematic monitoring of transfusion microbiology test kit performance. Transfus Med 2007;17:404-12. Stramer SL. Current risks of transfusion-transmitted agents. A review. Arch Pathol Lab Med 2007;131:702-7.

22. Ferreira MC, Nel TJ. Differential transmission of human immunodeficiency virus (HIV) via blood components from an HIV-infected donor. Transfusion 2006;46:156-7. 23. Mitchell R, Dow BC, Barr A, et al. False positive anti-HIV tests on blood donations. Lancet 1988;332:297-8. 24. Jackson JB, Hanson MR, Johnson GM, et al. Long-term follow-up of blood donors with indeterminate human immunodeficiency virus type 1 results on Western blot. Transfusion 1995;35:98-102. 25. Sayre KR, Dodd RY, Tegtmeier G, etal. False-positive human immunodeficiency virus type 1 Western-blot tests in noninfected blood donors. Transfusion 1996;36:45-52. 26. Janssen RS, Aberle-Grasse J, Rawal BD, et al. New testing strategy to detect early HIV-1 infection for use in incidence estimates and for clinical and prevention purposes. JAMA 1998; 280:42-8. 27. Busch MP, Aberle-Grasse J, Rawal BD, et al. Demographic correlates of HIV incidence among first-time blood donors (abstract). Transfusion 1998;38(Suppl):81S. 28. Adler MW. ABC of AIDS: Development of the epidemic. Br Med J 2001;322:1226-9. 29. Busch MP, Operskalski EA, Mosley JW, et al for the Transfusion Safety Study Group. Factors influencing HIV-1 transmission by blood transfusion. J Infect Dis 1996;174:26-33. 30. Crawford RJ, Mitchell R, Burnett AK, et al. Who may give blood? Br Med J (Clin Res Ed) 1987;294:572. 31. Busch MP, Stramer SL. The efficiency of HIV p24 antigen screening of US blood donors: Projections versus reality. Infusionstherapie Transfusionsmedizin 1998:25:194-7. 32. Busch MP. Transfusion-transmitted viral infections: Building bridges to transfusion medicine to reduce risks and understand epidemiology and pathogenesis. Transfusion 2006;46:1624-40. 33. Fiebig EW, Wright DJ, Rawal BD, et al. Dynamics of HIV viraemia and antibody seroconversion in plasma donors: implications for diagnosis and staging of primary HIV infection. AIDS 2003;17:1871-9. 34. Delwart EL, Kalmin ND, Jones TS, et al. First case of HIV transmission by an RNA-screened blood donation. Vox Sang 2004;86:171-7. 35. Simon F, Loussert-Ajaka I, Damond F, et al. HIV type 1 diversity in northern Paris, France. AIDS Res Hum Retrovir 1996;12:1427-33. 36. Parry JV, Murphy G, Barlow KL, et al. National surveillance of HIV1 subtypes for England and Wales: Design, methods, and initial findings. J Acquir Immune Defic Syndr 2001;26:381-8. 37. Fleury H, Recordon-Pinson P, Caumont A, et al. HIV type 1 diversity in France, 1999-2001: Molecular characterization of non-B HIV type 1 subtypes and potential impact on susceptibility to antiretroviral drugs. AIDS Res Hum Retrovir 2003;19:41-7. 38. Delwart EL, Orton S, Parekh B, et al. Two per cent of HIV-positive U.S. blood donors are infected with non-subtype B strains. AIDS Res Hum Retrovir 2003;19:1065-70. 39. Delwart E, Kuhns MC, Busch MP. Surveillance of the genetic variation in incident HIV, HCV, and HBV infections in blood and plasma donors: Implications for blood safety, diagnostics, treatment, and molecular epidemiology. J Med Virol 2006;78(Suppl 1):S30-35. 40. Wendel S. Rational testing for transmissible diseases. ISBT Science Series 2007;2:19-24. 41. Busch MP, Operskalski EA, Mosley JW, et al for the Transfusion Safety Study Group. Epidemiological background and long-term course of disease in human immunodeficiency virus type 1-infected blood donors identified before routine laboratory screening. Transfusion 1994;34:858-64.

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42. Kleinman SH, Niland JC, Azen SP, et al. Prevalence of antibodies to human immunodeficiency virus type 1 among blood donors prior to screening. The Transfusion Safety Study/NHLBI Donor Repository. Transfusion 1989;29:572-80. 43. Donegan E, Stuart M, Niland JC, et al. Infection with human immunodeficiency virus type 1 (HIV-1) among recipients of antibodypositive blood donations. Ann Intern Med 1990;113:733-9. 44. Busch MP, Donegan E, Stuart M, et al. Donor HIV-1 p24 antigenaemia and course of infection in recipients. Transfusion Safety Study Group (letter). Lancet 1990;335:1342. 45. Rawal BD, Busch MP, Endow R, et al. Reduction of human immunodeficiency virus-infected cells from donor blood by leukocyte filtration. Transfusion 1989;29:460-2. 46. Operskalski EA, Stram DO, Lee H, et al. Human immunodeficiency virus type 1 infection: Relationship of risk group and age to rate of progression to AIDS. Transfusion Safety Study Group. J Infect Dis 1995;172:648-55. 47. Blaxhult A, Granath F, Lidman K, et al. The influence of age on the latency period to AIDS in people infected by HIV through blood transfusion. AIDS 1990;4:125-9. 48. Learmont JC, Geczy AF, Mills J, et al. Immunologic and virologic status after 14 to 18 years of infection with an attenuated strain of HIV-1. A report from the Sydney Blood Bank Cohort. N Engl J Med 1999;340:1715-22. 49. Busch MP, Collier A, Gernsheimer T, et al. The Viral Activation Transfusion Study (VATS): Rationale, objectives, and design overview. Transfusion 1996;36:854-9. 50. Busch MP, Lee TH, Heitman J. Allogeneic leukocytes but not therapeutic blood elements induce reactivation and dissemination of latent human immunodeficiency virus type 1 infection: Implications for transfusion support of infected patients. Blood 1992;80:2128-35. 51. Collier A, Kalish L, Busch M, et al. Double-blind, randomized study of leukocyte-reduced red blood cell transfusions in patients with anemia and human immunodeficiency virus infection: Results of the Viral Activation Transfusion Study. JAMA 2001;285:1592-601. 52. Poiesz BJ, Ruscetti FW, Gazdar AF, et al. Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc Natl Acad Sci U S A 1980;77:7415-19. 53. Blattner W. Human retrovirology: HTLV. New York: Raven Press, 1990. 54. Brennan M, Runganga J, Barbara JAJ, et al. Prevalence of antibodies to human T cell leukaemia/lymphoma virus in blood donations in north London. Br Med J 1993;307:1235-9. 55. Flanagan P, McAlpine L, Ramskill SJ, et al. Evaluation of a combined HIV 1/2 and HTLV-I/II assay for screening blood donors. Vox Sang 1995;68:220-4. 56. Dow BC, Munro H, Ferguson K, et al. HTLV antibody screening using mini-pools. Transfus Med 2001;11:419-22. 57. Davidson F, Lycett C, Jarvis LM, et al. Detection of HTLV-I and -II in Scottish blood donor samples and archive donations. Vox Sang 2006;91:231-6. 58. Schreiber GB, Busch MP, Kleinman SH, et al. The risk of transfusion-transmitted viral infections. The Retrovirus Epidemiology Donor Study. N Engl J Med 1996;334:1685-90. 59. O’Brien SF, Yi Q-L, Fan W, et al. Current incidence and estimated residual risk of transfusion-transmitted infections in donations made to Canadian Blood Services. Transfusion 2007;47:316-5.

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60. Peeters M, Courgnaud V, Abela B, et al. Risk to human health from a plethora of simian immunodeficiency viruses in primate bushmeat. Emerg Infect Dis 2002;8:451-7. 61. Wolfe ND, Switzer WM, Carr JK, et al. Naturally acquired simian retrovirus infections in central African hunters. Lancet 2004;363:932-7. 62. Franklin IM, Dow BC, Jordan AD. Benefits of a blood donation archive repository: International survey of donor repository procedures and Scottish experiences. Transfusion 2007;47:1172-9. 63. Chang Y, Cesraman E, Pessin MS, et al. Identification of herpesviruslike DNA sequences in AIDS-associated Kaposi’s sarcoma. Science 1994;266:1865-9. 64. Allain JP. Emerging viral infections relevant to transfusion medicine. Blood Rev 2000;14:173-81. 65. Hecker M. Qiu D, Marquardt K, et al. Continuous cytomegalovirus seroconversion in a large group of healthy donors. Vox Sang 2004;86:41-4. 66. Franklin IM. Is there a right to donate blood? Patient rights; donor responsibilities. Transfus Med 2007;17:161-8. 67. Mach M, Stamminger R, Jahn G. Human cytomegalovirus: Recent aspects from molecular biology. J Gen Virol 1989;70:3117-46. 68. Pellett PE, Wright DJ, Engels EA, et al. Multicenter comparison of serological assays and estimations of human herpesvirus 8 seroprevalence among US blood donors. Transfusion 2003;43:1260-8. 69. Preiksaitis JK. The cytomegalovirus-“safe” blood product: Is leukoreduction equivalent to antibody screening? Transfus Med Rev 2000;14:112-36. 70. Blajchman MA, Goldman M, Freedman JJ, et al. Proceedings of a consensus conference: prevention of post-transfusion CMV in the era of universal leucoreduction. Transfus Med Rev 2001;15:1-20. 71. Vamvakas E. Is white-blood-cell reduction equivalent to antibody screening in preventing transmission of cytomegalovirus by transfusion: A review of the literature and meta-analysis. Transfus Med Rev 2005;19:181-99. 72. Dollard SC, Nelson KE, Ness PM, et al. Possible transmission of human herpesvirus-8 by blood transfsuion in a historical United States cohort, Transfusion 2005;45:500-3. 73. Hladik W, Dollard SC, Mermin J, et al. Transmission of human herpesvirus 8 by blood transfusion. N Engl J Med 2006;355:1331-8. 74. Prowse C, Ludlam A, Yap P. Human parvovirus B19 and blood products. Vox Sang 1997;72:1-10. 75. Manaresi E, Gallinella S, Venturoli S, et al. Detection of parvovirus B19 IgG: Choice of antigens and serological tests. J Clin Virol 2004;29:51-3. 76. Candotti D, Etiz N, Parysan A, et al. Identification and characterization of persistent human erythrovirus infection in blood donor samples. J Virology 2004;78:12169-78. 77. Lefrere JJ, Servant-Delmas A, Candotti D, et al. Persistent B19 infection in immunocompetent individuals: Implications for transfusion safety. Blood 2005;106:2890-5. 78. Heegard ED, Brown KE. Human parvovirus B19. Clin Microbiol Rev 2002;15:485-505. 79. Prowse C, Dow B, Pelly SJ, et al. Human parvovirus B19 infection in persons with haemophilia (letter). Thromb Haemost 1998;80:351. 80. Fryer JF, Delwart E, Hecht FM, et al. Frequent detection of the parvoviruses, PARV4 and PARV5, in plasma from blood donors and symptomatic individuals. Transfusion 2007;47:1054-61. 81. Prowse CV. An ABC of West Nile virus. Transfus Med 2003;13:1-7.

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82. Orton SL, Stramer SL, Dodd RY. Self-reported symptoms associated with West Nile virus infection in RNA-positive blood donors. Transfusion 2006;46:272-7. 83. Vamvakas EC, Kleinman S, Hume H, et al. The development of West Nile virus safety policies by Canadian Blood services: Guiding principles and a comparison between Canada and the United States. Transfus Med Rev 2006;20:97-109. 84. Montgomery SP, Brown JA, Kuehnert M, et al. Transfusion-associated transmission of West Nile virus, United States 2003-2005. Transfusion 2006:46:2038-46. 85. Stramer SL, Custer B, Busch MP, et al. Strategies for testing blood donors for West Nile virus. Transfusion 2006;46:2036-7. 86. Charrel RN, de Lamballerie X, Durand JP, et al. Prevalence of antibody against West Nile virus in volunteer blood donors living in southeastern France. Transfusion 2001;41:1320-1.

87. Writing Committee of the Second World Health Organization Consultation on Clinical Aspects of Human Infection with Avian Influenza A (H5N1) Virus. Update on avian influenza (H5N1) virus infection in humans. N Engl J Med 2008;358:261-73. 88. Likos AM, Kelvin DJ, Cameron CM, et al. Influenza viremia and the potential for blood-borne transmission. Transfusion 2007;47:1080-8. 89. Fischer SA, Graham MB, Kuehnert MJ, et al. Transmission of lymphocytic choriomeningitis virus by organ transplantation. N Engl J Med 2006;354:2235-49. 90. de Lamballerie X, Fulhorst CF, Charrel RN. Prevalence of antibodies to lymphocytic choriomeningitis virus in blood donors in southeastern France. Transfusion 2007;47:172-3. 91. Simmonds P. Transfusion virology: Progress and challenges. Blood Rev 1998;12:171-7.

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48

Transfusion Transmission of Parasites Bryan R. Spencer Manager of Blood Research, American Red Cross—New England Region, Dedham, Massachusetts, USA

The risk that a blood recipient in the United States will receive a component contaminated with parasites is, in absolute terms, small but not negligible. Estimates covering nearly 20 years are that roughly one per million transfusions might transmit parasitic infection.1,2 Given the large increase in international travel by US residents, the movement of large numbers of military personnel, and growing immigration, the opportunity for human trafficking of parasites that are endemic in tropical countries has never been greater. With the further development of molecular tools and improved diagnostics, these risks are being clarified. For some parasites, including Babesia and Leishmania species, the risk has probably been underappreciated given that transfusion transmission can easily go undetected, especially when the recipients are immunocompetent patients. The residual risk for some protozoan parasites appears to be on par with, or even greater than, transfusion-transmitted viral infections for which nucleic acid screening is performed. This chapter discusses the parasitic agents most likely to be found in blood donors, the risks from each, and the strategies for eliminating or diminishing these risks.

Chagas’ Disease Concern for transfusion transmission of Trypanosoma cruzi infection has increased in recent years, concurrent with the growing Hispanic population in the United States. Endemic in Central and South America and in Mexico, this protozoan hemoflagellate is transmitted in nature by triatomines, or reduviid bugs (also called “kissing bugs” for their tendency to bite on the face). Trypanosomes are not injected through the bite of the insect vector; rather, the insect vector deposits the infective metacyclic forms of the trypanosomes in its excreta during or soon after the blood meal. The sleeping host, in scratching the wound, can rub the Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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parasite into the wound or transfer it to mucous membranes in the eye or mouth. Triatomids competent for transmitting T. cruzi are widespread, including in the United States, where rare autochthonous transmission has been documented.3 The agent can also be transmitted readily by blood transfusion and solid-organ transplantation, and by congenital transmission.4 Rare outbreaks from contaminated food or drinks have also been reported.5 The typical clinical course following infection is an acute phase lasting 4 to 6 weeks, characterized by mild symptoms including fever, malaise, and edema of the face, as well as lymphadenopathy and hepatosplenomegaly.6 The fatality rate in the acute phase is usually less than 5%, and many cases go unrecognized. During this period, parasites are readily detectable in peripheral blood.4 In the ensuing indeterminate phase, however, carriers remain asymptomatic, and parasitemia tends to be low grade and intermittent.7 Up to decades later, 15% to 30% of carriers will develop serious sequelae involving the heart and gastrointestinal tract (mega-colon and mega-esophagus). Chagas’ disease kills an estimated 13,000 people annually, mostly from cardiac complications.4,8 Acute-stage treatment with benznidazole or nifurtimox is effective, but treatment during the chronic phase has only recently been shown to be effective. A 60-day course of benznidazole treatment eliminated infection in nearly two-thirds of chronically infected children,9 while a study in Argentina suggested that chronic-phase treatment with benznidazole might slow or prevent progression to cardiac disease.10 Despite considerable success in reducing insect transmission in the last two decades, Chagas’ disease remains a major public health problem in many countries, with an estimated 9 million infected and 25 million at risk for infection.11 The Southern Cone Initiative, inaugurated in six South American countries in 1991, sought to eliminate domestic infection by Triatoma infestans (the primary vector in several countries) and to interrupt transfusion transmission through universal donor screening.7 Estimated annual incidence of cases has declined by 90%, and Chile, Brazil, and Uruguay are all certified as having interrupted vector transmission.12 The success of this initiative inspired similar efforts in four Andean countries,13 Central America,14 and, the Amazon

Chapter 48: Transfusion Transmission of Parasites

region.15 Different species of vectors and different ecologic conditions apply across these regions, so no universal strategy can be applied uniformly. The adoption of universal blood screening in Latin American countries has dramatically altered the risk for transfusion transmission in countries where T. cruzi is endemic. Whereas in the early to mid-1990s only four of 17 Latin American countries in which data were compiled had achieved universal donor screening, 10 of 17 were at or above 99% coverage by 2002.16 During this period, estimates of cases of transfusion-transmitted T. cruzi in those countries dropped by nearly 75%, from 1996 to 536.16 Mexico alone accounts for two-thirds of recent estimated cases because of its large number of donors and relatively low screening coverage (27%), despite a moderately low seroprevalence in donors. A recent evaluation in Mexico demonstrated four cases of transfusion transmission and estimated that up to 1800 such cases occur in Mexico annually, on the basis of state-specific prevalence rates and donation data.17 Clearly, the risk in the United States and other countries receiving Latin American immigrants depends on the immigrants’ risk for acquisition of infection extending decades into the past. US census figures estimate that 41 million Hispanics live in the United States, of whom two-thirds are of Mexican origin,18 2.9 million are of Central American origin, and 2.2 million are of South American origin. Of these, an estimated 40%, 68%, and 70%, respectively, were foreign-born. Among the foreign-born, 40% or more arrived in the United States before 1990,18 and the subsequent intensification of control programs in T. cruzi-endemic countries. This suggests that a very large number of Hispanic immigrants might have been exposed long before entering the United States. Recent estimates project that between 70,000 and 100,000 Hispanic immigrants in the United States may be infected with T. cruzi.19 Canada and Australia are estimated to have more than 1000 T. cruzi-infected immigrants each, and Spain is estimated to have more than 10,000 undetected infections.20 Within the United States, 50% of Hispanic residents live in California and Texas, and a total greater than 25 million live in those two states plus Florida and New York. In all, about 80% of Hispanics in the United States live in nine states, but risk for transfusion transmission of T. cruzi can be found countrywide.18 There have been seven reported cases of T. cruzi transfusion transmission in the United States and Canada,21 and five cases of transmission associated with solid-organ transplantation.22,23 The actual number of transfusion cases is undoubtedly higher, because all seven cases have been detected in immunocompromised patients.21 Although platelet products have been implicated in six of these seven events, the study in Mexico17 revealed that two of four recipients who became T. cruzi-positive following transfusion had received whole blood from radioimmune precipitation assay (RIPA)-positive donors. An in-vitro study confirmed survival of T. cruzi up to 3 weeks in Whole Blood and Red Blood Cell (RBC) units,24 underlining that the risk is not limited to platelet products. The largest lookback study performed in the United States found zero positive

recipients of 18 tested; only three of the components were platelets.25 It remains unclear whether the apparently higher risk from platelets results from greater parasite survival and infectivity in this component or from the greater likelihood that recipients of platelets might be immunocompromised. Overall, data are lacking on infectivity estimates for different components, but it is estimated that roughly 20% of contaminated Whole Blood units will result in transfusion transmission.7,26 Preliminary look-back efforts following introduction of a screening test in the United States suggest infectivity as low as 10% for RBC units.27 There is no definitive estimate of the nationwide seroprevalence of T. cruzi antibodies in the United States, but a study of donors in Miami and Los Angeles indicated roughly 1 per 104 donations were from a seropositive donor, and in Los Angeles this risk appeared to be increasing over time.25 More recently, nearly 150,000 donors were screened as part of the licensure of a new commercial enzyme-linked immunosorbent assay (ELISA) for T. cruzi screening of blood donors. Conducted in three sites across California and Arizona, the repeat-reactive rates ranged from roughly 1:2000 in Los Angeles to 1:6000 in Tucson with half of all repeat-reactive donations testing positive by RIPA, a confirmatory assay, which at that time was not licensed by the Food and Drug Administration (FDA).28 Following FDA licensure of the screening test in December 2006,29 blood collection centers representing two-thirds of the US collections reported at least 582 RIPA-confirmed donors through August 8, 2008. Post-licensure results from two large blood centers covering a broad geographic area suggest an aggregate donor prevalence around 1:21,100.31 Figure 48-1 reflects that although the states of California and Florida account for the great majority of RIPApositive donors, the risk for transfusion transmission of T. cruzi is evident countrywide. Follow-up studies will help clarify the relationship between serologic status and parasitemia in donors, and the infectivity of components. Regulatory guidance is pending regarding donor screening for Chagas’ disease, but the FDA has in the past publicly declared its support for universal donor testing given the availability of appropriate screening tests.32 Meanwhile, AABB has issued recommendations for product management, recipient testing, and donor referral for medical evaluation for those blood centers choosing to implement Chagas’ screening in the absence of FDA requirements.32 Aside from testing, options for risk reduction include donor exclusion, pathogen removal, and pathogen inactivation. Canada has for years addressed risk by excluding donors on the basis of residence and travel history in areas where the disease is endemic, but intends to implement the newly licensed ELISA.33 In any case, deferral of donors on this basis is likely to be feasible only where such donors constitute a small portion of the donor pool. Leukocyte reduction filters have been shown to lower the concentration of trypanosomes in blood,34 but at least one case of transfusion transmission in platelets was from a leukocyte-reduced and irradiated product.21 A variety of photochemical treatment methods in platelets,35 plasma,36 and red cell components37 all show promise in reducing the parasite load by several logs.

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Tested donations Confirmed (582) RIPA positive donations ⬎⫽115 to ⬍197 ⬎⫽29 to ⬍115 ⬎⫽8 to ⬍29 ⬎⫽3 to ⬍8 ⬎⫽1 to ⬍3

Malaria Human malaria is caused by one of four species of protozoan parasites: Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. Malaria is a significant cause of global morbidity and mortality, responsible for an estimated 350 to 500 million clinical cases annually and over one million deaths; the disease is especially severe among young children in sub-Saharan Africa.38 Natural infection with malaria typically occurs through the bite of an infective female anopheline mosquito. Less frequently, parasites might be transmitted through blood transfusion, organ transplantation, parenteral exposure, or mother-to-child transmission.39,40 The United States recorded an average of three cases of transfusion-associated malaria per year between 1963 and 1989.41 An average of about one case per year—less than 0.1 per million donations—was the rate of occurrence from 1990 to 2005.42 This compares favorably to the historical estimate of zero to two cases per million donations in nonendemic areas, as contrasted with an incidence of 50 cases per million donations in areas where malaria is endemic.43 In malaria-endemic settings, where frequent boosting by infective mosquito bites help induce and maintain protective immune responses, severe infection is rare for those who survive beyond early childhood.44 In a nonimmune population, such as that of the United States, clinical malaria presents as a febrile illness with paroxysms, possibly at regular intervals, with accompanying flu-like symptoms. Complications can include severe anemia, hepatic involvement, cerebral alterations, renal failure, and shock.40 The case-fatality rate for malaria is generally

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Figure 48-1. Donors with confirmed T. cruzi infection are concentrated in states with high numbers of Hispanic residents, but the risk for transfusion transmission exists countrywide. Data through August 8, 2008.30

low—less than 1% in developed countries,45,46 but can surpass 10% in recipients of blood components.41 Once endemic throughout large parts of the country, malaria in the United States is now almost exclusively caused by imported infections.47 In a typical year, 1200 to 1500 malaria infections are diagnosed in the United States, more than 95% of which are found in US civilians who have visited malariaendemic areas and in foreign civilians from malaria-endemic countries. A comprehensive review of transfusion-transmitted malaria in the United States indicates that many of these infections were preventable, with ⬎60% having occurred because of deviations from the prevailing deferral guidelines.41 Of the cases in which donor exclusion guidelines were followed, P. malariae is 15 times more prevalent than its share of imported malaria overall in US travelers: 65% to 4%, respectively. This fact reflects the ability of P. malariae to remain at subpatent levels for lengthy periods, even up to 40 years.48 Indeed, because all malaria parasites remain viable in blood stored at 4ºC for at least 1 week, there exists risk from all four species.39 P. falciparum malaria has been transmitted in blood stored for 19 days, as well as in units of platelets.49 Interval to onset in transfusion cases is likely to vary by species, with P. falciparum being the quickest to develop at about 10 days, P. vivax taking 16 days, and P. malariae taking 40 days or more.39 Where a donor has been implicated, recent history in the United States implicates presumably semi-immune visitors from malaria-endemic areas. Over a 20-year period, only one US civilian without previous residence in malaria-endemic countries has been implicated in transfusion transmission, in contrast to 28 former residents of malaria-endemic countries.41

Chapter 48: Transfusion Transmission of Parasites

In the absence of an FDA-approved blood screening assay, prevention of transfusion-transmitted malaria in the United States has long relied on exclusion of presenting donors with elevated risk for malaria infection. The different categories, which follow guidelines of FDA50 and AABB,51 are as follows: 1. Donors who are permanent residents of countries where malaria is not endemic who travel to an area considered malariaendemic by the Centers for Disease Control and Prevention (CDC) are not accepted as donors of whole blood and blood components until 1 year following departure from the malariaendemic area, assuming they have remained free of malaria symptoms. 2. Prospective donors who have had malaria should be deferred for 3 years after symptoms have abated. 3. Immigrants, refugees, citizens, or residents of countries where malaria is endemic should not be accepted as donors of whole blood and blood components before 3 years after departure, assuming they have remained free of malaria symptoms. These guidelines do not apply to plasma, or plasma components or derivatives lacking intact red cells. In 2000, the FDA released draft guidance52 that contained the following modifications to the 1994 policy: 1. Clarified the term “resident” of a malaria-endemic country as applying to those who had resided in a malarious area for 5 years or longer. 2. Clarified that prospective donors who have had malaria must have received appropriate treatment before remaining asymptomatic for 3 years. 3. Stipulated that donors who might possess partial acquired immunity to malaria should not be accepted as donors of whole blood or blood components for a period of 3 years following their last visit to a malarious region. 4. Called for specific donor travel questions, including capture questions with follow-up of travel history. This 2000 draft guidance remains pending, and may be superseded by different recommendations discussed at a March 15, 2001 Blood Products Advisory Committee meeting.53 Advances in diagnostics have created opportunities for nonmalarious countries to implement strategies that lessen the impact of malaria deferrals while minimizing risk of transfusion transmission. In Europe, regulations54 published by the Council of Europe in 2006—and updated since then—endorse the use of validated immunologic tests to shorten the deferral period of donors with potential malaria risk who test negative. Those donors who have lived for 6 months or more in a malariaendemic area are acceptable as blood donors if they test antibody-negative at least 4 months after their last visit to such an area, as opposed to being permanently deferred. Likewise, donors who report a travel history ⬍6 months duration to malariaendemic area may be accepted as blood donors if they test antibody-negative at least 4 months after their last visit to such an area, in contrast to a 1-year (no malaria symptoms reported) or 3-year deferral (malaria-like symptoms within 6 months of return from malarious area).54

Since 2001, England has used an enzyme immunoassay (EIA) based on three recombinant P. falciparum antigens and one P. vivax antigen (all to erythrocytic merozoite stage) to shorten the deferral period for at-risk donors to 6 months for those testing negative.55 Australia also seeks to reinstate deferred donors on the basis of this same test,56 and several other countries have malaria donor screening tests under evaluation.43 Although not all four Plasmodia species are screened for in the assay adopted by England, it accounts for the two species causing most cases of transfusion transmission and does appear to have some crossreactivity with other species.57 Immunofluorescent antibody tests for malaria antibody detection tend to be sensitive, but have the limitations of being time-consuming and subjective. Direct methods for detection of malaria infection are also available. Most widely used for malaria diagnosis worldwide is Giemsa- or Wright-stained blood films, although their use is not practical for mass screening in areas where malaria is not endemic, where labor costs are prohibitive, and the expertise is lacking. Assays detecting circulating parasite antigens such as histidine-rich protein 2 and lactate dehydrogenase offer sensitivity as low as 100 to 1000 parasites per microliter,43 still insufficient to prevent transfusion transmission. A variety of nucleic acid amplification test (NAT) techniques allow for detection of one or more malaria species. Conventional58 and semi-nested polymerase chain reaction (PCR)59 screening of blood bank samples have detection thresholds on the order of 10⫺3 parasites/µL. Evaluation of the costs of screening blood donors by PCR supports a strategy targeting only those donors with identified risk factors for malaria by questionnaire, not one based on universal screening.60 Newer NAT techniques such as loop-mediated isothermal amplification assay offer sensitivity and specificity equivalent to real-time PCR, but with shorter turnaround time and lower costs.61 With an infectious dose theoretically as low as one to 10 parasites per unit of blood, however, even the most sensitive NAT detection methods cannot reduce the risk to zero.

Babesiosis Awareness of the risk for transfusion-transmitted babesiosis is growing, though historically it has been underappreciated. Transmitted by ticks, over 100 species of these intra-erythrocytic protozoan parasites infect a large number of vertebrate species worldwide.62 In the United States, most reported cases of human babesiosis are caused by Babesia microti, especially in the Northeast and Upper Midwest; in Europe, B. divergens is considered responsible for most human illness from babesial infection. Heightened scrutiny, together with growing phylogenetic analyses, is documenting the human risk in new areas, as well as demonstrating a greater diversity of infecting species than heretofore appreciated. Clinical manifestation of Babesia infection covers a broad spectrum, ranging from chronic, silent infection to fulminant, malaria-like illness. Most B. microti infections appear to be subclinical, but mild flu-like symptoms including fever, headache,

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and myalgias can occur 1 to 6 weeks following a tick bite.62 Risk for severe disease is higher in the elderly, the immunocompromised, and the asplenic. Complications can include thrombocytopenia; hemolytic anemia; and renal, heart, or respiratory failure.62 The case fatality rate can range from 5% to 10% for B. microti63 to 29% for B. divergens.64 All reported cases of B. divergens have occurred in asplenic hosts,64 which almost certainly contributes to this disparity. Since the first case of human babesiosis was recorded in the United States in 1968,65 over 1500 cases have been reported in the country. The primary agent B. microti is transmitted by the black-legged deer tick (Ixodes scapularis, formerly known as I. dammini), the vector of Lyme disease. Most US cases are reported in the Northeast and the Upper Midwest, with the former region extending as far south as New Jersey. Sporadic cases of human babesiosis caused by other Babesia species have been reported from Washington state and northern California, Missouri, and Kentucky since the early 1990s.66-68 In Europe, about 30 human cases of babesiosis have been reported since the first report in 1957, a fatal case in an asplenic Yugoslav farmer.69 Most European cases are attributed to B. divergens, also a bovine pathogen, with cases reported from several countries in Western Europe, the former Yugoslavia, and the former USSR.64 Risk for human infection correlates geographically with the presence of infected cattle populations and areas infested by the tick vector, Ixodes ricinus; little is understood about the role that spleenintact human populations and silent carriage of B. divergens might play in terms of risk to the blood supply.64 Greater awareness and scrutiny, together with molecular and biologic characterization of patient isolates of Babesia parasites, have yielded a more complex picture than historically thought, one still being elucidated. The predominant cause of human babesiosis in the United States—B. microti—has been denominated a diverse species complex composed of three distinct clades with identical morphology, but partly distinct vertebrate hosts and potentially differing levels of pathogenicity for humans.70 Also in the United States, some, but not all, of the isolates from the Pacific Northwest have been grouped as a new species, Babesia duncani, that has been found in humans, dogs, and wildlife in the Western United States.71 Babesia isolates from human cases in Missouri and Kentucky67,68 and also from rabbits on Nantucket Island72 that were originally attributed to B. divergens are now considered to be distinct from the agent of most human babesiosis in Europe.73 These isolates, along with another from an asplenic man whose parasitemia surpassed 40%, are considered “divergens-like” on the basis of morphologic, serologic, and phylogenetic analysis.74 This means that at least three distinct species of Babesia protozoa are known to cause human illness in the United States. As underappreciated as the risk for B. microti might be, even less is known about the risk posed to human health and the blood supply by the newly described species and isolates. In Europe a similar picture is emerging. Although most cases of human babesiosis are attributed to B. divergens, a new

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variant—designated EU1—has been isolated from asplenic patients in Italy and Austria. A near-identical isolate (EU3) was found in an asplenic German patient. Both organisms are phylogenetically closest to B. divergens and B. odocoilei (a parasite of white-tailed deer).75,76 Also, the mounting entomologic77 and serologic78 evidence for human cases of B. microti in Europe has been confirmed by the first documented autochthonous case of human babesiosis in Europe caused by this agent, likely through blood transfusion.79 Finally, in East Asia, the first case of autochthonous human babesiosis in Japan has recently been confirmed, a result of transfusion-acquired B. microti-like infection.80 Asymptomatic, B. microti-like infection has been documented in Taiwan.81 The first case of human babesiosis has been reported from Korea, with the isolated parasite designated KO1 and appearing to be related to Babesia species that infect sheep.82 The two most salient features of B. microti infection contributing to transfusion risk are the probability that most infections are clinically silent83 and the ability of immunocompetent persons to carry infection for lengthy periods.84 There is no certain measure of what proportion of infections are asymptomatic, but seropositivity rates of blood donors or the general population tend to be orders of magnitude higher than the rates of symptomatic cases reported to public health departments. For example, the highest reported incidence of babesiosis in any part of Connecticut in 2002 was 62.7 cases per 100,000 population (0.06 %) in the town of Waterford.85 However, a survey of blood donors in the region of the state of highest endemicity (southeast corner) indicated a point prevalence more than 20 times higher, at 1.4%.86 Of 19 seropositive donors, 10 were confirmed parasitemic by PCR. Early research confirmed the ability of the parasite to persist for several months in untreated individuals.84 In in-vitro studies, the parasite survives storage at 4ºC and remains infective for up to 21 days,87 and one case of transfusion transmission occurred after the storage of blood for 35 days.88 The implications of longterm carriage of B. microti are amply demonstrated in a case in Minnesota, where an asymptomatic donor infected recipients from each of four donations over a 6-month period.89 In the United States, an estimated 60 transfusion cases of Babesia infection have occurred since the first report in 1980, with an estimate that 10 cases occur annually, many unpublished.90 Most cases of transfusion transmission have involved RBCs, although transmission through platelet concentrates has also been reported.91 In transfusion cases, symptoms typically develop 2 to 8 weeks following transfusion, and include fever, hemolytic anemia, and thrombocytopenia.92 Transfusion transmission of B. microti has been documented once each in Canada,93 Germany,79 and Japan,94 the only countries besides the United States to report transfusion cases of Babesia. Only the cases in Germany and Japan reflect autochthonous transmission, however. In Canada the donor acquired infection while on holiday in the United States. In the Pacific Northwest, the variant designated WA-1, since named B. duncani, has also been implicated in two cases of transfusion transmission.95

Chapter 48: Transfusion Transmission of Parasites

Few studies have directly measured the risk for transfusion transmission of Babesia parasites. Given the absence of symptoms in healthy individuals, studying paired donor-recipient specimens is the most secure way of estimating the risk in areas of endemicity. One study estimated a risk of 1 in 617 units (0.17%) of RBCs might be at risk for transmitting B. microti in the state of Connecticut, with the risk from platelets estimated as zero.96 In another Connecticut study, analysis of paired donorrecipient specimens involving chronically transfused patients indicated one possible B. microti seroconversion in nearly 2000 evaluable transfusions.97 Infectivity of different blood components is not easy to determine given that the likelihood of transmitting infection to and detecting clinical infection in the recipient are both likely to depend on the dose of parasites, the length of storage, and the recipient’s immune status. In the aforementioned case in Minnesota, one of three recipients of platelets became infected with B. microti, while all three surviving recipients of red cells became infected.89 In another case, of six individuals transfused with RBCs from an asymptomatic donor, two neonates and one elderly adult became infected, while two other neonates and a child remained aparasitemic.98 Look-back studies in Connecticut found that 26% of recipients of RBC units became infected, compared to only 11% of those receiving platelet products.99 Traditional diagnosis of Babesia infection relies on microscopic examination of Giemsa-stained blood films. This method, however, requires an experienced microscopist, and a high enough index of clinical suspicion to perform the examination. Immunofluorescent assay diagnosis is both sensitive and specific for measuring exposure, but is too time consuming for mass screening, and involves subjectivity on the reader’s part. Inoculation of blood from suspected human cases into laboratory animals offers both high sensitivity and specificity, but its utility is limited to research investigation. Real-time PCR appears to be up to three times as sensitive as conventional nested PCR for donors with variable serologic status.100 Human babesiosis has historically been treated with clindamycin and quinine (CQ), but atovaquone with azithromycin has emerged as the first-line recommendation in the United States because of fewer collateral effects.101 Treatment with CQ continues to be recommended for severe cases, although partial or whole blood exchange transfusion might be performed in such cases.101,102 In the absence of a licensed screening assay, prevention of transfusion-transmitted babesiosis relies exclusively on asking donors if they have ever had babesiosis. This strategy is insufficient because only donors whose Babesia infection was patent and properly diagnosed will self-report a risk. Because the majority of B. microti infections are silent or remain undiagnosed,103 parasitemic donors can escape detection. Seasonal deferral in areas of endemicity would adversely impact the blood supply, and would not address the issues of nonresidents who travel to Babesia-endemic areas93 nor the transfer of blood components from areas where the infection is endemic to areas where it is not.

The predictive value of self-reported exposure to ticks in relation to serologic status has proven low in Wisconsin and Connecticut donors.104 As with other protozoan infections, pathogen inactivation methods hold promise in reducing the risk from transfusiontransmitted babesiosis. A range of photochemical treatments currently under development indicate reductions by 4 to 5 logs in plasma105 and platelets.106 Electrophilic agents that disrupt nucleic acid replication have shown potency in inactivating B. microti by bio-assay (hamster inoculation) and by PCR.107

Leishmaniasis Leishmania species are a large group of protozoan parasites with broad distribution worldwide.108 Leishmania parasites are transmitted in nature by the bite of a female phlebotomine sandfly, but can also be transmitted via blood components and, rarely, congenitally or sexually.109 Clinical manifestations can vary widely, ranging from asymptomatic infection to severe illness with visceral, cutaneous, or mucosal involvement. The visceral form (kala-azar) is characterized by fever, wasting, hepatosplenomegaly, and pancytopenia; if untreated it is usually lethal.110 Cutaneous forms involve progressive skin lesions that become ulcerative, sometimes with mucosal involvement. Although Leishmania species are endemic in about 88 countries, the public health burden is hardly uniform. More than 90% of visceral cases appear in Bangladesh, Brazil, India, Nepal, and Sudan, and about 90% of cutaneous leishmaniasis occurs in Afghanistan, Brazil, Peru, Iran, Saudi Arabia, and Syria.111 The World Health Organization (WHO) estimates that 2 million new infections occur annually—about 1.5 million are cutaneous leishmaniasis (CL), and half a million are visceral leishmaniasis (VL); overall, as many as 12 million people worldwide are infected.111 The distribution is expanding because of myriad factors that include ecological disturbances, growing urbanization in developing countries, and the growing numbers of people immunocomprised by human immunodeficiency virus (HIV) infection or other reasons.109 The United States is home to both phlebotomine sandflies and Leishmania parasites, but autochtonous transmission is limited to infrequent and isolated outbreaks.112 The infective form of Leishmania parasites in the sandfly is the flagellated promastigote; in humans and other vertebrate hosts the parasite takes the form of an oval amastigote that is typically found in phagocytic vacuoles of macrophages and other mononuclear phagocytes.113 Diagnostic methods include microscopic visualization of amastigotes in the tissue aspirates or biopsies from spleen, marrow, or lymph nodes, or in the peripheral blood buffy coat. The duration of parasite circulation in the blood can vary across the ⬎20 Leishmania species, but in any case rarely lasts more than 1 year.114 The immunologic response differs according to the clinical syndrome, with VL leading to a stronger and more enduring antibody production than CL.114 The conventional treatment of leishmaniasis has been pentavalent antimony, but

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new strategies include lipid formulations of amphotericin B, injectable paromomycin, and miltefosine.115 The risk for transfusion transmission of Leishmania may be underappreciated and its occurrence underreported, with 10 probable or confirmed cases having been reported in the literature116 over the past 60 years. Half of these cases were in infants, and nine of 10 were in children 6 years of age or younger.108 The average incubation period was over 7 months, with fever and hepatosplenomegaly being the most common symptoms.108 Studies from areas of endemicity, as well as studies of US military personnel returning from operations in southwest Asia, highlight the risk. Viscerotropic L. donovani and L. tropica isolated from US armed service personnel have been shown to survive at least 25 days in Whole Blood or RBCs stored at 4ºC, at least 5 days in platelet concentrates stored at 24ºC, and at least 35 days in glycerolized RBCs frozen at –70ºC. Further, L. tropica-spiked Whole Blood stored at 4ºC for 30 days retained infectivity to healthy mice. Fresh frozen plasma did not support parasite survival.117 Because transfusion transmission would likely be mistaken for sandfly transmission in Leishmania-endemic areas, and because infection may remain subpatent in immunocompetent individuals, assessing the risk for transmission in blood is a challenge. The few studies involving blood donors indicate nonnegligible rates of detectable parasitemia. In one study in southern France, 76 of 565 blood donors were seropositive by Western blot, and direct detection via kinetoplast DNA amplification and/or direct culture from buffy coat samples were positive in 16 donors. The investigators concluded that L. infantum circulates intermittently and at low density, and that serologic screening of donors would be insufficiently specific to maintain blood availability.118 Another study in the Balearic Islands of Spain found similar results. From 656 healthy blood donors, 2.4% to 7.6% were seropositive by different methods, and 22% of a subset of donors yielded positive cultures. Further, nine of 18 donors who were positive by nested PCR remained positive 12 months later.119 Numbers of this magnitude suggest that the actual transfusion risk might be quite a bit higher than indicated by published reports, which represent cases detected in largely immunosuppressed individuals. The options available to blood banks to prevent distribution of Leishmania-contaminated blood are limited. Targeted donor exclusion is practiced selectively in the United States, specifically relating to the risk of armed services personnel and other travelers to theaters of war harboring Leishmania species upon return. To prevent this, AABB established a 12-month deferral during Operation Desert Storm in Iraq in the early 1990s, and again more recently.116 Given the broad distribution of Leishmania across 88 countries, travel-based deferrals might prejudice blood availability if strictly applied, while further lengthening and complicating the donor health history questionnaire. Because serologic status correlates poorly with asymptomatic infection,120,121 antibody tests hold little promise for donor screening. Photochemical inactivation of different Leishmania species has shown 4-log reduction of amastigote and promastigote forms

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in apheresis platelets,122 and another study demonstrated 5- to 6-log reductions in both platelets and plasma.123 Early results of another method using phototreatment alone shows promise in red cells.124 Further, filtration of leukocytes at both the point of collection and the bedside has been shown to dramatically lower free and intracellular Leishmania parasites.125

Toxoplasmosis Toxoplasma gondii is an obligate, intracellular protozoan that can grow in any mammalian or avian organs or tissues.113 Felines (the definitive host) become infected in nature by consuming intermediate rodent hosts with infective Toxoplasma cysts in the brain or skeletal muscle. The parasite is ubiquitous in nature and can infect a wide variety of animals, including sheep, cattle, and pigs, developing into cysts that remain infective for years. Human infection can occur through many modes of transmission. Most commonly, they include consumption of raw or undercooked meat containing Toxoplasma cysts, and accidental ingestion of T. gondii oocysts from soil or cat litter contaminated with excreted oocysts. Water is increasingly implicated as a vehicle for transmission.126 There are rare reports of infection acquired via solid-organ transplantation and blood transfusion.113 Finally, congenital transmission can occur when the mother acquires primary infection during pregnancy, but the probability of transmission to the fetus (and clinical outcome) depends on the timing of infection.127 Up to one-third of the world’s population is estimated to be infected with T. gondii. About 90% of primary infections are subclinical, and appear to be lifelong. Illness in those with patent infections is usually non-specific and self-limiting, typically involving fever and isolated swollen lymph nodes. More rarely, infection might lead to myocarditis, pneumonitis, hepatitis, or encephalitis.128 Toxoplasmosis is more frequently severe in immunocompromised hosts, and it has emerged as a common opportunistic infection of persons with AIDS.129 Estimates of seroprevalence across countries vary from less than 10% to greater than 90%, and the acquisition of infection depends on an array of local and household factors that include hygiene and sanitation, source and preparation of food, exposure to felines, and climatic factors influencing the survivability of oocysts in nature.128 In the United States, the National Health and Nutrition Examination Survey (NHANES) from 1988 to 1994 indicated an age-adjusted seroprevalence of 22.5% for those above 12 years of age.129 The subsequent NHANES (1999-2004), also drawn from a nationally representative sample, demonstrated a decline in seroprevalence greater than one-third for US-born residents between 12 and 49 years of age. Notably, foreign-born residents living in the United States were three times as likely as US-born residents to be antibody-positive, 24.8% vs 8.2%, respectively.130 Transfusion transmission of toxoplasmosis was reported nearly 40 years ago, in a case where patients with acute leukemia were transfused with leukocytes from donors with chronic

Chapter 48: Transfusion Transmission of Parasites

myelogenous leukemia.131 Although few reports of transfusion transmission are recorded in the literature, the risk has been well documented in recipients of cardiac transplant,128 bone marrow transplant,132 and more recently, small-bowel and other solidorgan transplantation.133 Limited seroprevalence studies have been conducted in healthy blood donors, indicating a broad range of antibody prevalence: 7.4% in Durango, Mexico134; 9.6% in northeast Thailand135; 20% in Turkey136 and southern India137; and 75% in northeastern Brazil.138 Antibody presence is long-lived and does not necessarily denote infectivity. Little information is available on the long-term kinetics of antibody development and patent parasitemia. Advances in diagnostic methods include nested PCR139 and rapid, real-time PCR,140 but these assays are used principally for diagnosis of congenital and ocular toxoplasmosis and with immuncompromised patients.128 Parasite isolation or detection by PCR is rarely useful in immunocompetent patients,141 indicating low probability of detectable parasitemia. One study in Brazil performed real-time PCR on buffy coat samples from individuals classified by serology as chronically infected, and found 3.6% (4 of 110) were positive.140 The mechanisms regulating the persistence of T. gondii infection and the conversion from quiescent to active infection are still being elucidated.142 Although PCR has been established as effective at diagnosing ocular toxoplasmosis in immunocompetent patients,128 the presence of low-grade or intermittent parasitemia in healthy individuals remains little studied. However, the ability of the parasite to survive 50 days at 4ºC,131 and the isolated reports of transfusion transmission in the literature, both establish an element of risk. Prevention of transfusion-transmitted toxoplasmosis is not feasible with either donor exclusion or serologic screening. In most places, discarding units from seropositive donors would heavily prejudice blood availability, with unclear indications that positive antibody status of the donor implies risk for parasitemia. In high-prevalence countries, many blood recipients are likely to have been previously exposed. In an immunocompetent recipient, transfusion transmission is likely to go undetected. Given the parasite’s ability to readily invade and replicate in leukocytes,143,144 leukocyte filtration might diminish the risk in similar fashion as with cytomegalovirus. Whether inactivation treatments being evaluated for other protozoa might be of use for Toxoplasma remains unexplored.

lymphatic filariasis and O. volvulus are particularly severe, with an estimated prevalence of 120 million and 37 million infections, respectively.145 These organisms share similar life cycles and are all transmitted by hematophagous arthropods. In each, adult female worms produce larvae called microfilaria, which for most species circulate in the bloodstream, sometimes with periodicity timed to their primary insect vector’s feeding habits.113 The microfilaria are the infective form for insects, but when transmitted by transfusion are incapable of propagating further.146 The lymphatic forms of filariasis, which cause elephantiasis, have been targeted by WHO as potentially eradicable, primarily by eliminating the human reservoir of microfilaria that infect the mosquito vectors through repeated mass administration of curative drugs.147,148 Results from low- to moderate-prevalence areas in Egypt149 and from moderate- to high-prevalence parts of Papua New Guinea150 indicate dramatic success in lowering human infections and even, surprisingly, reversing the pathology associated with infection. There is little published information on the risk for transfusion transmission of filariasis. In most filariasis-endemic areas, the risk for vector-acquired infection would be orders of magnitude greater than that from transfusion for the average individual, given the relative degree of exposure to infective insect vectors and the limited rate of blood transfusion. However, limited studies from Nigeria have shown prevalence of microfilaria of 3.5% with Loa loa151; 15.6% with M. perstans and 1.3% with L. loa152; and 1.3% with unspecified microfilaria.153 There has been at least one report of an American blood donor being found with microfilaria.146 Isolated case reports of transfusion transmission exist from Italy154 and India,155 in both cases indicating that the outcome in blood recipients might often be no more severe than mild allergic reaction in response to microfilarial antigens. The microfilaria of both W. bancrofti156 and L. loa157 survive routine storage conditions for blood. Those from M. ozzardi158 and from B. malayi and W. bancrofti159 have been successfully recovered following cryopreservation in research laboratories. One article from Spain reports an evaluation of alternative strategies for screening for L. loa microfilaria in donors from Central and West Africa.160 This example is the exception, however, as most countries appear to consider the risk to blood recipients too small to merit donor or component screening.

Conclusion Microfilariasis Filarial worms are arthropod-borne macroparasites that can be caused by a number of different organisms. Wuchereria bancrofti and Brugia spp. cause lymphatic filariasis; Loa loa, Onchocerca volvulus, and Mansonella streptocerca cause nonlymphatic, subcutaneous filariasis; and M. ozzardi and M. perstans cause nonlymphatic infections of different body cavities and are typically asymptomatic or mild.113 The filariases occur in more than 80 countries, and the health and socioeconomic burden from

The risk for transfusion transmission of parasites in the United States is not high, but might well be greater than the prevailing estimated risks of one per million units. Until the recent licensure of a screening assay, it appears likely that hundreds of individuals have donated annually while infected with T. cruzi, the result of demographic changes in the United States and its donor population. Even assuming low-end estimates of infectivity, that implies dozens of cases of transfusion-transmission annually. The clinical impact of such events might be

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uncertain, but at least in immunocompromised blood recipients the risk is serious. Perceived risk for Babesia species in blood might not be widespread, but the combination of asymptomatic infection in healthy donors, the lack of a licensed screening assay, and the mobility of both people and blood components implies risks that are higher and geographically less circumscribed than appreciated. Silent infection in semi-immune donors contributes to risk for malaria transmission in blood components, but recent years have seen an average of one case per year. Serologic or direct detection assays can both contribute to enhanced safety against these and other parasitic agents. Broadly effective methods such as pathogen removal or inactivation also appear promising.

Disclaimer The author has disclosed no conflict of interest.

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49

Bacterial Contamination of Blood Products Yara A. Park1 & Mark E. Brecher2 1

Assistant Professor, Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA 2 Director, McLendon Clinical Laboratories, University of North Carolina Hospitals, and Professor and Vice Chair for Clinical Services, Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Bacterial contamination of blood components is a persistent but often overlooked problem in transfusion medicine. Before recent developments in skin preparation, diversion, and testing for bacterial contamination, the incidence of platelet bacterial contamination was approximately 1 in 1000.1 Although recent public attention has focused on transfusion-transmitted viral infection, improved methods of screening through donor questioning and testing have greatly reduced the transmission of hepatitis viruses and retroviruses. Given the reduction of viral transmission via allogeneic blood, the risk of bacterial contamination has emerged as the greatest residual threat of transfusion-transmitted disease. This chapter provides a quick overview of risks associated with blood components. The main focus is on approaches to minimize or eliminate the risk of bacterial contamination.

Yersinia cases (Table 49-1). Of the reported deaths, one was caused by a coagulase-negative Staphylococcus strain and seven were caused by a variety of gram-negative organisms. These organisms are all capable of growth at 1 to 6ºC. Sepsis associated with the transfusion of gram-negative bacterially contaminated RBCs is typically severe and rapid in onset. Patients frequently develop high fevers (temperatures as high as 109ºF have been observed) and chills during or immediately following transfusion. From 1987 to 1996, 20 cases of Yersinia-infected RBC units in 14 states were reported to the Centers for Disease Control and Prevention (CDC).8 Twelve of the 20 recipients died in 37 days or less following transfusion. The median time from transfusion to death was 25 hours. Of the seven who developed disseminated intravascular coagulation, six died.

Allogeneic RBC Units

Transfusion-Transmitted Bacterial Infection of Red Blood Cells Sepsis associated with the transfusion of bacterially contaminated Red Blood Cell (RBC) components is generally regarded as a very rare event. From 1995 to 2004, 25 fatalities thought to be secondary to contamination of Whole Blood or RBC units were reported to the US Food and Drug Administration (FDA) (Fig 49-1).2 The risk of death from a bacterially contaminated RBC transfusion in the United States has been estimated to be 0.13 per million.3 Reports from New Zealand have indicated a Yersinia enterocolitica transfusion-transmitted incidence rate of 1 in 65,000, with a fatality rate of 1 in 104,000 RBC units transfused.4 Unrecognized cases, underreporting, and regional variation may account for observed differences in the incidence. Passive reporting studies from the United States,3 France,5 and United Kingdom (UK)6 of bacterially contaminated RBCs that caused symptoms of infection show a relative paucity of Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

Asymptomatic donors with transient Yersinia bacteremia are presumed to be the source of contamination. Implicated donors are typically found to have infection elevated antibody titers [immunoglobulin M (IgM) or IgG] to Y. enterocolitica, implying a recent infection.8-10 The possibility of contamination of RBC components is directly related to their storage time; most cases of Yersinia contamination of RBC units occur after storage for 25 days.11 Serratia marcescens was linked to an outbreak caused by contamination of RBCs in Denmark and Sweden.12,13 The contamination was thought to involve the manufacturing process, because the sterile bag sets were autoclaved and put in a clean but not sterile outer plastic package. It was thought that S. marcescens present in the dust in the factory contaminated the outside of the containers, and in the presence of moisture and a nutrient (the plasticizer diethylhexylphthalate), the bacteria proliferated and somehow gained entry into the bag.14 Prospective bacterial cultures of Whole Blood or RBC units, however, have shown a much higher incidence of bacterial contamination (2 to 4 per 1000 units). The organisms commonly cultured are Staphylococcus or Propionibacterium spp., which generally proliferate poorly during storage at 1 to 6ºC.

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Section IV: Part II

Escherichia coli 12.0%

Staphylococcus aureus 4.0%

Serratia marcescens 12.0%

Staphylococcus epidermidis 16.0%

Klebsiella sp 12.0%

Pseudomonas fluorescens 8.0% Pseudomonas aeruginosa 8.0%

Gram neg rod (other) 4.0% Pantoea agglomerans 4.0% Enterobacter cloacae 4.0% Yersinia enterocolitica 8.0% Serratia liquefaciens 8.0%

Figure 49-1. Transfusion fatalities by organism in RBC units or whole blood, as reported to the United States Food and Drug Administration from 1995-2004 10 yrs, 25 cases (data from Niu et al2).

Table 49-1. Summary of Organisms from RBC Transfusions Identified in the BACON, SHOT, and BACTHEM Studies* Organism

Gram positive Coagulase-negative Staphylococci Streptococcus spp. Staphylococcus aureus Enteroccocus faecalis Bacillus cereus Proprionibacterium acnes Subtotal

United States BACON

United Kingdom SHOT

France BACTHEM (29 isolates, 25 implicated units)

Total

2(1)

2

2 (1 or 50%)

2 (0 or 0%)

3 4† 2‡ 1§ 2 1 13 (0 or 0%)

7(1) 4 2 1 2 1 17 (1 or 6%)

2(2) 1

1

2(1)

1(1)

5(3) 1 2(1) 1(1) 5(1) 2(1) 3 1 1 21 (7 or 33%) 38 (8 or 21%)

Gram negative Serratia liquefaciens Serratia marcescens Yersinia enterocolitica Enterobacter sp. Acineterbacter sp. Pseudomonas sp. Escherichia coli Klebsiella pneumoniae Proteus mirabilis Subtotal

3 (2 or 67%)

2 (1 or 50%)

1 1(1) 5(1)‡ 2(1) 3§ 1 1 16 (4 or 25%)

Total

5 (3 or 60%)

4 (1 or 25%)

29 (4 or 14%)

*Numbers of fatalities and the percent of the total are reported in parentheses. In two cases, two isolates of Streptococcus sp. were isolated from the implicated bag. ‡ In one case, Staphylococcus aureus and Acineterbacter baumannii were both isolated from the implicated bag. § In one case, Enterococcus faecalis and Escherichia coli were both isolated from the implicated bag. Used with permission from Brecher and Hay.7 †

Autologous RBC Units Although autologous blood is generally considered a “safer” blood component, there have been at least six reported cases of bacterial contamination of autologous RBC units, five

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from Y. enterocolitica15-18 (and personal communication, Susan Cookson, CDC, September 23, 1996) and one from Serratia liquefaciens.19 Fortunately, all recipients survived, possibly because of preformed immunity. Upon retrospective

Chapter 49: Bacterial Contamination of Blood Products

Enterococcus sp 1.7% Clostridium perfringens 1.7% Gram pos rod (other) 8.0%

Streptococcus 6.7% Staphylococcus aureus 6.7%

Bacillus sp 1.7%

Klebsiella sp 18.3%

Figure 49-2. Transfusion fatalities by organism in platelets, as reported to the United States Food and Drug Administration from 1995-2004 10 yrs, 60 cases (data from Niu et al2).

Escherichia coli 15.0%

questioning, all patients infected by Yersinia recalled gastrointestinal symptoms in the days before donation. In the case of Serratia contamination, the patient’s infected toe ulcer was presumed to be the source.

Transfusion-Transmitted Bacterial Infection of Plasma, Cryoprecipitate, and Derivatives Cell-free products, such as plasma and cryoprecipitate, are stored in the frozen state and thus are rarely associated with significant contamination. However, Pseudomonas cepacia and P. aeruginosa have been cultured from cryoprecipitate and plasma thawed in contaminated waterbaths.20,21 Human serum albumin is a good culture medium and preserves viability of contaminants. The heating step (60ºC for 10 hours) in the manufacturing of albumin is performed to inactivate certain viruses, not to ensure bacterial sterility.22,23 This would require autoclaving (superheated under pressure), which would cause albumin to denature. On occasion, specific lots of albumin product have been found to be contaminated with bacteria, typically Pseudomonas spp.24 These lots have produced endotoxic shock, transient bacteremias, and febrile reactions in recipients. Two patients in different hospitals developed Enterobacter cloacae septicemia after receiving albumin.24,25 Cultures of unopened product grew Stenotrophomonas multophilia and Enterococcus gallinarum in addition to E. cloacae. This resulted in a worldwide recall of certain lots of 5%, and 25% albumin. It is suspected that cracks in the glass bottles were responsible for the contamination. Manufacturing problems, therefore, are a source of bacterial risk from these derivatives.

Staphylococcus epidermidis 18.3%

Serratia marcescans 8.3%

Gram neg rod (other) 1.7% Pasturella multocida 1.7% Pseudomonas aeruginosa 3.3% Morganella/Providencia 1.7% Salmonella sp 3.3% Enterobacter sp 8.3%

Transfusion-Transmitted Bacterial Infection of Platelets Source of Contamination Skin commensal organisms such as Staphylococcus epidermidis and Bacillus cereus are the organisms most often implicated in platelet bacterial contamination.26,27 These organisms typically do not grow at 0 to 6ºC, but survive and multiply readily at 20 to 24ºC, the storage temperature of platelets. Fatalities caused by platelet contamination tend to be predominantly gram-negative organisms (Fig 49-2). Potential sources of these organisms include 1) donor-related transient bacteremia; 2) incomplete disinfection or skin core removal by the collection needle; and 3) contamination of the collection bag, tubing, or anticoagulant. In some cases, retrograde flow from vacuum tubes used in collection or even the recipient have been implicated as a source of contamination.28,29

Clinical Presentation The clinical sequelae resulting from transfusion of bacterially contaminated platelets range from asymptomatic to mild fever (which may be indistinguishable from a nonhemolytic transfusion reaction) to acute sepsis, hypotension, and death. The clinical picture is much more varied and often less severe than that of patients infected by transfusion of bacterially contaminated RBCs.30 Sepsis caused by transfusion of contaminated platelets is vastly underrecognized and underreported. Those patients who receive the greatest number of platelets (patients being treated with chemotherapy who are both thrombocytopenic and immunosuppressed) are those most at risk of sepsis (Table 49-2). In such patients fever can be readily

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and 1 in 61,000 for apheresis units.40 The University Hospitals of Cleveland reported a fatality rate of 1 in 48,000 per PRP-WBD platelets.41 However, it is widely suspected that platelet bacterial sepsis is frequently unrecognized and, thus, is underreported. From October 1, 1995 to September 30, 2004, there were 60 transfusion-transmitted fatalities caused by contamination of platelets reported to the FDA (2) (Tables 49-4 and 49-5).

attributed to other infectious causes. Broad-spectrum antibiotics should be considered for any patient who develops fever within 6 hours of platelet transfusion.31,32

Incidence Sepsis resulting from transfusion of bacterially contaminated platelets is the most common transfusion-transmitted disease. Platelets are stored at room temperature, 20 to 24ºC, making them an excellent growth medium. Multiple aerobicculture surveillance studies have demonstrated that 1 in 1000 to 3000 platelet units are bacterially contaminated (Table 49-3). Based on the fact that approximately 10 million platelet units are transfused every year in the United States,39 it has been estimated that 2000 to 4000 bacterially contaminated platelets units are transfused every year. Despite the estimates of contamination, the actual septic transfusion reaction rates were much lower at approximately 1 in 25,000 platelet units (range 1 in 13,000 to 100,000).3,5,40 Observations from hospitals actively pursuing platelet-related sepsis support these estimates. Johns Hopkins found a fatality rate of 1 in 17,000 for pooled platelet-rich plasma, whole-blood-derived (PRP-WBD) platelets

Table 49-4. Fatalities from Platelet Transfusion in the United States from 1995 to 2004 (data from Nin et al2)

Table 49-2. Factors Affecting Outcome of Transfusion of Bacterially Contaminated Blood Components ● ● ● ● ● ●

Virulence of the organism Immune status and general condition of the recipient Concentration and bolus dose of bacteria transfused Timely recognition and therapeutic intervention Intensity of patient monitoring—ie, inpatient vs outpatient Medicines the patient is receiving—ie, antibiotics

Used with permission from Krishan and Brecher.31

Organisms

Number of Deaths

Gram-Positive (n
22) Staphylococcus epidermidis Staphylococcus aureus Streptococcus group G Clostridium perfringens Streptococcus group F / Eikenella One unidentified gram-positive rod

11 4 2 1 1 1

Gram-Negative (n
38) Klebsiella pneumoniae Escherichia coli Serratia marcescans Enterobacter aerogenes Enterobacter cloacae Pseudomonas aeruginosa Salmonella spp. Bacillus spp. Enterobacter agglomerans Klebsiella oxytoca Morganella/Providencia Pasturella multocida Two unidentified gram-negative rods

10 9 5 2 2 2 2 1 1 1 1 1 1

Table 49-3. Selected Culture Studies of Whole-Blood-Derived Platelet Concentrates (PC) and Apheresis Platelets (Aph) Country

Product

Sample volume (mLs)

Method

Day of Sampling

“N”

Initial Positive

Confirmed Positive

Rate per 1000 Units

Ref

Belgium

PC Aph PC

NS

BacT/Alert

NS

BacT/Alert

0

294 (1.7%) 98 (1.4%) 140 (1.8%)†

143 (0.81%) 12(0.17%)* NS

8.1 1.7 18

Claeys et al33

2.5-5.0

17675 6885 7644 1949 13,454 3,553 4,995 23,390 5,147 16,290 10,065

34 (1.8%)† 35 (0.26%) 21 (0.59%) NS NS NS NS NS

21 (0.16%) 16 (0.45%) 4 (0.08) 12 (0.05%) 3 (0.06%) 4 (0.02%) 7 (0.07%)

18 1.6 4.5 0.8 0.5 0.6 0.2 0.7

Belgium

Brazil US US Canada

Aph PC Aph PC PC Aph PC

2

BacT/Alert

0

5 NS

Broth Plate

6–12 4–5

2

Bactec

1 3

*Extrapolated from units available for retesting (125 PCs and 27 Aph). † Based on the successful subculture of the culture bottle [originally 144 (1.9%) and 38 (2.0%), respectively].

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Schelstaete et al34 Wendel et al35 Leiby et al36 Dykstra et al37 Blajchman et al38

Chapter 49: Bacterial Contamination of Blood Products

Prevention

bacterially screened units. Of the 20 septic reactions, three were fatal and involved a Staphylococcus species. All of the units involved with septic fatalities and 13 of the units implicated in septic reactions were associated with platelets transfused on the fifth day after collection. If the units implicated in the septic transfusion reactions (which were culture negative) were considered to have tested as false-negative, the false-negative rate would be 1 in 74,807.46 The effects of the AABB standard are difficult to quantify. On the basis of 2006 platelet utilization data, in the United States in one year, approximately 2.8 million platelet units were transfused (1.5 million apheresis platelets and 1.3 million WBD platelets).39 Using different fatality projections, anywhere from eight fatalities per year, based on the BaCon study,3 up to 40 fatalities per year, based on a 1% fatality rate46 would be expected. From 1995 to 2004, 60 deaths caused by bacterially contaminated platelets were reported to the FDA, averaging six deaths per year before the initiation of the standard.2 Using the ARC data for 2004 through 2006 with additional data from Blood Systems, Inc. (1 probable septic transfusion reaction in 209,654 collections tested) and the New York Blood Center (no septic transfusion reactions in 150,241 collections tested), the passively reported septic reaction rate with platelets since testing is approximately 1 in 230,000.47 This is compared to an approximate rate of 1 in 40,000 reported by ARC for a 10-month period in 2003.44 This shows a decrease in the risk of septic reactions from platelets since the institution of the AABB standard, but these data are limited by virtue of being passively reported.47

Both the College of American Pathologists (CAP) and AABB have instituted steps to require the detection of bacteria in platelet products. In December 2002, the CAP added a phase I item to the Laboratory Accreditation Checklist regarding the detection of bacteria.42 The checklist item, TRM.44955, asked “Does the laboratory have a system to detect the presence of bacteria in platelet components?” The question was revised in December 2004 to ask “Does the laboratory have a validated system to detect the presence of bacteria in platelet components?” With this revision, the CAP ceased allowing swirling to be an acceptable method of detection, but still allows the use of surrogate testing methods such as pH or glucose measurements. In March 2004, AABB instituted a new standard that requires blood banks or transfusion services to have steps in place to limit and detect bacterial contamination in all platelet products.43 Since that time, blood centers and transfusion services have been implementing a variety of interventions to accomplish this goal. The American Red Cross (ARC) began routine aerobic cultures using the BacT/ALERT (bioMérieux, Durham, NC) at that time and in their first 10 months of testing, 226 of 350,658 platelet collections initially tested positive.44 Sixty-eight of these were confirmed positives for a rate of bacterial contamination of 1 in 5157. In the first two years of testing by the ARC, the confirmed positive rate was 1 in 5399 apheresis platelet units.45 Despite universal testing of all platelet products, in the 2-year period from March 2004 through May 2006, the ARC reported 20 septic transfusion reactions caused by transfusion of

Table 49-5. Summary of Organisms from Platelet Transfusions Identified in the BaCon, SHOT, and BACTHEM studies* Organism

Gram positive Bacillus cereus Coagulase-negative Staphylococci Streptococcus spp. Staphylococcus aureus Proprionibacterium acnes Subtotal Gram negative Klebsiella spp. Serratia spp. Escherichia coli Acinetobacter Enterobacter spp. Providencia rettgeri Yersinia enterocolitica Subtotal Total

United States BACON

United Kingdom SHOT

France BACTHEM

Total

1

4 (1)

2

7( 1)

9 3 (1) 4

8 (1) 2 3 (1)

5

17 (1 or 6%)

14 (3 or 21%)

20 (1) 5 (1) 6 (1) 3 41 (4 or 10%)

3 10 (0 or 0%)

2 (1) 1 (1) 1 11 (5 or 45%)

2 (1)

2 (1) 1 (1) 1 1 1

3 (2 or 67%)

6 (2 or 33%)

2 (1) 3 (3) 8 (2) 1 4(2) 1(1) 1 20 (9 or 45%)

28 (6 or 21%)

17 (5 or 29%)

16 (2 or 13%)

58 (13 or 22%)

2 (2) 5 (1)

4 (2)

*Numbers of fatalities and the percent of the total are reported in parentheses. Used with permission from Brecher and Hay.7

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Strategies to Reduce the Risk of Posttransfusion Sepsis Several approaches to the reduction of risk of posttransfusion sepsis can be grouped into four major categories: bacterial avoidance, growth inhibition, detection, and elimination.

Bacterial Avoidance Donor Screening Histories of recent dental procedures, gastrointestinal or genitourinary procedure, and breast feeding (which can be associated with skin cracks) may all be associated with bacteremia and are cause to defer a potential donor. Unfortunately, asking donors about symptoms suggestive of infections are problematic. For example, 13% of donors have had gastrointestinal symptoms in the 30 days before donation.48 In addition, retrospective questioning of donors implicated in Yersinia red cell sepsis showed that only half had any gastrointestinal symptoms in the preceding 30 days.8-10 Therefore, donor questioning about gastrointestinal symptoms does not appear to be a specific predictor of Yersinia bacteremia. Skin Preparation Despite excellent technique, one cannot ensure a sterile venipuncture, because organisms harbored in sebaceous glands and hair follicles cannot be completely removed or killed, and skin fragments drawn up into the collection bag during the initial phase of donation can provide a source of infectious organisms.49,50 Scarring or dimpling of the venipuncture site from prior donation has also been recognized as a risk factor for bacterial contamination, because these areas frequently are difficult to disinfect.51 In one case, phlebotomy at a dimpled venipuncture site of an apheresis donor resulted in three episodes of platelet contamination with gram-positive organisms; sepsis occurred in four recipients of those platelets. Iodine solutions have been shown to be the most effective in reducing the donor skin bacterial burden (Table 49-6). Skin of donors who are allergic to iodine is often cleansed with a chlorhexidine solution or double isopropyl alcohol skin

disinfection. In general, tincture of iodine, povidone iodine, or chlorhexidine are used for skin preparation. Table 49-7 summarizes the effectiveness of the most commonly used disinfection methods.53

Diversion Diversion of the first few milliliters of whole blood from the primary container has been shown to reduce the amount of bacterial contamination from the skin. A study performed on 22,000 blood donations by the Red Cross in the Netherlands, in which the first 10 mL of donor blood were diverted from the primary bag, showed that 16 of the first 5-mL aliquots were bacterially contaminated, while only two of the second 5-mL aliquots were positive after culture.54 A second study from the Netherlands compared the bacterial contamination rates of whole blood collections with and without the removal of the first 10 mL. The diversion of the first 10 mL showed a significant decrease in bacterial contamination (18,263 collections with 0.39% contamination without diversion compared with 7,115 collections with 0.21% contamination with diversion, p
0.05).55 It must be remembered that diversion is most effective at decreasing contamination with skin flora.56,57 A majority of bacteria-related fatalities involve gram-negative organisms, which are not interdicted by diversion. Using the ARC data for collections between 2004 and 2006, it is possible to compare the true-positive rates with and without diversion. The authors report on the contamination rate of one-arm collections and two-arm collections. Table 49-7. Percentage and Mean Count of Donors with Bacteria after Skin Disinfection CFU/Plate

Povidine Iodine 2

IPA and Iodine

IPA 2 Sponge

IPA 2 Swab

0 10 100 1000 Mean count

39% 57% 75% 96% 175

79% 93% 100% 100% 3

0% 69% 86% 97% 161

29% 50% 68% 89% 237

Used with permission from Brecher and Hay.7 (Data from McDonald et al.53)

Table 49-6. Percentage of Donors with Bacteria Growth after Skin Disinfection Bacterial Colonies per Plate

Povidone Iodine

Isopropyl Alcohol and Iodine Tincture

Chlorhexidine Gluconate

Green Soap and Isopropyl Alcohol

0 1-10 11-100 100 p value compared to povidone iodine

34-49% 35-43% 10-14% 0-13%

63% 34% 2% 1%
0.001

60% 25% 12% 3% 0.3

0% 17% 47% 36%
0.001

Used with permission from Goldman et al.52

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Chapter 49: Bacterial Contamination of Blood Products

The two-arm collection set-ups involved a diversion pouch that was located on the venous return line, not the inlet line. The one-arm collections utilized a correctly placed diversion pouch. Table 49-8 shows the decrease in the contamination rate of skin flora with the use of a correctly placed diversion pouch.45

Apheresis Platelet vs Whole-Blood-Derived Platelet Concentrates Therapeutic doses of platelets can be obtained from a single donor through an apheresis procedure or from whole blood donation. Four to six platelets concentrates from whole blood donations must be pooled to make a therapeutic dose; therefore, it would be expected that pooled platelets obtained from multiple donors would be at higher risk of bacterial contamination. From 1986 to 1998, Johns Hopkins Hospital increased the use of apheresis platelets from 51.7% to 99.4% and saw a threefold reduction in septic transfusion reaction involving platelets, from 1 in 4818 transfusions to 1 in 15,098 transfusions.58 A survey of AABB-accredited blood centers, hospital blood banks, and transfusion services (in total, 900 institutions) in late 2004 examined platelet usage, supply, and testing methods.59 Between 2003 and 2004 there was an 11.3% reduction in the use of WBD platelets and 77.2% of platelets transfused by the institutions surveyed were apheresis platelets. Not only do apheresis platelets decrease the risk of exposing a recipient to a contaminated unit, the type of platelet used appears to affect the type of testing performed to detect bacteria in the United States. Over 90% of apheresis units were tested with culture, whereas most WBD platelets were tested using a variety of methods including staining with microscopic examination and the use of markers of increased metabolism such as pH and glucose. In practice, many institutions have felt that culturing of individual WBD platelets to be impractical. A new system, Acrodose PL (Pall, Covina, CA) is now available for prestorage pooling of WBD platelets. The Acrodose system allows institutions to pool WBD platelets and then store them for the life of the individual platelets. By using prestorage pooling, the volume of the product is acceptable for culturing with either the BacT/ALERT or enhanced bacterial detection

Table 49-8. Comparison of One- and Two-Arm Apheresis Platelet Collections Two-arm procedures Rate of skin 17.2 (106 of 617,595 contaminants per collections) 105 cultures Rate of nonskin 5.5 (10 of 617,595 organisms per collections) 105 cultures Rate of true 22.7 (140 of 617,595 positives per collections) 105 cultures

One-arm procedures*

OR (95% CI)

7.8 (30 of 386,611 collections)

2.2 (1.5-3.3)

4.1 (16 of 386,611 collections)

1.3 (0.7-2.4)

11.9 (46 of 386,611 1.9 (1.4-2.7) collections)

*The diversion pouch was in the correct place on the one-arm procedures only.45

system (eBDS, Pall, East Hills, NY). Platelets collected in CPD or CP2D collections bags, both leukocyte-reduced and non-leukocyte-reduced, can be pooled with the Acrodose system. This would allow users of PRP-WBPC to transition from surrogate testing to the more sensitive method of culturing. Prepooled whole-blood-derived platelets prepared by the buffy coat method have been available in Europe since the early 1990s. Recently, Canada has been in the process of transitioning from the platelet-rich plasma method to the buffy coat method. The experience in Europe is that the confirmed positive culture rate of prepooled buffy coat platelets is equivalent to apheresis platelets.60 This may be a result of the overnight incubation in the presence of white cells.

Growth Inhibition Optimizing Storage Time In 1991, the Blood Products Advisory Committee (BPAC) of the FDA reviewed all cases of Yersinia sepsis from RBC units reported to either the FDA or the CDC during the late 1980s. At that time all reported cases of RBC-associated Yersinia sepsis in the United States had occurred in units older than 25 days. As a result of timing necessary for the bacteria to attain a lethal concentration, the BPAC proposed reducing the storage time of RBCs from 42 to 25 days. This recommendation was subsequently rejected for several reasons. 1. A questionnaire distributed at the time revealed that 20% of RBC units in stock at over 1500 blood banks and transfusion services were more than 28 days old.61,62 Discarding such units would have severely compromised the nation’s blood supply. 2. A shorter outdate would require recruitment of new donors. It was estimated that the addition of a quarter of million donations per year would be required to replace the loss resulting from shorter outdates. This would involve additional risk, because first-time donors are more likely to carry disease because their blood has not been repeatedly tested, as has the blood of repeat donors. 3. Units less than 25 days old can also cause sepsis, so that decreasing the allowable storage time would lessen the problem but not eliminate it.11,63,64 4. Older units are less likely to transmit viruses such as human immunodeficiency virus (HIV) and human T-cell lymphotropic virus types I and II.65,66 Longer platelet storage time has also been associated with increased probability of contamination. In 1983, in the United States platelet storage for WBD platelets was transiently approved for 7 days. This 7-day storage was based on acceptable in-vitro function, in-vivo recovery, and survival data. However, because of anecdotal reports of bacterial proliferation over the extended storage time, the shelf life was returned in 1986 to 5 days. Unfortunately, merely decreasing the shelf life did not eliminate the problem of bacterial contamination. It has been shown that even a moderate inoculate [10 to 50 colony-forming units (CFU/mL)] of certain bacteria such as B. cereus and P. aeruginosa in platelets has a minimal lag phase with a doubling time of 1 to 2 hours.67 This can lead to a bacterial load of 108 in just 1 to

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2 days. The lowest safest limit of bacteria and the time to clinical symptoms are unknown. In one well-documented outbreak of Salmonella choleraseus in seven patients linked to one common repeat donor with an occult chronic osteomyelitis, the time to the onset of illness ranged from 5 to 12 days (mean 8.6 days). In all cases, the platelet units were stored for less than 1 day.68 In a similar situation, the CDC reported in 2006 a multistate outbreak of Pseudomonas fluorescens bloodstream infections. All cases could be traced to contaminated heparin flushes.69 A total of 28 patients had delayed onset of P. fluorescens infections, ranging from 84 to 421 days after their last potential exposure. Only by severely reducing the storage time of platelets to 1 to 2 days would one significantly impact the risk of bacterial overgrowth. However, operational changes affect the storage age of available platelets. With increased complexity of disease-marker testing, the availability of 1-day-old platelets has decreased. For example, in the United States in 1982 the mean age of distributed platelets was 1.6 days, in 1983 (after extension of the dating period to 5 days) it was 2.0 days, and in 1992 (after addition of increased laboratory testing) it was 2.5 days. In 1983, only 5% of issued platelets were greater than 3 days old. In 1992, just 10% were older than 3 days. But with the introduction of centralized testing by the ARC, the mean age of issued platelets increased 2.7 days with 20% older than 3 days.70 With the addition of nucleic acid testing for HIV and hepatitis C virus (HCV), additional delays occurred. Not only can this decrease available shelf life of an already precariously limited supply of platelets, it can also decrease the availability of fresh platelets, which are the most hemostatic and the least likely to be bacterially contaminated.

Optimizing Storage Temperature Bacterial proliferation in red cells or whole blood is generally limited to a few gram-negative organisms capable of proliferation at 1 to 6ºC (so-called psychrophilic organisms, most notably Y. enterocolitica, Serratia spp., and Pseudomonas fluorescens). Freezing red cells with glycerol would prevent the proliferation of bacteria but would be associated with significant decreased availability and increased cost.

Platelets are typically stored at 20 to 24ºC, principally because of a potential concern of bacterial contamination and progressive decline in platelet function (storage lesion) if stored for longer periods at these temperatures. Recently, with the addition of an early culture, the use of 7 day platelets has resumed in many parts of the world (see the discussion of international practice and specifically the United States Post Approval Surveillance Study of Platelet Outcomes, Release Tested (PASSPORT) later in the chapter). Although storage of platelets at 4ºC results in a significantly lower rate of bacterial growth, it also causes a temperature-induced activation of platelets and rapid decline in functional ability and in-vitro viability. Studies of cold-storage platelets with the addition of specific second-messenger stimulators have reported that platelets stored at 4ºC were bacteriostatic and retained partial functional ability and viability compared with control platelets.71,72 If a practical method for storing platelets at 4ºC or in a frozen state were perfected, it would have the potential to reduce the risk of bacterial contamination of this blood component.

Addition of Antibiotics The addition of antibiotics to blood components has been considered but has not been adopted because of the added risk of an adverse drug reactions (trading one infrequent reaction for another) and the potential for the development of antibiotic resistance if small amounts of antibiotics were infused with every transfusion.73

Bacteria Detection A simple low-cost method to detect bacterial overgrowth would be a valuable tool for screening units before release from the blood bank. Several methods for bacterial detection are available and more are in development (Fig 49-3). Unlike viral contamination of blood components, which is detected from a sample obtained at the time of donation, bacterial contamination of blood components frequently requires time for the organisms to proliferate before being detectable. Therefore, knowledge of growth characteristics of bacteria in blood components must be considered for successful implementation of a detection strategy (Figs 49-4 and 49-5).

Time 12-30 hours

Selected methods BacT/ALERT

24-30 hours
1 hour

Pall BDS Verax PGD Dielectrophoresis Acridine orange


1 hour Minutes

 Mixed results  Reliable

Gram‘s stain

Minutes

Antibiotic probe

1 hour

rRNA probe


1 hour

Reagent strips

Minutes

Swirling

Seconds

Endotoxin

Hours 0

1

2

3

4

5

6

7

Bacterial concentration log10 CFU/mL

780

8

9

Figure 49-3. Methods of detection of bacterial contamination of blood components. Areas of light gray shading represent concentrations (CFU/mL) at which some, but not all, bacteria are detected by each method. Dark gray shaded areas represent ranges over which each method is reliable. The approximate time from beginning to end of the assay is reported.

Chapter 49: Bacterial Contamination of Blood Products

showed slower and more varied growth.75 This study concluded that an assay capable of detecting 102 CFU/mL on Day 3 of storage would detect a vast majority of bacterially contaminated platelet units. In the case of red cell contamination, inoculation experiments of whole blood with Y. enterocolitica have demonstrated an initial rapid decline in the number of viable organisms followed by a resumption of growth after a lag phase of approximately 3 to 14 days.11,76-80 During this lag phase, Yersinia typically cannot be recovered from samples obtained from the bag.

Timing of Detection Using an automated detection system (Bactec, Becton Dickinson, Cockeysville, MD) Blajchman et al cultured WBD platelets concentrates on Days 1 (16,290 platelet concentrates) and 3 (10,065 platelet concentrates) following preparation.38 Of the 16,290 platelet concentrates cultured on the day of collection, 4 units were found to be positive; however, an additional 3 units that were culture negative on the day of collection were culture positive after 2 additional days of storage. On the basis of these results the authors concluded that cultures from the day of collection may be inadequate to detect all contaminated platelet concentrates. Bacterial growth characteristics were reported for 165 platelet units, each inoculated on the day of collection with one of the following organisms: B. cereus, P. aeruginosa, Klebsiella pneumoniae, S. marcescens, S. aureus, and S. epidermidis.72 All examples of B. cereus, P. aeruginosa, K. pneumoniae, S. marcescens, and S. aureus had concentrations 102 CFU/mL by Day 3 following inoculation. By Day 4, all units with these organisms contained 105 CFU/mL. Units contaminated with S. epidermidis

Visual Inspection of Red Blood Cells to Detect Color Change It is known that in bacterially contaminated RBC units, the attached tubing segments almost invariably remain sterile even when the organism is cultured from the blood bag.9,10 An observation first noted by Kim et al and subsequently confirmed by multiple authors is a darkening of the color of unit as compared to the attached segments in additive solution RBC units.80-83 Compared with sterile units, the contaminated units became noticeably darker in color 1.5 to 2 weeks after the organism was

10 9

Figure 49-4. Growth curves of 4 units of AS-3 RBCs inoculated with Serratia liquefaciens (2 units inoculated to 4.2 CFU/mL) and Yersinia enterocolitica (2 units inoculated to 11.6 CFU/mL). Bacteria was not detectable by culture until Days 3 to 6. Isolates were from strains actually implicated in posttransfusion sepsis (the isolates were kindly provided by M. Arduino, Centers for Disease Control and Prevention, Atlanta, GA). Used with permission from Brecher et al.74

Log10 CFU/mL

8 7 6 Yersinia 1 Yersinia 2 Serratia 1 Serratia 2

5 4 3 2 1 0

5

10

15

20

25

30

35

Days of storage

14 12

Figure 49-5. Growth curves of six bacteria species (Serratia marcescens, n  7; Klebsiella pneumoniae, n  21; Staphylococcus epidermidis, n  21; Pseudomonas sp., n  15; Bacillus cereus, n  9; and Staphylococcus aureus, n  22) in 95 platelet units. All bacteria were inoculated on Day 0 at 10 to 50 CFU/mL. Used with permission from Brecher and Hay.7

Log10 CFU/mL

10 8 6 4 2 0

Serratia marcescens Bacillus cereus

Day 1

Day 2

Klebsiella pneumoniae Staphylococcus aureus

Day 3

Day 4

Staphylococcus epidermidis Pseudomonas

Day 5

Day 6

781

Section IV: Part II

detected in culture. This color change was very apparent when the contaminated units were compared to the attached segments of tubing, which did not darken. The sensitivity of visual identification of bacterial contamination in this manner is approximately 108 CFU/mL. One study found bacterial concentrations in the range of 1.8  104 to 1.6  109 CFU/mL at the time the whole blood units were first identified by visual inspection as potentially contaminated.83

Microscopic Examination with Gram’s Stain and Acridine Orange Microscopic examination has been used to detect bacterial contamination in both red cell components and platelets. Several authors have reported the use of the Gram’s stain for microscopic examination of blood components.84,85 One institution implemented a surveillance program that included pretransfusion Gram’s stains of platelet pools and apheresis units.80 In platelets aged 1 to 5 days, the sensitivity and specificity of this method were 80% and 96% to 99%, respectively. In 4- to 5-day-old platelets, the sensitivity increased to 100%, with a specificity of 99.3%. The true-positive results were associated with bacterial concentrations 106 CFU/mL. Based on their analysis, these authors concluded that the use of the pretransfusion Gram’s stain, combined with culture of 4- to 5-day-old units, was more cost-effective than discarding WBD platelets stored for 5 days. However, Barrett et al84 reported a higher rate of false positives. In their study, eight of 5334 platelet units yielded positive Gram’s stain results, six of which were negative by culture.84 Other investigators have reported on the use of acridine orange as a stain for bacteria.76,86 Surrogate Markers Brecher et al87 described a decrease in PO2 and an increase in PCO2 (with an associated decrease in pH) in platelets that supported the growth of S. epidermidis. The units, which had initial PO2 levels of 79 to 137 mmHg, often showed PO2 levels below 20 mmHg by the time the organisms reached a plateau concentration of 107 to 108 CFU/mL. Other groups have explored this method.88-91 Although this technique has the added advantage of being potentially noninvasive, it detects contamination only at high levels. Because platelets (and white cells) also produce carbon dioxide, this complicates attempts to detect bacterial contamination by measurements of this analyte. Because proliferating bacteria consume glucose and generate acid, several investigators have explored the use of dipsticks to indicate metabolic changes consistent with bacterial proliferation.92,93 Prospective surveillance of 3000 platelet concentrates at the M.D. Anderson Cancer Center in using reagent strips found 12 platelets concentrates with a glucose concentration or pH outside the reference range.94 Two of 12 platelet concentrates were found to be culture positive for B. cereus. The remaining 10 platelet concentrates did not demonstrate bacterial proliferation upon culture. Many users of WBD platelets employ the surrogate markers because culture would require a large proportion of the unit’s

782

volume. Unfortunately, the rate of detection of the surrogate markers is much less than bacterial culture. The 2004 survey of AABB institutions showed the true-positive rate for institutions using culture was 1 in 4028 compared to a true-positive rate of 1 in 18,535 for institutions using a nonculture method.59 This represents a 4.6-fold difference in the true-positive rate between culture and nonculture methods.

Endotoxin Assay Investigators have used the Limulus amebocyte lysate assay for detection of endotoxin in blood components.11,77 This method is limited to detection of endotoxin-producing organisms such as Y. enterocolitica. DNA/RNA Techniques Several amplification methods have been described for the detection of bacterial contamination. Schmidt et al95 reported on the use of RNA polymerase chain reaction (PCR) to detect S. aureus, E. coli, B. cereus, and K. pneumoniae in pooled platelet concentrates. At an inoculum of 10 CFU/mL, the PCR testing detected all four bacteria at 12, 16, 20, and 24 hours after spiking. With a lower inoculum of 10 CFU/bag, the PCR testing detected 60% of E. coli, 80% of B. cereus, 90% of K. pneumoniae, and 100% of S. aureus 12 hours after spiking. Another study compared DNA PCR with the automated culture system BacT/ALERT.96 A total of 2146 platelet concentrates were tested with both methods. When comparing to the culture method, the PCR had a sensitivity and specificity of 100%. To date, because of the complexities associated with such tests, nucleotide-based amplification has not been routinely applied to bacterial screening of platelets. One group described the use of a nonamplified chemiluminescence-linked universal bacterial rRNA probe.67,87 This method uses an acridinium ester-labeled single-stranded DNA probe complementary to highly conserved bacterial rRNA regions. Although a multicenter trial confirmed the effectiveness of this technique in detecting platelet samples contaminated with one of four bacterial species, the company developing this assay chose to not pursue further assay development. Rapid Immunoassay In September 2007, the FDA approved a new rapid immunoassay for the detection of aerobic and anaerobic bacteria in leukocytereduced apheresis platelets as an adjunct quality control device. The Platelet PGD Test (Verax Biomedical, Worcester, MA) is a qualitative immunoassay that differentiates between grampositive and gram-negative bacteria. As approved, it is intended to be an adjunct test after the use of an FDA-cleared bacterial culture method. The Platelet PGD Test can detect B. cereus, C. perfringens, E. aerogenes, E. coli, K. pneumoniae, P. aeruginosa, S. aureus, S. epidermidis, and S. agalactiae at a level 105 CFU/ mL and S. marcescens at a level of 8.6  105 CFU/mL. The test takes approximately 25 minutes to perform and is optimally performed after at least 72 hours of platelet storage. The system is approved only for leukocyte-reduced apheresis platelets and has not been validated in WBD platelets.

Chapter 49: Bacterial Contamination of Blood Products

Bacterial Culture There are currently two FDA-approved culture systems for bacteria detection in platelets in the United States. One uses an automated liquid culture system that includes broth bottles containing colorimetric sensors that change color as a consequence of increasing CO2 produced by bacterial proliferation. The system (BacT/ALERT) monitors both the rate of change of the colorimetric sensor and the absolute color change of the sensor. The bottles are inoculated with a needle, rendering the system not completely closed. This creates the possibility of introducing bacteria into the bottle. The method reliably detects contamination of platelets inoculated to 10 CFU/mL and in many cases 5 CFU/mL (eg, B. cereus, S. marcescens, C. perfringens, S. epidermidis, S. pyogenes, E. coli, K. pneumoniae, S. aureus, and viridans streptococci) in 12 to 26 hours. The ARC experience for 2004 through 2006 with the BacT/ ALERT testing of 1,004,206 apheresis platelets showed a truepositive rate of 30.3% of all initially positive cultures.45 A truepositive is defined as concordance between the initial culture and an additional confirmatory sample, a second 4 to 5 mL taken from the unit or its co-component. The false-positive rate was 57%, 32.4% caused by sampling contamination and 24.5%

caused by instrument signal error. The remainder of the initially positive cultures (12.7%) was called indeterminate, defined as the component not being available for confirmatory testing. In total, 612 donations had an initial positive culture result. Ninetyseven platelet units had been transfused at the time of the positive results; and only one of the 97 was confirmed positive, for coagulase-negative Staphylococcus. This unit was transfused on Day 2 and the patient did not have signs of sepsis. No septic reactions were noted with the units associated with an indeterminate or the false-positive culture. Blood Systems, another large collection system in the United States, also reported their first 2 years of experience with culturing apheresis platelets and WBD platelets.97 With the apheresis platelets, 198 of 122,971 collections initially tested positive and 21 were confirmed positive to give a confirmed positive rate of 1 in 5568. No confirmed positives were transfused. A total of 50 false-positive results were seen, caused by either machine reading error or contamination, and 26 cases were indeterminate. For WBD platelets, a total of 13,579 were cultured with 12 initial positives, one true positive, and 11 false positives. Data from ARC and Blood Systems are summarized in Table 49-9 and Fig 49-6.

Table 49-9. Summary of Experience with Bacterial Culture at ARC and Blood Systems* True Positive (%)

False Positive (%)

Indeterminate (%)

Gram positive Staphylococcus sp. Bacillus sp. Streptococcus sp. Diptheroids/Corynebacterium Other

77 (86.5) 43 (48.3) 7 (7.9) 21 (23.6) 1 (1.1) 5 (5.6)

200 (98.5) 53 (26.1) 117 (57.6) 5 (2.5) 10 (4.9) 15 (7.4)

59 (92.2) 15 (23.4) 28 (43.8) 3 (4.7) 8 (12.5) 5 (7.8)

Gram negative Fungal

12† (13.4) 0 (0)

2 (1.0) 1 (0.5)

2 (3.1) 3 (4.7)

Total

89 (100)

203 (100)

64 (100)

98

*Used with permission from Pietersz et al. † True-positive gram negatives included Serratia marcescens (n  4), Klebsiella sp. (n  3), Escherichia coli (n  3), Citrobacter diversus (n  1) and unspecified gram-negative rod (n  1).

True-positive organisms N
207 isolates

Figure 49-6. True-positive (confirmed) organisms, isolated from 1,237,177 apheresis cultures (tested with 4 mL in an aerobic BacT/ALERT bottle). Examples of organisms isolated only once or twice were grouped in the “other” category. These included example(s) of Micrococcus sp., Diptheroids/Corynebacterium, Enterococcus avium (n  2), Granulicatella adiacens, Citrobacter sp., Lactobacillus sp., and Enterobacter aerogenes. Used with permission from Pietersz et al.98

Serratia sp. 2.4% Klebsiella sp. 3.4% Escherichia coli 5.8% Bacillus sp. 5.3% Staphylococcus aureus 5.3%

Listeria sp. 1.9% Other 3.4%

Coagulase-negative Staphylococcus sp. 52.9%

Streptococcus sp. 19.4%

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Section IV: Part II

The second culture system is the enhanced Pall Bacterial Detection System (eBDS). The eBDS system measures the oxygen in the headspace gas of the sample pouch. Because the system measures oxygen consumption, it will not detect obligate anaerobes. One study reported on the use of the eBDS system with 118,067 apheresis and WBD units from 23 US blood centers.98,99 Investigators found 118 initially reactive (1 in 992) and 23 confirmed positive (1 in 5133) units. All true-positive units contained Staphylococcus spp. and Streptococcus spp. Ninety-five falsepositive units were identified (1 in 1243). Of the false positives, 18 were caused contamination and 56 had no bacteria present. There was one report of a missed detection with S. epidermidis. In a study by Fourneir-Wirth et al, 5 to 50 CFU/mL bacteria was inoculated into apheresis platelet units and tested in the eBDS.100 No false-positive results were seen. After 18 hours of incubation, 61 of 63 units tested positive and after 24 hours, all 63 tested positive. In the earlier incubation, two cases of B. cereus were missed.

with apheresis platelets collected with caridianBCT (Lakewood, CO) and Baxter (Deerfield, IL) equipment. This study allows centers to extend the expiration of these apheresis platelets to 7 days if the units are cultured with both an aerobic and an anaerobic bottle. The University of North Carolina recently reported its experiences with regard to inventory impact as a result of the extended shelf life.109 In total from March through August 2006, of the 1688 platelets transfused, 139 were older than 5 days. Since the implementation of 7-day platelets, the mean day of issue was 4.02 as compared to 3.44 before the extended shelf life. Additionally, the percent of outdating decreased from 2.8% to 1.3% with the extended shelf life. In the study period, 6 units were removed from inventory for an initial positive culture, none of which was confirmed. Only one allergic reaction was noted to a 7-day platelet, but no other adverse events were reported. Due to the higher than expected cluture positive rate with outdated platelets, the passport study has been suspended.

Sampling and Bottle Types for Culture The BacT/ALERT system can be used either with one aerobic bottle or with one aerobic and one anaerobic bottle. In 2004, approximately half of hospital blood banks used only the aerobic bottle, which is inoculated with 4 mL of the platelet product.59 Although most bacteria involved in septic transfusion reactions are aerobic, certain aerobic bacteria grow faster in the anaerobic bottle, most likely because of the difference in the nutrient broth.101-107 One study used a strain of Staphylococcus lugdunensis to perform a variety of spiking experiments to investigate the growth characteristics in the two types of bottles.108 The strain of S. lugdunensis was implicated in a fatal septic reaction to a platelet unit. Using a 4-mL inoculum, the anaerobic bottle became positive at a mean time of 18.73 hours as compared to the aerobic bottle at 21.07 (p
0.001). As the number of organisms inoculated into each bottle was increased, the difference in time to reactivity decreased (Fig 49-7). Because most centers use 4 mL per bottle, the use of an anaerobic bottle could reduce the time to detection and possibly prevent the transfusion of contaminated units. In 2005, the FDA approved the Post Approval Surveillance Study of Platelet Outcomes, Release Tested (PASSPORT) for use

Causes of False-Negative Results Even though great strides have been made in the prevention of platelet septic transfusion reactions, there are still missed cases. Missampling, micropunctures, leakage, postsampling handling, and contaminated tubes or container surfaces are all possible causes of contamination.110 WBD platelets have presented a problem because of the difficulty with culture, but with the introduction of the Acrodose prestorage pooling system, more contaminated WBD platelets can be interdicted before transfusion. With the recent FDA clearance of the Verax system, contaminated units that are culture negative might be interdicted immediately before transfusion (although this would be an offlabel use if used with WBD platelets). Reduced morbidity and mortality of platelet transfusion is also achieved with the optimal and correct use of blood components.47

Bacterial Elimination Filtration Leukocyte reduction of blood components is widely used in North American, Europe, Asia, and Australia. Several countries,

35 BPA

BPN

Hours

30

25

20

15

0

1

2

3

4

5

6 mLs

784

7

8

9

10

Figure 49-7. Time to reactivity of the aerobic (BPA) and the anaerobic (BPN) bottles inoculated at 1.5 CFU/mL with increasing volume. At lower inocula, the reactive BPN bottles are faster than the BPA bottles; at higher inocula, the time to 11 reactivity were equivalent. Used with permission from Brecher and Hay.108

Chapter 49: Bacterial Contamination of Blood Products

including Canada, England, Portugal, France, Ireland, and Norway, have switched to a completely leukocyte-reduced blood supply (so-called “universal leukocyte reduction”).111,112 Because of concern regarding the accumulation of cytokines, leukocyte and platelet breakdown products, and possibly immunogenic white cell fragments in stored blood, emphasis has been placed on prestorage rather than poststorage leukocyte reduction.113-115 A theoretic risk of prestorage filtration is that early removal of the phagocytic leukocytes, which would normally remove low levels of bacteria present in these products, might lead to an increase in bacterial contamination of blood components and septic complications. In the case of Y. enterocolitica (the organism that has been most extensively studied), prestorage leukocyte reduction by filtration actually is associated with a decrease in bacterial growth in inoculated RBCs.4,76,77,116-118 (Table 49-10). The mechanism by which leukocyte reduction removes bacteria is multifactorial. Bacteria that have been phagocytosed but not killed are removed with the white cells. Alternatively, organisms may be adsorbed to leukocytes or activated by complement, to then be bound indirectly to the charged filter fibers, or the bacteria may directly adhere to the filter fibers. AuBuchon and Pickard showed with a panel of bacterial organisms, differences in the affinity for bacteria by direct adherence of red cell leukocyte reduction filters and platelet leukocyte reduction filters.119 In general, red cell reduction filters are most efficient, removing 88% to 100% of organisms, while platelet filters remove only 67% to 97% of organisms. Unlike the decrease in Y. enterocolitica growth seen with filtered, prestorage leukocyte-reduced red cells, leukocyte reduction by filtration failed to decrease bacterial growth in platelets spiked with bacteria.120-121

Prolonged Room Temperature Hold of Red Blood Cells When whole blood is placed immediately at 4ºC, bacterial growth occurs more rapidly than if held at 10ºC for 24 hours before 4ºC storage.123 Bacterial growth of most pathogens is inhibited by cold temperatures, but host mechanisms present in fresh whole

blood are also inhibited at 4ºC. With plasmid-encoded complement resistance, Yersinia can be complement sensitive when blood is transiently stored at 20ºC.73 Because phagocytosis and complement activation are impaired at 4ºC, it may be advantageous to allow red cells to remain in contact with plasma for several hours before separation and storage of components.

Pathogen Inactivation Several techniques to inactivate pathogens in blood components using chemicals are under active investigation or are implemented in select countries (see Chapter 51).

International Comparison There is wide variation in the way different countries approach the problem of bacterial contamination of platelets. A recent survey of 12 different countries reveals the many approaches to preventing septic transfusion reactions.98 The responses are summarized in Table 49-11.

Transfusion-Transmitted Syphilis Treponema pallidum is a thin-walled, motile, spiral gram-negative rod or spirochete that cannot be visualized with Gram’s stain and does not grow on bacteriologic media or cell culture. Although it is a bacterium, it is often treated as a distinct entity, different from other transfusion-transmitted bacterial organisms and thus is addressed separately. Only 25% of patients with primary syphilis have a reactive serologic test for syphilis and the test does not become routinely positive until the fourth week after the onset of symptoms; therefore, donors infected with T. pallidum may be asymptomatic with negative serology during periods of spirochetemia.124,125 Although the organism does not survive prolonged storage at 4ºC, it may live for 1 to 5 days at these cold temperatures.126,127 Therefore, a rare infection may be associated with transfusion of a fresh RBC unit from a donor who was in the seronegative phase at the time of

Table 49-10. Selected Studies of Yersinia enterocolitica Growth and Prestorage Leukocyte Reduction by Filtration Inoculating Concentration CFU/mL

Filtered Growth/Total (%)

Control Growth/Total (%)

Filter Type

p Value*

Reference

100 65

0/10 (0) 2/10 (20)

10/10 (100) 8/8 (100)


0.001 0.002

Brecher et al104 Brecher et al72

10/150 0.3-132 20-30,000 1.5

3/8 (37) 3/24 (12) 6/30 (20) 2/6 (33)

8/8 (100) 16/24 (67) 22/30 (70) 6/6 (100)

Sepacell PL-5N Leukotrap and Leukotrap RC (Pall RC300) Pall BPF4 Sepacell R-500 Cellselect, NPBI Leukotrap RC (Pall RC300)

0.03
0.001
0.001 0.06

Brecher et al102 Hoppe73 Brecher et al105 Brecher et al103

Total

16/88 (18)

70/86 (81)


0.001

* Two-sided Fisher’s exact test.

785

Country

Routine Screening for Bacterial Contamination

Method Used for Screening

Australia

5% of platelet concentrates (PCs) are tested; 100% tested beginning in April 2008

Predicted that outdating will increase 1-5% with 100% testing, BacT/ALERT; cultures taken 24 hours after collection for apheresis using a 24-hour hold before sampling platelets and 48 hours after for pooled platelets; both aerobic and anaerobic bottles

Austria

Approximately 5% of apheresis platelets are tested for quality control (QC)

BacT/ALERT; both aerobic and anaerobic bottles

No effect because testing duration is 7 days and platelet shelf life is 5 days

Canada

Approximately 98% of apheresis platelets and 4-unit buffy coat (BC) platelet pools are tested; once all whole-blood derived units are BC, all will be tested

BacT/ALERT; aerobic bottle only; tested 24-30 hours after collection

No effect because no quarantine after the culture sample is drawn

Denmark

Currently, 2/3 of PCs are tested; the National Board of Health will hopefully recommend that all be tested in the near future

BacT/ALERT or corresponding culture methods; sampled 3-30 hours Shelf life increased from 5 to 7 days with culture; outdating after collection of whole blood (0-24 hours after pooling of BC or decreased from 15% to 5% collection of apheresis units)

France*

None

Not applicable

Not applicable

Germany

Done for QC only

BacT/ALERT with both aerobic and anaerobic bottles

Not applicable

Ireland

On all platelets since 2004

BacT/ALERT; for platelet pools—platelets incubated 36 hours before Modest improvement in logistics because of retesting at Day 4 of culture; for apheresis platelets—incubated 12 hours before platelets collected on caridianBCT Trima and then shelf life culture; use both aerobic and anaerobic extended 2 days

Japan

No, platelet shelf life only 72 hours

Not applicable

Not applicable

Netherlands

On all platelets since 2001

BacT/ALERT; both aerobic and anaerobic bottles; sampled at preparation and issued as “negative to date” as soon as culture begins

Outdating decreased because of extension of shelf life from 5 to 7 days

Norway

On all platelets at major hospitals since 1998

BacT/ALERT using only aerobic bottles sampled the day after culture Outdating decreased from 18% to 11% based on extending shelf of the starting material (either whole-blood-derived or apheresis life to 6.5 days platelets); no quarantine after culture

United Kingdom In England, only for QC or tested in order to extend the shelf life over holidays; in Scotland, Wales, and in Northern Ireland all PCs are routinely tested

If used to extend shelf life: sampled on Day 3 into a BacT/ALERT aerobic bottle and quarantined for 48 hours

Impact of Testing on Logistics and Outdating

Extending the shelf life has assisted with supply over extended holidays

If used in retrospective national monitoring study: BacT/ALERT aerobic and anaerobic bottles are inoculated and incubated for 7 days United States

No governmental mandate but required by AABB and CAP

Two systems FDA approved and available for testing: BacT/ALERT, No or modest impact on availability as well as no increase in eBDS, all units sampled after 24-36 hour hold; whole blood outdating derived platelets tested with surrogate markers such as glucose or pH

* Pathogen inactivation is being used on Reunion Island because of the risk of chikungunya transmission; being studied in other French locations and will be implemented gradually.

Section IV: Part II

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Table 49-11. Comparison of Methods Used in Different Countries to Address the Bacterial Contamination of Platelets98

Chapter 49: Bacterial Contamination of Blood Products

donation. Platelets stored at 20 to 24ºC, provide a more hospitable temperature for T. pallidum; however, this organism does not thrive with the high oxygen tension in modern platelet storage bags. Since 1969, only three cases of transfusion-transmitted syphilis have been reported in the literature.128-130 The extremely low rate of transfusion-transmitted syphilis infection likely results from 1) donor questioning targeting high-risk behavior; 2) the cardiolipin-based assay, although an insensitive test in the acute postinfectious setting, does pick up a number of infected donors; 3) refrigerator storage, which results in the death of spirochetes; 4) antibiotics given to many patients at the time of platelet transfusion, which would be bactericidal for any viable organisms; and 5) donors excluded for a positive test for HIV, HCV, or hepatitis B because of the high correlation between infection with T. pallidum and viruses such as these, even though the donors may have been in the seronegative phase of syphilis at the time of donation.131-136 Because syphilis testing plays only a minor role in protecting the blood supply and is associated with a high degree of false-positive reactions, elimination of syphilis testing has been advocated. The counter argument is that such testing provides a surrogate marker for individuals at risk of other sexually transmitted diseases and, therefore, should be retained.

Transfusion-Transmitted Lyme Disease Lyme disease, a tick-borne illness, caused by the spirochete Borrelia burgdorferi is theoretically transmittable through blood, although no reported human transfusion-transmitted cases have been identified.137 The tick that carries B. burgdorferi, Ixodes scapularis, also carries Babesia microti and Anaplasma phagocytophilum, both of which are transmissible by transfusion. In a murine model of Lyme disease, approximately 50% of mice transfused blood from a spirochetemic donor mouse became infected.138 Although there have been cases of blood donors being diagnosed with Lyme disease shortly after their donation, no spirochetes could be identified in the recipient of the units.139 This might be unrecognized because of vague symptoms of Lyme disease as well as the lack of characteristic erythema migrans at the site of tick bite.137,140 Additionally, spirochetes are rarely identified even in patients with active disease, which may in turn limit the transmission through transfusion.139

Conclusion Bacterial contamination of blood components is the most common cause of transfusion-transmitted infectious disease. Most cases of posttransfusion sepsis involve platelets that must be stored at room temperature. With the introduction of diversion techniques and bacteria detection of platelets, the rate of clinically significant septic reaction has substantially decreased and the availability of platelets to patients has been enhanced.

Disclaimer Y. Park has disclosed no conflicts of interest. M. Brecher has disclosed relationships with bioMérienx, Pall, Fenwal, Verax and Cerus.

References 1. Bacterial contamination of blood components. Association Bulletin #96-6. Bethesda, MD: AABB, 1996. 2. Niu MT, Knippen M, Simmons L, et al. Transfusion-transmitted Klebsiella pneumoniae fatalities, 1995 to 2004. Transfus Med Rev 2006;20:149-57. 3. Kuehnert MJ, Roth VR, Haley NR, et al. Transfusion-transmitted bacterial infection in the United States, 1998 through 2000. Transfusion 2001,41:1493-9. 4. Theakston EP, Morris AJ, Streat SJ, et al. Transfusion transmitted Yersinia enterocolitica infection in New Zealand. Aust N Z Med 1997;27:62-7. 5. Perez P, Salmi LR, Follea G, et al. Determinants of transfusion-associated bacterial contamination: Results of the French BACTHEM case-control study. Transfusion 2001,41:862-71. 6. Serious Hazards of Transfusion (SHOT) report for 2004-2005. Manchester, UK: SHOT Office, 2005. [Available at http://www.shotuk.org/home.htm.] 7. Brecher ME, Hay SN. Bacterial contamination of blood components. Clin Microbiol Rev 2005;18:195-204. 8. Update: Yersinia enterocolitica bacteremia and endotoxin shock associated with red blood cell transfusion—United States, 1991. MMWR Morb Mortal Wkly Rep 1991;40:176-8. 9. Tipple MA, Bland LA, Murphy JJ, et al. Sepsis associated with transfusion of red cells contaminated with Yersinia enterocolitica. Transfusion 1990;30:207-13. 10. Red blood cell transfusions contaminated with Yersinia enterocolitica—United States, 1991-1996, and initiation of a national study to detect bacteria-associated transfusion reactions. MMWR Morb Mortal Wkly Rep 1997;20;46:553-5. 11. Arduino MJ, Bland LA, Tipple MA, et al. Growth and endotoxin production of Yersinia enterocolitica and Enterobacter agglomerans in packed erythrocytes. J Clin Microbiol 1989;27:1483-5. 12. Hogman CF, Fritz H, Sandberg L. Post-transfusion Serratia marcescens septicemia (editorial). Transfusion 1993;33:189-91. 13. Heltberg O, Show F, Gerner-Smidt P et al. Nosocomial epidemic of Serratia marcescens septicemia ascribed to contaminated blood transfusion bags. Transfusion 1993;33:221-7. 14. Szewzyk U, Szewzyk R, Stenstrom TA. Growth and survival of Serratia marcescens under aerobic and anaerobic conditions in the presence of materials from blood bags. J Clin Microbiol 1993;31:1826-30. 15. Richards C, Kolins J, Trindale CD. Autologous transfusion-transmitted Yersinia enterocolitica (letter). JAMA 1992;268:1541-2. 16. Sire JM, Michelet C, Mesnard R et al. Septic shock due to Yersinia enterocolitica after autologous transfusion (letter). Clin Infect Dis 1993;17:954-5. 17. Haditsch M, Binder L, Gabriel C et al. Yersinia enterocolitica septicemia in autologous blood transfusion. Transfusion 1994;34: 907-9.

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18. Benavides A, Nicol K, Koranyi K, et al. Yersinia septic shock following an autologous transfusion in a pediatric patient. Tranfus Apher Sci 2003;28:19-23. 19. Duncan KL, Ransley J, Elterman M. Transfusion-transmitted Serratia liquifaciens from an autologous blood unit (letter). Transfusion 1994;34:738-9. 20. Rhame FS, McCullough JJ, Cameron S, et al. Pseudomonas cepacia infections caused by thawing cryoprecipitate in a contaminated water bath (abstract). Transfusion 1979;19:653. 21. Casewell MW, Slater NGP, Cooper JE. Operating theatre waterbaths as a cause of Pseudomonas septicemia. J Hosp Infect 1981;2: 237-40. 22. McClelland DB. Safety of human albumin as a constituent of biologic therapeutic products. Transfusion 1998;38:690-9. 23. Albumin recall. Lancet 1996;348:1026. 24. Steere AC, Tenney JH, Mackel DC, et al. Pseudomonas species bacteremia caused by contaminated normal human serum albumin. J Infect Dis 1977;135:729-3. 25. Wang SA. Tokars JI. Bianchine PJ, et al. Enterobacter cloacae bloodstream infections traced to contaminated human albumin. Clini Infect Dis 2000;30:35-40. 26. Klein HG, Dodd RY, Ness PM, et al. Current status of microbial contamination of blood components: Summary of a conference. Transfusion 1997;37:95-101. 27. Halpin TJ, Kilker S, Epstein J, et al. Bacterial contamination of platelet pools—Ohio, 1991. MMWR Morb Mortal Wkly Rep 1992;41:36-7. 28. Blajchman MA, Thornley J, Richardson H, et al. Platelet transfusioninduced Serratia marcescens sepsis due to vacuum tube contamination. Transfusion 1979;19:39-44. 29. Engstrand M, Engstrand L, Hogman CF, et al. Retrograde transmission of Proteus mirabilis during platelet transfusion and the use of arbitrarily primed polymerase chain reaction for bacteria typing in suspected cases of transfusion transmission of infection. Transfusion 1995;35:871-3. 30. Morro JF, Braine HG, Kickler TS, et al. Septic reactions to platelet transfusions: A persistent problem. JAMA 1991;266:555-8. 31. Krishnan LS, Brecher ME. Transfusion transmitted bacterial infection. Hematol Oncol Clini of North Am 1995;9:167-85. 32. Chiu EKW, Yuien KY, Lie AKW, et al. A prosective study of symptomatic bacteremia following platelet transfusion and its managment. Transfusion 1994;34:950-4. 33. Claeys H, Verhaegle B. Bacterial screening of platelets (abstract). Vox Sang 2000;78(Suppl 1):374. 34. Schelstaete B, Bijnens B, Wuyts G. Prevalence of bacteria in leucodepleted pooled platelet concentrates and apheresis platelets: A 3-year experience (abstract). Vox Sang 2000;78(Suppl 1):373. 35. Wendel S, Fontao-Wew Orleansendel R, Germano S, et al. Screening of bacteria for blood component production in a Brazilian blood bank (abstract). Vox Sang 2000;78(Suppl 1):376. 36. Leiby DA, Kerr KL, Campos JM, et al. A retrospective analysis of microbial contaminants in outdated random-donor platelets from multiple sites. Transfusion 1997;37:259-63. 37. Dykstra A, Jacobs M, Yomtovian R. Prospective microbiologic surveillance (PMS) of random donor (RDP) and single donor apheresis (SDP) platelets (abstract). Transfusion 1998;38(Suppl): 104S. 38. Blajchman MA, Ali A, Lyn P, et al. Bacterial surveillance of platelets concentrates: Quantitation of bacterial load (abstract). Transfusion 1997;37(suppl):74S.

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39. Department of Health and Human Services. The 2007 National Blood Collection and Utilization Survey Report. Washington, DC: DHHS, 2008. 40. Ness P, Braine H, King K, et al. Single-donor platelets reduce the risk of septic platelet transfusion reactions. Transfusion 2001;18:11-24. 41. Engelfriet CP, Reesink HW, Blajchman MA, et al. Bacterial contamination of blood components. Vox Sang 2000;78:59-67. 42. Shulman, IA. College of American Pathologists Laboratory Accreditation checklist item TRM.44955. Arch Pathol Lab Med 2004;128:958-63. 43. Silva MA, ed. Standards for blood banks and transfusion services. 24th ed. Bethesda,MD: AABB, 2006. 44. Fang CT, Chambers LA, Kennedy J, et al. Detection of bacterial contamination in apheresis platelet products: American Red Cross experience, 2004. Transfusion 2005;45:1845-52. 45. Eder AF, Kennedy JM, Dy BA, et al. Bacterial screening of apheresis platelets and the residual risk of septic transfusion reactions: The American Red Cross experience (2004-2006). Transfusion 2007;47:1134- 42. 46. Palavecino EL, Yomtovian RA, Jacobs MR. Detecting bacterial contamination in platelet products. Clin Lab 2006;52:443-56. 47. Yomtovian R, Tomasulo P, Jacobs MR. Platelet bacterial contamination: Assessing progress and identifying quandaries in a rapidly evolving field. Transfusion 2007; 47:1340-6. 48. Grossman BJ, Kollins P, Lau PM, et al. Screening blood donors for gastrointestinal illness: A strategy to eliminate carriers of Yersinia entercolitica. Transfusion 1991;31:500-1. 49. Gibson T, Norris W. Skin fragments removed by injection needles. Lancet 1958;ii:983-5. 50. Lilly HA, Lowbury EJL, Wilkins MD. Limits to progressive reduction of resident skin bacteria by disinfecion. J Clin Pathol 1979;32:382-5. 51. Anderson KC, Lew MA, Gorgone BC, et al. Transfusion-related sepsis after prolonged platelet storage. Am J Med 1986;81:405-11. 52. Goldman M, Roy G, Frechette N, et al. Evaluation of donor skin disinfection methods. Transfusion 1997;37:309-12 53. McDonald CP, Lowe P, Roy A, et al. Evaluation of donor arm disinfection techniques. Vox Sang 2001;80:135-41. 54. Olthuis H, Putlaert C, Verhagen C, et al. A simple method to remove contaminating bacteria during venipuncture (abstract). Vox Sang 1996;70:(Suppl):113. 55. de Korte D, Vlaar R, Marcelis J, et al. Reduction of the degree of bacterial contamination for whole blood collections (abstract). Transfusion 2000;40 (Suppl):36S. 56. Bruneau C, Perez P, Chassaigne M, et al. Efficacy of a new collection procedure for preventing bacterial contamination of whole-blood donations. Transfusion 2001;41:74-81. 57. DeKorte D, Marcelis JH, Verhoeven AJ, et al. Diversion of first blood volume results in a reduction of bacterial contamination for wholeblood collections. Vox Sang 2002;83:13-16. 58. Ness PM, Braine HG, Barrasso C, et al. Single donor platelets reduce the risk of septic transfusion reactions (abstract). Transfusion 1999;39(Suppl):89S. 59. Silva MA, Gregory KR, Carr-Greer MA et al. Summary of the AABB Interorganizational Task Force on Bacterial Contamination of Platelets: Fall 2004 impact survey. Transfusion 2006;46:636-41. 60. Schrezenmeier H, Walther-Wenke G, Müller TH, et al. Bacterial contamination of platelet concentrates: Results of a prospective multicenter study comparing pooled whole blood-derived platelets and apheresis platelets. Transfusion 2007;47:644-52.

Chapter 49: Bacterial Contamination of Blood Products

61. CCBC. FDA committee endorses education and research to combat rare bacterial reaction: Rejects operational changes for now. CCBC Newsletter 1991;May 10:1-4. 62. AABB. FDA Blood Products Advisory Committee supports educational efforts on Yersinia enterocolitica. Blood Bank Week 1991;20:1-3. 63. Jensenius M, Hoel T, Heier HE. Yersinia enterocolitica septicemia after blood transfusion. Tidsskr Nor Laegeforen 1995;115:940-2. 64. Jacobs J, Jamaer D, Vandeven J, et al. Yersinia enterocolitica in donor blood: A case report and review. J Clin Microbiol 1989;27:1119-21. 65. Donegan E, Lenes BA, Tomasulo PA, et al. Transmission of HIV-1 by component type and duration of shelf storage before transfusion (letter). Transfusion 1991;30:851-2. 66. Donegan E, Lee H, Operskalski EA, et al. Transfusion transmission of retroviruses: Human T-lymphotropic virus types I and II compared with human immunodeficiency virus type 1. Transfusion 1994;34:478-83. 67. Brecher NE, Hogan JJ, Boothe G, et al. The use of a chemiluminescence-linked universal bacterial ribosomal RNA gene probe and blood gas analysis for the rapid detection of bacterial contamination in white cell reduced and nonreduced platelets. Transfusion 1993;33:450-7. 68. Rhame FS, Root RK, MacLowry JD, et al. Salmonella septicemia from platelet transfusions. Ann Intern Med 1973;78:633-41. 69. Update: Delayed onset Pseudomonas fluorescens bloodstream infections after exposure to contaminated heparin flushes—Michigan and South Dakota, 2005-2006. MMWR Morb Mortal Wkly Rep 2006;55:961-3. 70. Connor J, Currie LM, Allan H, et al. Recovery of in vitro functional activity of platelet concentrates stored at 4oC and treated with second-messenger effectors. Transfusion 1996;36:691-8. 71. Currie LM, Harper JR, Allan H, et al. Inhibition of cytokine accumulation and bacterial growth during storage of platelet concentrates at 4 degrees C with retention of in vitro functional activity. Transfusion 1997;37:18-24. 72. Brecher ME, Holland PV, Pineda A, et al. Bacterial growth in inoculated platelets: Implications for bacterial detection and the extension of platelet storage. Transfusion 2000;40:1308-12. 73. Hoppe PA. Interim measures for detection of bacterially contaminated red cell components (editorial). Transfusion 1992;32:199-201. 74. Brecher ME, Foster M, Mair D. Glucose and haemolysis as a rapid soreen for contamination of red blood cells with Yersinia and Serratia. Vox Sang 2001;81:136-8. 75. Brecher ME, Holland PV, Pineda A, et al. Growth of bacteria in inoculated platelets: Implications for bacterial detection and the extension of platelet storage. Transfusion 2000;34:750-5. 76. Kim DM, Brecher ME, Bland LA, et al. Prestorage removal of Yersinia enterocolitica from red cells with white cell-reduction filters. Transfusion 1992;32:658-62. 77. Buchholz DH, AuBuchon JP, Snyder EL, et al. Removal of Yersinia enterocolitica from AS-1 red cells. Transfusion 1992;32:667-72. 78. Gibb AP, Martin Km, Davidson GA, et al. Modeling the growth of Yersinia enterocolitica in donated blood. Transfusion 1994;34:304-10. 79. Pietersz RNI, Reesink HW, Pauw W, et al. Prevention of Yersinia enterocolitica growth in red blood cell concentrates. Lancet 1992;340:755-6. 80. Kim DM, Brecher ME, Bland LA, et al. Visual identification of bacterially contaminated red cells. Transfusion 1992;32:221-5. 81. Franzin L, Gioannini P. Growth of Yersinia species in artificially contaminated blood bags. Transfusion 1992;32:673-6. 82. Bradley RM, Gander RM, Patel SK, et al: Inhibitory effect of 0ºC storage on the proliferation of Yersinia enterocolitica in donated blood. Transfusion 1992;37:691-5.

83. Pickard C, Herschel L, Seery P, et al. Visual identification of bacterially contaminated red blood cells (abstract). Transfusion 1998;38(Suppl):12S. 84. Barrett BB, Andersen JW, Anderson KC. Strategies for the avoidance of bacterial contamination of blood components. Transfusion 1993;33:228-33. 85. Yomtovian R, Lazarus HM, Goodnough LT, et al. A prospective microbiologic surveillance program to detect and prevent the transfusion of bacterially contaminated platelets. Transfusion 1993;33: 902-9. 86. Chongokolwatana V, Morgan M, Feagin JC, et al. Comparison of microscopy and a bacterial DNA probe for detecting bacterially contaminated platelets (abstract). Transfusion 1993;33(Suppl):50S. 87. Brecher ME, Boothe G, Kerr A. The use of chemiluminescencelinked universal bacterial ribosomal RNA gene probe and blood gas analysis for the rapid detection of bacterial contamination in white cell-reduced and nonreduced platelets. Transfusion 1993;33:450-7. 88. Arpi M, Bremmelgaard A, Abel Y, et al. A novel screening method for the detection of microbial contamination of platelet concentrates: An experimental pilot study. Vox Sang 1993;65:335-6. 89. Hogman CF, Gong J. Studies of one invasive and two noninvasive methods for detection of bacterial contamination of platelet concentrates. Vox Sang 1994;67:351-5. 90. Cortus M, Chong, Carmen R, Wenz B. A new system to detect bacterial contamination in platelet concentrates (abstract). Transfusion 2000;440(Suppl):36S. 91. Wenz B, Delgiacco G, Cortus MA, et al. A system designed to detect bacterial contamination in platelet concentrates (abstract). Vox Sang 2000;78 (Suppl):65. 92. Wagner S, Robinette D. Evaluation of swirling, pH, and glucose tests for the detection of bacterial contamination in platelet concentrates. Transfusion 1996;36:989-93. 93. Burstain JM, Brecher ME, Workman K, et al. Rapid identification of bacterially contaminated platelets using reagent strips: Glucose and pH analysis as markers of bacterial metabolism. Transfusion 1997;37:255-8. 94. Mhawech PY, Werch J, Stager C, et al. Detecting bacterial contamination in platelet concentrates using reagent strips-application in a major cancer center blood bank (abstract). Transfusion 1999;39 (Suppl):36S-37S. 95. Schmidt M, Hourfar MK, Nico SB, et al. A comparison of three rapid bacterial detection methods under simulated real-life conditions. Transfusion 2006;46:1367-73. 96. Mohammadi T, Pietersz RNI, Vandenbroucke-Grauls C, et al. Detection of bacteria in platelet concentrates: Comparison of broad-range real-time 16S rDNA polymerase chain reaction and automated culturing. Transfusion 2005;45:731-6. 97. Kleinman SH, Kamel HT, Harpool DR, et al. Two-year experience with aerobic culturing of apheresis and whole blood-derived platelets. Transfusion 2006;46:1787-94. 98. Pietersz RNI, Engelfreit CP, Reesink HW, et al. Detection of bacterial contamination of platelet concentrates. Vox Sang 2007;93: 260-77. 99. Holme S, Bunch C, Selman B. Bacterial contamination in stored platelets: Performance of the Pall eBDS system under routine use conditions (abstract). Vox Sang 2005;89(Suppl 1):194. 100. Fournier-Wirth C, Deschaseaux M, Defer C, et al. Evaluation of the enhanced bacterial detection system for screening of contaminated platelets. Transfusion 2006;46:220-4.

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101. Brecher MR, Hay SN, Rose AD, et al. Evaluation of BacT/Alert plastic culture bottles for use in testing whole blood-derived leukoreduced platelet-rich-plasma platelets with a single contaminated unit. Transfusion 2005;45:1512-17. 102. Brecher ME, Hay SN, Rothenberg SJ. Validation of BacT/Alert plastic culture bottles for use in testing whole blood-derived leukoreduced platelet-rich-plasma-derived platelets. Transfusion 2004;44:1174-8. 103. Brecher ME, Hay SN, Rothenberg SJ. Evaluation of a new generation of plastic culture bottles with an automated microbial detection system for nine common contaminating organism found in PLT components. Transfusion 2004;44:359-63. 104. Brecher ME, Hay SN, Rothenberg SJ. Monitoring of apheresis platelet bacterial contamination with an automated liquid culture system: A university experience. Transfusion 2003;43:974-8. 105. Brecher ME, Heath DG, Hay SN, et al. Evaluation of a new generation of culture bottle using an automated bacterial culture system for detecting nine common contaminating organism found in platelet components. Transfusion 2002;42:774-9. 106. Brecher ME, Means N, Jere CS, et al. Evaluation of an automated culture system for detecting bacterial contamination of platelets: An analysis with 15 contaminating organism. Transfusion 2001;41:477-82. 107. McDonald CP, Hartley S, Orchard K, et al. Evaluation of the 3D BacT/Alert automated culture system for the detection of microbial contamination of platelet concentrates. Transfus Med 2002;12:303-9. 108. Brecher ME, Hay SN. Investigation of an isolate of Staphylococcus lugdunensis implicated in a platelet fatality: A possible advantage to the use of an anaerobic bottle. Transfusion 2007;47:1390-4. 109. Hay SN, Immel CC, McClannan LS, et al. The introduction of 7-day platelets: A univeristy hospital experience. J Clin Apher 2007;22:283-6. 110. Beckers EAM. Effects of bacterial testing: What risks are remaining? ISBT Science Series 2007;2:30-4. 111. Dzik S, AuBuchon J, Jeffries L, et al. Leukocyte reduction of blood components: Public policy and new technology. Transfus Med Rev. 2000;14:34-52. 112. Leukocyte reduction. Association Bulletin 99-7. Bethesda, MD: AABB,1999. 113. Blajchman MA. The effect of leukodepletion on allogenic donor platelet survival and refractoriness in an animal model. Semin Hematol 1991;28(Suppl):14-17. 114. Brecher ME, Pineda AA, Torloni AS, et al. Prestorage leukocyte depletion: Effect on leukocyte and platelet metabolites, erythrocyte lysis, metabolism and in vivo survival. Semin Hematol 1991;28(Suppl):3-9. 115. Heddle NM, Klama L, Singer J, et al. The role of the plasma from platelet concentrates in transfusion reactions. N Engl J Med 1994;331:625-8. 116. Wenz B, Burns ER, Freundlich LF. Prevention of growth of Yersinia enterocolitica in blood by polyester fiber filtration. Transfusion 1992;32:663-6. 117. Kim DM, Estes TJ, Brecher ME, et al. WBC filtration, blood gas analysis and plasma hemoglobin in Yersinia enterocolitica contaminated red cells (abstract). Transfusion 1992;32(Suppl):41S. 118. Hogman CF, Gong J, Hambraeus A, et al. The role of white cells in the transmission of Yersinia enterocolitica in blood products. Transfusion 1992;32:654-7. 119. AuBuchon JP, Pickard C. White cell reduction and bacterial proliferation (letter). Transfusion 1993;33:533-4. 120. Buchholz DH, AuBuchon JP, Snyder EL, et al. Effects of white cell reduction on the resistance of blood components to bacterial multiplication. Transfusion 1994;34:852-7.

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121. Sherburne B, McCullough A, Dzik WH, et al. Bacterial proliferation in platelet concentrates is unaffected by pre-storage leukocyte depletion (abstract). Blood 1991;78(Suppl):350a. 122. Wenz B, Ciavarella D, Freundlich L. Effect of prestorage white cell reduction on bacterial growth in platelet concentrates. Transfusion 1993;33:520-3. 123. Pietersz RNI, Reesink HW, Dekker MA, et al. Elimination of Yersinia enterocolitica by a 20h hold of whole blood and removal of leukocytes by filtration (abstract). Transfusion 1992;32(Suppl):253S. 124. Seidl S. Syphilis screening in the 1990s. Transfusion 1990;30:773-4. 125. Spangler AS, Jackson JH, Fiumara NJ, et al. Syphilis with a negative blood test reaction. JAMA 1964;189:87-90. 126. Van der Sluis JJ, Onvlee PC, Kothe FCHA, et al. Transfusion syphilis, survival of Treponema pallidum in donor blood I. Report of an orientating study. Vox Sang 1984;47:197-204. 127. Van der Sluis JJ, ten Kate FJW, Vuzevski VD, et al. Transfusion syphilis, survival of Treponema pallidum in donor blood II. Dose dependence of experimentally determined survival times. Vox Sang 1985;49:390-9. 128. Soendjojo A, Boedisantoso M, Ilias MI, et al. Syphilis d’emblee due to a blood transfusion. Br J Venereal Dis 1982;58:149-50. 129. Risseeuw-Appel IM, and Kothe FC. Transfusion syphilis: A case report. Sex Transm Dis 1983;10:200-1. 130. Chambers RW, Foley HT, Schmidt PJ. Transfusion of syphilis by fresh blood components. Transfusion 1969;9:32-4. 131. Quinn TC, Cannon RO, Glasser D, et al. The association of syphilis with risk of human immunodeficiency virus infection in patients attending sexually transmitted disease clinics. Arch Intern Med 1990;150:1297-302. 132. Nelson KE, Vlahov D, Cohn S, et al. Sexually transmitted diseases in a population of intravenous drug users: Association with seropositivity to the human immunodeficiency virus (HIV). J Infect Dis 1991;164:457-63. 133. Potterat JJ. Does syphilis facilitate sexual acquisition of HIV? (letter) JAMA 1987;258:473. 134. Otten MW Jr, Zaidi AA, Peterman TA, et al. High rate of HIV seroconversion among patients attending urban sexually transmitted disease clinics. AIDS 1994;8:549-53. 135. Rosenblum L, Darrow W, Witte J, et al. Sexual practices in the transmission of hepatitis B virus and prevalence of hepatitis delta virus infection in female prostitutes in the United States. JAMA 1992;267:2477-81. 136. Thomas DL, Cannon RO, Shapiro CN, et al. Hepatitis C, hepatitis B, and human immunodeficiency virus infections among nonintravenous drug-using patients attending clinics for sexually transmitted diseases. J Infect Dis 1994;169:990-5. 137. Cable RG, Leiby DA. Risk and prevention of transfusiontransmitted babesiosis and other tick-borne diseases. Curr Opin Hematol 2003;10:405-11. 138. Gabitzsch ES, Piesman J, Dolan MC, et al. Transfer of Borrelia burgdorferi s.s. infection via blood transfusion in a murine model. J Parasitol 2006;92:869-70. 139. Cable R, Krause P, Badon S, et al. Acute blood donor co-infection with Babesia microti and Borrelia burgdorferi (abstract). Transfusion 1993;33(Suppl):50S. 140. Cable RG, Trouern-Trend J. Tick-borne infections. In: Linden JV, Bianco C, eds. Blood safety and surveillance. New York: Marcel Dekker, 2001:399-422.

50

Prion Diseases Marc L. Turner Professor of Cellular Therapy, University of Edinburgh, and Clinical Director/Consultant Haematologist, Edinburgh Blood Transfusion Centre, Royal Infirmary of Edinburgh, Edinburgh, Scotland, United Kingdom

Prion diseases, or transmissible spongiform encephalopathies (TSEs), comprise a spectrum of diseases in animals and humans. In animals, these diseases include scrapie in sheep and goats, chronic wasting disease in deer and elk, and transmissible mink encephalopathy. Bovine spongiform encephalopathy (BSE) was first described in cattle in the United Kingdom (UK) in the early 1980s and developed into an epidemic of more than 180,000 bovine cases, spreading also to a range of other animals including domestic and exotic cats and exotic ungulates. Sporadic or classical Creutzfeldt-Jakob disease (CJD), first described in the early 1920s,1 occurs at an incidence of around 1 in 1 million per year. It presents at a mean age of 68 years with a rapidly progressive dementia, leading to death after about 6 months. Kuru, described in the Fore people of Papua New Guinea,2 comprised an endemic disease presenting with cerebellar ataxia and a more prolonged clinical course. The disease is thought to have been spread through cannibalistic funeral rites. Although these cultural practices died out toward the end of the 1950s, there are still occasional people who develop new clinical disease, pointing to the potentially very prolonged incubation periods of these diseases. Iatrogenic transmission of sporadic CJD has occurred via neurosurgical instrumentation and electroencephalogram (EEG) electrodes, corneal and dura mater grafts, and cadaveric pituitary-derived growth and follicularstimulating hormones.3 Those patients infected through direct central nervous system inoculation tend to manifest an incubation period of around 2 years and a rapidly progressive dementia reminiscent of sporadic CJD. Those infected through peripheral inoculation demonstrate a more prolonged and variable incubation period (mean 13-15 years) and a clinical syndrome similar to that of Kuru, suggesting that the route of infection has a significant impact on the incubation period and clinical manifestation of disease.

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

Finally, there are a group of rare inherited human prion diseases—including familial CJD, Gerstmann-Sträusler-Scheinker (GSS) disease, and fatal familial insomnia—related to polymorphisms within the gene for prion protein.4 The pathology of these disorders is characterized by the accumulation of abnormal prion protein (PrPTSE) within the central nervous system associated with neuronal degeneration and reactive gliosis, giving rise to the characteristic spongiform appearance from which these diseases took their original name.5 There is patchy evidence of peripheral accumulation of PrPTSE in patients with sporadic and familial forms of CJD, but little substantive evidence of infectivity in peripheral blood and tissues or of transmission by blood components, plasma derivatives, or cellular, tissue, or organ transplants.6 Variant CJD was first described in 1996.7 The disease is characterized by a relatively early onset compared to sporadic disease (median 28 years, range 12-74 years), a clinical presentation consisting of behavioral disturbance,8 dysesthesia and ataxia followed by progressive neurologic deterioration,9 and a prolonged clinical phase (median 14 months, range 6 to 48 months).10 The pathologic features are also characteristic, with the presence of PrPTSE not only within the central nervous system, but also in the follicular dendritic cells of peripheral lymphoid tissues including tonsils, spleen, lymph nodes, and gut-associated lymphoid tissue. PrPTSE has been demonstrated in two appendix samples removed 8 months and 2 years before the onset of clinical disease.11 The epidemiologic, pathologic, and experimental data are consistent with variant CJD having arisen from the oral transmission of BSE from infected cattle. To date, 167 cases have been described in the UK, 23 in France, four in Ireland, three each in the US and Spain, two each in the Netherlands and Portugal, and one each in Canada, Japan, Saudi Arabia, and Italy.12 Two of the Irish patients, two of those in the US, and those in Canada and Japan are thought to have been infected during travel in the UK. The overall incidence of clinical cases appears to be diminishing, with current mathematical models projecting a maximum likelihood of 70 [95% confidence interval (CI), 10-190] further clinical

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cases in the UK.13 However, the possibility of a cohort of subclinically infected individuals14 and four cases of transmission of variant CJD prions by blood components described in 2004 and 200615-17 raise concern that secondary and higher order transmissions by blood components, plasma derivatives, and cellular, tissue, or organ transplantation could extend the outbreak, particularly if compounded by other potential routes of transmission such as surgery and interventional medical procedures. Although blood service organizations have taken several precautionary measures,18,19 the persisting uncertainties surrounding the nature, level, and distribution of infectivity; the prevalence of subclinical disease; and the overall transmissibility of the disease undermine any ability to accurately judge the magnitude of this risk and evaluate the impact of current and proposed risk management strategies.

The Molecular Basis of Prion Diseases Prion diseases are associated with a change in the secondary and tertiary conformation of a widely expressed glycoprotein termed prion protein (PrPC). PrPC is a 30- to 35-kD protein with two N-linked glycosylation sites and a glycosylphosphatidylinositol anchor. The secondary structure consists of around 40% alphahelix and 3% to 4% beta-pleated sheet. Prion infection is associated with a conformational transformation, resulting in an increase in the proportion of beta-pleated sheet. This, in turn, engenders a change in the physicochemical and biological properties of the molecule (PrPTSE), including an increased resistance to degradation of biological and physical agents (PrPRES).20 The precise etiology of the conformational change remains unclear, with some authorities favoring a process of homodimerization and others preferring one of nuclear polymerization. The prion hypothesis21,22 proposes that PrPTSE is the direct cause of infectivity, although it is acknowledged that the relationship between PrPTSE, PrPRES, infectivity, and tissue damage is not straightforward. Some authorities suggest that a small nucleic acid-based agent may be the causative agent.23,24

The Nature, Concentration, and Distribution of Infectivity The current understanding of the pathogenesis of CJD is largely predicated on animal studies. Biochemical and biological assays point to the accumulation of high concentrations of PrPTSE and infectivity in the central nervous system in all forms of CJD. Animal models of peripherally transmitted prion diseases,25,26 point to follicular dendritic cells (FDCs)27,28 in the germinal centers of lymphoid tissue as the key cell in the establishment of infection and not B lymphocytes as previously suggested.29,30 The presence of abnormal prion accumulation in the FDCs in tonsil, spleen, and lymph nodes of all patients with variant CJD thus far examined postmortem suggests a similar pathophysiology

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for CJD in humans following primary (eg, cattle BSE to humans) or secondary and higher order (human to human) transmission.31 The exact nature of infectivity in peripheral blood and tissues is uncertain. Recent work suggests that in brain homogenates, the maximum specific infectivity is associated with proteinaseresistant particles of 300 to 600 kD (ie, oligomers of 14 to 28 PrP molecules).32 There are also data suggesting that proteinase sensitive forms of PrPTSE could be infectious. Further work in this area, particularly relating to the physicochemical characteristics of the infectious prion in plasma is a priority. The World Health Organization (WHO) has reviewed the data on the distribution of infectivity and PrPTSE in peripheral tissues and organs in human prion diseases and in naturally and experimentally infected animal prion diseases.6 Although the highest levels of infectivity are confined to neurologic tissues, lower levels of infectivity and/or PrPTSE have been demonstrated in a wide range of other tissues. Two sets of rodent studies are informative with regard to the likely concentration and distribution of infectivity in peripheral blood. The first studies were carried out in mice infected with the Fukuoka-1 strain of GSS disease33,34 and provided the data on which the original risk assessments were based.35 The second set of studies was carried out in hamsters infected with the 263K strain of scrapie and has led to a review of blood infectivity assumptions.36,37 Overall, the current working assumption is that the level of infectivity in the peripheral blood during the incubation period of a peripherally transmitted prion disease such as variant CJD is likely to be in the order of magnitude of 10 infectious doses/mL. In humans the data on peripheral blood infectivity in sporadic and familial CJD is open to interpretation38 and it has not yet proven possible to detect infectivity in the peripheral blood of patients with variant CJD, despite the fact that the disease is clearly clinically transmissible. Moreover, it is known that the distribution of PrPc varies significantly between rodents and humans.39-41 There are, therefore, important caveats regarding the extrapolation of these estimates to humans and it is probably safer to consider a plausible range of infectivity in the peripheral blood of humans for the purposes of risk assessment. The spatial distribution of infectivity also varies between models. Brown et al33,34 mimicked to a certain extent clinical blood separation processes and demonstrated a four- to fivefold higher concentration of infectivity in the buffy coat (per unit volume) compared to plasma. Gregori et al studied purified blood components and concluded that red cells and platelets have very little infectivity, and that approximately 40% of infectivity is associated with leukocytes, with most of the remainder residing within the plasma.36,42 The temporal development of infectivity in peripheral tissues during the incubation period is similarly uncertain. In rodent models, a variety of patterns of change in infectivity and PrPTSE concentration have been observed.43,44 In humans, the pattern of development of infectivity in peripheral blood and tissues during CJD infection is unknown, although, as noted previously,

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abnormal PrP has been detected in an appendix sample removed 2 years before development of clinical variant CJD.

Transmissibility of CJD by Blood and Tissues In general, one would expect that transmission of infection within a species would be more efficient than that between different species.45 The transmission of infection by blood drawn from a donor during both the incubation period and clinical phase of disease has been demonstrated in sheep naturally infected with scrapie and experimentally infected with BSE with an efficiency of approximately 50%, suggesting that the level of infectivity might be lower than that seen in the rodent models.46-48 Although there are a handful of reports of patients who have developed sporadic CJD following exposure to blood components,49 plasma derivatives,50-53 or organ transplantation,50 these reports have not established a convincing link with sporadic CJD in the donors. To the contrary, a large number of epidemiologic case control,54-64 look-back,65,66 and surveillance67-70 studies over the past 25 years have demonstrated no clear evidence of transmission of sporadic CJD by blood transfusion or plasma derivatives. It should be borne in mind, however, that CJD is a rare disease and occasional cases of transmission by blood components could have been missed. The Transfusion Medicine Epidemiology Review was established in the UK in 1996 in order to monitor potential linkage between CJD in donors and recipients.71,72 Individuals who develop variant CJD are reported to the UK Blood Services by the National CJD Surveillance Unit (NCJD-SU) to establish whether they have previously been blood donors. If they have been donors, the recipients are traced and notified. In the reverse arm, NCJD-SU determines whether individuals who have developed variant CJD have themselves received blood. If they have been transfusion recipients, the UK Blood Services trace and notify the donors. Individuals identified in this way are considered to be “at risk for public health purposes.”73 There are clear ethical and social tensions in adopting a notification strategy when the level of risk is uncertain, psychologic and social detriment may ensue, and no practical benefit accrues to the notified individuals.74,75 No linkage has been identified between donors and recipients with sporadic or familial CJD in this study. However, 18 UK blood donors have gone on to develop variant CJD and of their 66 traceable recipients (26 of whom are informative in the sense of having survived more than 5 years after transfusion), three have developed variant CJD and one showed evidence of subclinical infection at postmortem examination (having died of an unrelated condition).15-17 In France, three blood donors have developed variant CJD, although of their 18 traceable recipients, none have developed variant CJD at the time of this writing. Eleven of the above-noted UK blood donors also contributed 25 plasma donations to pools from which a total of 178 plasma batches were manufactured before the UK began importing plasma in 1999. No recipients of implicated products have thus

far developed clinical variant CJD—although again, following risk assessment, many of these people are now regarded as “potentially at risk for public health purposes.”73,76 Cells, tissues, and organs are much less frequently transplanted and many are derived from brain-stem dead or nonheart-beating donors. In the absence of routine cadaveric testing it is, therefore, not possible to know whether such individuals may have been incubating CJD at the time of their deaths.77 There are no clearly substantiated reports of individuals having developed CJD as a result of cellular, tissue, or organ transplantation. However, both prion diseases and these transplant procedures are relatively uncommon events and given that a significant tissue mass is transplanted, the concentration of infectivity that would be required to transmit infection is well below the level of sensitivity of current assay methods. Therefore, a precautionary assumption is that these tissues may be capable of transmitting disease should the donor be infected.

The Prevalence and Distribution of Subclinical Disease It is the prevalence and epidemiologic distribution of pre- or subclinical disease that drives the risk of secondary transmission, rather than the incidence of clinical disease. Although the incidence of sporadic CJD is known, the incubation period is uncertain and the prevalence of pre- or subclinical disease is unknown. Iatrogenic and familial forms of CJD are fortunately rare; however, it appears likely that preclinical cases will occur in these forms of disease. Those individuals in at-risk groups can often be identified by family or medical history and excluded as donors. The prevalence and distribution of subclinical variant CJD is also unknown; however, the available data provide some important hints. First, the median age of onset of disease has not altered over the past decade in the way one would expect where a cohort of individuals has been exposed to the risk of infection over a discrete period.7,12,78 These data suggest the existence of age-related susceptibility and/or exposure between the ages of 10 and 20 years,13,79,80 a cohort who would now be between 20 and 40 years old. The patients who have developed variant CJD in France have an older age profile (median age 37 years) than in those in the UK: the reason for this difference is unknown. The data generated by retrospective study of tonsil and appendectomy samples has also been revealing.14,81,82 Three out of 12,674 samples were found to be positive for abnormal prion protein on Western blot,83 where the specificity is thought to be high,84 but the sensitivity uncertain.85 Mathematical modeling suggests a maximum likelihood of 3000 infected people (95% CI, 520-6810) in the UK, mainly in the 10- to 30-year age group.13 This is at variance with the number of individuals projected to develop clinical disease and suggests a probability of subclinical infection of 0.93 (95% CI, 0.70-0.97).13,86 The potential existence of a cohort of individuals in the population with long-term

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subclinical infection87,88 is consistent with experimental animal data89,90 suggesting that methionine/valine heterozygosity or valine homozygosity at codon 129 of the PRNP gene predisposes to a prolonged period of subclinical disease. Although all clinical cases of variant CJD thus far have been methionine homozygous at codon 129,91 the second reported case of transfusion-transmitted variant CJD prions was shown to be methionine/valine heterozygous at codon 129.16 Two of the three patients who tested positive in the retrospective study of tonsils and appendices have also recently been shown to be valine homozygous at this locus,92 suggesting that codon 129 genotype (among other things) may influence the propensity of an infected individual to develop clinical disease. Long-term studies on patients who developed Kuru or iatrogenic CJD support this possibility. Individuals who were homozygous for methionine at codon 129 developed clinical disease sooner than those of other codon 129 genotypes, who have developed disease more sporadically and with longer incubation periods (in some cases, over 40 years from their likely infection).93 Taken together, these data suggest that the underlying prevalence of subclinical disease in the UK could be around 1 in 10,000 (range 1 in 1000 to 1 in 20,000) and that there could be further waves of clinical disease in individuals with other codon 129 genotypes. There are no specific data on which to estimate the prevalence in other countries. However, from the relative incidence of clinical disease, one might expect prevalence in Ireland to be approximately 20% of that in the UK, that in France to be approximately 10%, and that in the Netherlands to be around 5%.

Approaches to Risk Management In the face of the uncertainties and extrapolated estimates, a precautionary set of assumptions is that it is likely that there exists a proportion of healthy people with subclinical variant CJD in the UK and other Western European countries, that infectivity is present in the peripheral blood of these individuals, and that transmission of disease is possible through blood and tissue products, but the exact magnitude of these risks is unclear. The best risk management approaches are those that are likely to have some beneficial impact over the widest range of plausible scenarios. However, the effectiveness of such precautionary strategies may be partial or unknown and the potential for risk substitution and ethical dilemma needs to be considered.

Donor Selection Residence in the UK (and, to a lesser extent, Ireland and France) is clearly a risk factor for variant CJD and many countries exclude prospective donors who have lived in such countries.94 The stipulated period of residence varies widely, depending on the negative impact on the blood donor base and the perceived differential risk.95 It has proved more difficult to delineate subpopulations who may be considered to be at higher risk of subclinical vCJD than the general population. Individuals considered to be “presumed

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infected” or “at risk for public health purposes” are deferred from blood transfusion and tissue transplantation.73 Although blood transfusion by itself is thought unlikely to give rise to a self-sustaining outbreak, it is possible that transfusion in combination with other potential routes of transmission such as surgical or invasive medical procedures could result in such an event. Confirmation of clinical transmission of variant CJD via blood transfusion led to the introduction of deferral of donors in the UK who themselves have definitely or probably received blood components in order to reduce the risk of tertiary and higher order transmissions, at the cost of a 5% to 10% loss in blood donations. However, surveys have documented considerable inconsistency in donor recall of blood transfusion, undermining confidence in the effectiveness of such measures. Donor exclusion criteria are blunt risk management tools that have the potential for significant negative impact on individual donors and on the blood and tissue supply.

Donor Screening Assays The development of peripheral blood assays aimed at the detection of subclinical CJD in donor populations is hampered by the absence of an antibody response or of detectable DNA associated with transmission of the infectious agent. Surrogate assays providing nonspecific evidence of neurologic damage96,97 or alteration in peripheral tissue physiology98,99 have thus far proved insufficiently sensitive and specific for subclinical CJD. PrPTSE detection remains the most promising route to development of a preclinical screening assay,100-101 despite caveats about the precise relationship with infectivity. Achieving the required levels of sensitivity and specificity has, however, proved to be a major challenge. It has been estimated that the threshold of detection may need to be as low as 106 molecules of PrPTSE/ mL in peripheral blood.102 Immunoblotting with proteinase K digestion and gel electrophoresis has been enhanced using phosphotungstic acid precipitation and chemiluminescence,103 but is probably still insufficiently sensitive to detect PrPTSE in blood. Capillary immunoelectrophoresis following proteinase K digestion and competitive antibody binding was the first method to describe detection of PrPTSE in blood,104 but has proved complex and difficult to reproduce in other laboratories.105 A number of attempts have now been made to move away from the use of proteinase K resistance as the defining principle.106 Several assays use denaturation of PrPTSE aggregates, the best characterized of which is the conformation dependent immunoassay.107 Several PrPTSE-specific monoclonal antibodies have now been described, of which 15B3 is the best characterized.108 Several groups have developed assays predicated on differential binding of other ligands including polyanionic compounds,109 synthetic polypeptides,6 streptomycin/calyx-6-arene,6 and palindromic peptides.110 Some of these approaches are now showing sufficient sensitivity to detect infectivity in the peripheral blood of TSE-infected animals. Finally, two approaches appear to amplify PrPTSE and/or infectivity. Protein folding cyclic amplification

Chapter 50: Prion Diseases

3,000,000 donors vCJD1/10,000 Assay 99% effective

Figure 50-1. The impact of sensitivity and specificity of an assay for variant CJD on test results from a donor population.

True positives 99% prevalence 297 donors

uses repeated cycles of incubation and sonication to amplify PrPTSE up to 107-fold,44 whereas in-vitro cell culture has also proved capable of amplifying murine scrapie.111 Such amplification might prove a valuable adjunct to other detection methods. There are three key concerns relating to the introduction of donor screening assays. 1. The specificity of an assay is dependent not only on its technical properties, but also on the population in which it is deployed. An assay with a satisfactory positive predictive value in the context of patients with suspect disease may have a very poor positive predictive value in the context of general population screening. The potential impact of an assay with a low specificity on the false-positive rate of a healthy donor population is illustrated in Fig 50-1. Overall, there is a clear need for assays employing different analytical principles, one or more of which can be used for primary screening and others as supplementary or confirmatory assays to help control the false-positive rate. 2. The validation of such assays is problematic.112-114 The standard approach to validation of microbiological screening assays uses large numbers of samples from patients known to have the disease in question. Patients with variant CJD in particular are fortunately rare and it will be problematic to obtain large volumes of blood for ethical and practical reasons. It is likely, therefore, that it will be necessary to carry out the development and validation of peripheral blood screening assays on spiked brain homogenates and endogenously infected animal models, raising concerns about the relevance of these models to the human setting. 3. Consideration will need to be given to the impact of introduction of a screening assay on donors. There are likely to be difficulties in the discrimination of false from true positives and, indeed, in understanding the “meaning” of a true-positive result in terms of the implications for the donor. Donors will have to be informed that they will be screened and notified of any positive result. The psychological and social impact on the individual is likely to be high and the negative impact on the blood supply could be profound. Further, detailed consideration needs to be given to the practical and ethical implications of introducing an assay and the provision of adequate supporting resources.115-117

Blood Component Processing Whole blood is now rarely transfused, the majority of components being collected via apheresis or derived from whole blood

False negatives 1% prevalence 3 donors

True negatives 99% (1-prevalence) 2,969,703 donors

False positives 1% (1-prevalence) 29,997 donors

donation and processed into red cell concentrate (in additive solution), platelet concentrate, and fresh frozen plasma. In addition, in the UK, Ireland, France, and some other countries, all clinical blood components are subject to universal leukocyte reduction, which removes 3-4 log10 leukocytes.118-120 The rodent studies of Brown et al33,34 and Gregori et al36 suggest that it is likely that component processing brings about some reduction in overall infectivity because of reduction in the amount of residual leukocytes and plasma, but it is unlikely that this will significantly affect transmissibility. Several companies are working on the development of prion reduction filters for red cell components that offer the possibility of a further reduction in infectivity.121,122 Current risk management models suggest that a 3 log10 reduction in infectivity with use of the device would be needed in association with leukocyte reduction in order to effect a reduction in the risk of transmission. Potential detrimental effects include additional blood loss in the dead volume of the filter and the possibility of alterations to red cell antigenicity or membrane properties. There are concerns about the evaluation of the efficacy of these technologies given that they have developed using spiked brain homogenates and endogenous infectivity animal models. The UK and Irish Blood Services have developed a series of quality, efficacy, and operational specifications and are commissioning independent evaluation and clinical safety studies.

Plasma Derivative Manufacture It is unlikely that the virus inactivation steps used in the manufacture of plasma derivatives will have a significant impact on prion infectivity.123 However, work based on consideration of the likely physicochemical characteristics of the prion agent and the partitioning effects of plasma fractionation suggests that a significant reduction in infectivity may occur during the fractionation process, resulting in a relatively low risk of transmission by plasma derivatives.124 Studies of spiked brain homogenates support this prediction, but are unlikely to accurately reflect the physicochemical nature of the infectious agent in plasma.125-128 A smaller number of endogenous infectivity studies have been carried out in animal models that again support the general premise, but are unable to demonstrate more than a 1- to 2-log reduction because of low concentrations of infectivity in the starting material.34 Several manufacturers are using nanofiltration as a second

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virus reduction step, and these filters have, at least experimentally, demonstrated prion removal, mainly using brain homogenate spikes. Prion-binding affinity chromatography systems are under development but are probably still some way from routine application. There has been no documented case of transmission of sporadic CJD by plasma derivatives. Surveillance of patients exposed to large volumes of plasma products have also not shown any evidence of transmission. Some of these products are known to have been manufactured from pools that contained plasma from a donor who subsequently developed variant CJD. Most such individuals are considered to be “potentially at risk for public health purposes.”129

Cellular, Tissue, and Organ Transplantation Many types of cells, tissues, and organs are transplanted.130 As noted previously, the concentration of infectivity in such tissues is uncertain, although broadly speaking, a very low concentration would suffice to cause transmission of disease if the donor were infected. Deferral of transfused donors has proved to be complex because of the nature of the donor population(s), the shortage of some tissues, and the lifesaving nature of the procedure— particularly of organ transplantation. A feasibility study has been initiated to look at the possibility of testing tonsil from cadaveric donors for PrPTSE accumulation. Broadly speaking, any processing that reduces the amount of cellular material and also avoids pooling is likely to be of benefit. There are ongoing studies of the applicability of, for example, washing method for bone processing. Agents that proved effective in the decontamination of surgical instrumentation are largely inapplicable to cells or organs although some might be applicable to acellular tissues such as heart valves, bone, or tendons. Finally, it is possible that some of the compounds found to mitigate TSE infectivity in vitro and/or in vivo could be applicable to these products.131,132 Substantial further research and development work will need to be carried out before results of these investigations can be translated to the clinical setting.

Ethical, Legal, and Societal Considerations Many of the issues raised above pose not only scientific and medical challenges, but also wider ethical, legal, and societal questions. Early uncertainty over the possibility of secondary transmission of CJD by blood transfusion or plasma derivatives led to the implementation of risk management strategies based on the precautionary principle, perhaps best articulated by the Krever Commission in Canada133,134: “Preventive action should be taken when there is evidence that a potentially disease-causing agent is or may be blood borne, even when there is no evidence that recipients have been affected. If harm can occur, it should be assumed that it will occur. If there are no measures that will entirely prevent the harm, measures that may only partially prevent transmission should be taken.”

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The accumulating evidence of the existence of a cohort of individuals subclinically infected with variant CJD prions in the UK, along with recent reports of clinical transmission by blood transfusion, speak to the prudence of these measures. However, important uncertainties persist and the effectiveness of current risk reduction measures including donor selection procedures, component processing, and plasma fractionation remains unclear. New technologies such as prion reduction filters and prion screening assays are under development and the precautionary principle would speak to the need to implement these where possible. However, such measures are not without potential countervailing risks and ethical problems.135 Donor deferral criteria can be considered discriminatory, and the introduction of a donor screening assay could have a serious impact on the psychological health of individuals who test positive, leading to difficulties in balancing non-malevolenceficence with regard to the patient against non-maleficence with regard to the donor. Donor deferral and screening measures could have a serious negative impact on the sufficiency of blood and tissue supply, leading to a failure of beneficenceficence.136 Component processing technologies such as universal leukocyte reduction137 and prion reduction filters138 offer the opportunity to circumvent these problems, but at significant financial cost, raising issues of equity of access to health-care resources. Finally, compulsory notification of people at potentially increased risk of exposure to CJD infection through blood components or plasma derivatives raises the issue of respect for autonomy.139 In this context, continued efforts should be made to improve the evidence base regarding clinical blood transfusion and tissue transplantation140; to promote a conservative approach to the use of human blood and tissue products141; to ensure that patients are appropriately informed142,143; and to facilitate public understanding of, and engagement with, the uncertainties and ethical dilemmas inherent in managing this risk.144

Acknowledgments I would like to thank Professor Bob Will, Dr. Lorna Williamson, and Dr. Patricia Hewitt for their comments on this manuscript. Any errors are the responsibility of the author alone.

Disclaimer The author has disclosed no conflicts of interest.

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44. Saa P, Castilla J, Soto C. Presymptomatic detection of prions in blood. Science 2006;313:92-94. 45. Hill AF, Collinge J. Prion strains and species barriers. Contrib Microbiol 2004;11:33-49. 46. Houston F, Foster JD, Chong A, et al. Transmission of BSE by blood transfusion in sheep. Lancet 2000;356:999-1000. 47. Hunter N, Houston F. Can prion diseases be transmitted between individuals via blood transfusion? Evidence from sheep experiments. Dev Biol (Basel) 2002;108:93-8. 48. Hunter N, Foster J, Chong A, et al. Transmission of prion diseases by blood transfusion. J Gen Virol 2002;83:2897-905. 49. Klein R, Dumble LJ. Transmission of Creutzfeldt-Jakob disease by blood transfusion (letter). Lancet 1993;341:768. 50. Creange A, Gray F, Cesaro P, et al. Creutzfeldt-Jakob disease after liver transplantation. Ann Neurol 1995;38:269-72. 51. Creange A, Gray F, Cesaro P, Degos JD. Pooled plasma derivatives and Creutzfeldt-Jakob disease (letter). Lancet 1996;347:482. 52. de Silva R. Pooled plasma derivatives and Creutzfeldt-Jakob disease (letter). Lancet 1996;347:967. 53. Patry D, Curry B, Easton D, et al. Creutzfeldt-Jakob disease (CJD) after blood product transfusion from a donor with CJD. Neurology 1998;50:1872-3. 54. Kondo K, Kuroiwa Y. A case control study of Creutzfeldt-Jakob disease: Association with physical injuries. Ann Neurol 1982;11:377-81. 55. Davanipour Z, Alter M, Sobel E, et al. A case-control study of Creutzfeldt-Jakob disease. Dietary risk factors. Am J Epidemiol 1985;122:443-51. 56. Harries-Jones R, Knight R, Will RG, et al. Creutzfeldt-Jakob disease in England and Wales, 1980-1984: A case-control study of potential risk factors. J Neurol Neurosurg Psychiatry 1988;51:1113-9. 57. Esmonde TF, Will RG, Slattery JM, et al. Creutzfeldt-Jakob disease and blood transfusion. Lancet 1993;341:205-7. 58. Wientjens DP, Davanipour Z, Hofman A, et al. Risk factors for Creutzfeldt-Jakob disease: A reanalysis of case-control studies. Neurology 1996;46:1287-91. 59. van Duijn CM, Delasnerie-Laupretre N, Masullo C, et al. Casecontrol study of risk factors of Creutzfeldt-Jakob disease in Europe during 1993-95. European Union (EU) Collaborative Study Group of Creutzfeldt-Jakob disease (CJD). Lancet 1998;351:1081-5. 60. Will RG, Alperovitch A, Poser S, et al. Descriptive epidemiology of Creutzfeldt-Jakob disease in six European countries, 1993-1995. EU Collaborative Study Group for CJD. Ann Neurol 1998;43:763-7. 61. Collins S, Law MG, Fletcher A, et al. Surgical treatment and risk of sporadic Creutzfeldt-Jakob disease: A case-control study. Lancet 1999;353:693-7. 62. Wilson K, Code C, Ricketts MN. Risk of acquiring Creutzfeldt-Jakob disease from blood transfusions: Systematic review of case-control studies. Br Med J 2000;321:17-9. 63. Zerr I, Brandel JP, Masullo C, et al. European surveillance on Creutzfeldt-Jakob disease: A case-control study for medical risk factors. J Clin Epidemiol 2000;53:747-54. 64. Riggs JE, Moudgil SS, Hobbs GR. Creutzfeldt-Jakob disease and blood transfusions: A meta-analysis of case-control studies. Mil Med 2001;166:1057-8. 65. Heye N, Hensen S, Muller N. Creutzfeldt-Jakob disease and blood transfusion. Lancet 1994;343:298-9. 66. Operskalski EA, Mosley JW. Pooled plasma derivatives and Creutzfeldt-Jakob disease (letter). Lancet 1995;346:1224.

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67. Brown P. Can Creutzfeldt-Jakob disease be transmitted by transfusion? Curr Opin Hematol 1995;2:472-7. 68. Lee CA, Ironside JW, Bell JE, et al. Retrospective neuropathological review of prion disease in UK haemophilic patients. Thromb Haemost 1998;80:909-11. 69. Evatt B. Prions and haemophilia: Assessment of risk. Haemophilia 1998;4:628-33. 70. Evatt B, Austin H, Barnhart E, et al. Surveillance for CreutzfeldtJakob disease among persons with hemophilia. Transfusion 1998;38:817-20. 71. Hewitt P, Llewelyn CA, Mackenzie J, Will RG. Creutzfeldt-Jakob disease and blood transfusion: Results of the UK Transfusion Medicine Epidemiology Review study. Vox Sang 2006;91:221-30. 72. National Blood Service, Scottish National Blood Transfusion Service, Welsh Blood Service, Northern Ireland Blood Transfusion Service, National CJD Surveillance Unit. Transfusion medicine epidemiology review. [Available at http://www.cjd.ed.ac.uk/TMER/TMER.htm (accessed May 16, 2008).] 73. Health Protection Agency. Creutzfeldt-Jakob disease (CJD). London, UK: HPA, 2007. [Available at http://www.hpa.org.uk (accessed May 16, 2008).] 74. Boixiere A, Hergon E, Duchange N, et al. [Informing the transfused patient of the possible transmission of variant Creutzfeldt-Jakob disease by blood transfusion]. Presse Med 2004;33:1533-7. 75. Hewitt PE. Implications of notifying donors and recipients. Vox Sang 2004;87(Suppl 2):1-2. 76. Bird SM. Recipients of blood or blood products “at vCJD risk.” Br Med J 2004;328:118-9. 77. Warwick RM, Eglin R. Should deceased donors be tested for vCJD? Cell Tissue Bank 2005;6:263-70. 78. Will R. Variant Creutzfeldt-Jakob disease. Folia Neuropathol 2004;42(Suppl A):77-83. 79. Boelle PY, Cesbron JY, Valleron AJ. Epidemiological evidence of higher susceptibility to vCJD in the young. BMC Infect Dis 2004;4:26. 80. Cooper JD, Bird SM. Predicting the incidence of variant CreutzfeldtJakob disease from UK dietary exposure to bovine spongiform encephalopathy for the 1940 to 1969 and post-1969 birth cohorts. Int J Epidemiol 2003;32:784-91. 81. Hilton DA, Ghani AC, Conyers L, et al. Accumulation of prion protein in tonsil and appendix: Review of tissue samples. Br Med J 2002;325:633-4. 82. Frosh A, Smith LC, Jackson CJ, et al. Analysis of 2000 consecutive UK tonsillectomy specimens for disease-related prion protein. Lancet 2004;364:1260-2. 83. Hilton DA, Ghani AC, Conyers L, et al. Prevalence of lymphoreticular prion protein accumulation in UK tissue samples. J Pathol 2004;203:733-9. 84. Hilton DA, Sutak J, Smith ME, et al. Specificity of lymphoreticular accumulation of prion protein for variant Creutzfeldt-Jakob disease. J Clin Pathol 2004;57:300-2. 85. Hilton DA. Pathogenesis and prevalence of variant Creutzfeldt-Jakob disease. J Pathol 2006;208:134-41. 86. Cooper JD, Bird SM, de Angelis D. Prevalence of detectable abnormal prion protein in persons incubating vCJD: Plausible incubation periods and cautious inference. J Epidemiol Biostat 2000;5:209-19. 87. Hill AF, Collinge J. Subclinical prion infection. Trends Microbiol 2003;11:578-84. 88. Hill AF, Collinge J. Subclinical prion infection in humans and animals. Br Med Bull 2003;66:161-70.

Chapter 50: Prion Diseases

89. Wadsworth JD, Asante EA, Desbruslais M, et al. Human prion protein with valine 129 prevents expression of variant CJD phenotype. Science 2004;306:1793-6. 90. Bishop MT, Hart P, Aitchison L, et al. Predicting susceptibility and incubation time of human-to-human transmission of vCJD. Lancet Neurol 2006;5:393-8. 91. Head MW, Bunn TJ, Bishop MT, et al. Prion protein heterogeneity in sporadic but not variant Creutzfeldt-Jakob disease: UK cases 1991-2002. Ann Neurol 2004;55:851-9. 92. Ironside JW, Bishop MT, Connolly K, et al. Variant CreutzfeldtJakob disease: Prion protein genotype analysis of positive appendix tissue samples from a retrospective prevalence study. Br Med J 2006;332:1186-8. 93. Collinge J, Whitfield J, McKintosh E, et al. Kuru in the 21st century—an acquired human prion disease with very long incubation periods. Lancet 2006;367:2068-74. 94. Ault A. FDA bans UK blood donation. Nat Med 1999;5:720. 95. Ponte M. Insights into the management of emerging infections: Regulating variant Creutzfeldt-Jakob disease transfusion risk in the UK and the US. PLoS Med 2006;3:e342. 96. Otto M, Wiltfang J, Schutz E, et al. Diagnosis of Creutzfeldt-Jakob disease by measurement of S100 protein in serum: Prospective case-control study. Br Med J 1998;316:577-82. 97. Zerr I, Bodemer M, Gefeller O, et al. Detection of 14-3-3 protein in the cerebrospinal fluid supports the diagnosis of Creutzfeldt-Jakob disease. Ann Neurol 1998;43:32-40. 98. Miele G, Manson J, Clinton M. A novel erythroid-specific marker of transmissible spongiform encephalopathies. Nat Med 2001;7:361-4. 99. Fagge T, Barclay GR, Macgregor I, et al. Variation in concentration of prion protein in the peripheral blood of patients with variant and sporadic Creutzfeldt-Jakob disease detected by dissociation enhanced lanthanide fluoroimmunoassay and flow cytometry. Transfusion 2005;45:504-13. 100. Cervenakova L, Brown P. Advances in screening test development for transmissible spongiform encephalopathies. Expert Rev Anti Infect Ther 2004;2:873-80. 101. Brown P, Cervenakova L. The modern landscape of transfusionrelated iatrogenic Creutzfeldt-Jakob disease and blood screening tests. Curr Opin Hematol 2004;11:351-6. 102. Brown P, Cervenakova L, Diringer H. Blood infectivity and the prospects for a diagnostic screening test in Creutzfeldt-Jakob disease. J Lab Clin Med 2001;137:5-13. 103. Wadsworth JD, Joiner S, Hill AF, et al. Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay. Lancet 2001;358:171-80. 104. Schmerr MJ, Jenny AL, Bulgin MS, et al. Use of capillary electrophoresis and fluorescent labeled peptides to detect the abnormal prion protein in the blood of animals that are infected with a transmissible spongiform encephalopathy. J Chromatogr A 1999;853:207-14. 105. Cervenakova L, Brown P, Soukharev S, et al. Failure of immunocompetitive capillary electrophoresis assay to detect disease-specific prion protein in buffy coat from humans and chimpanzees with Creutzfeldt-Jakob disease. Electrophoresis 2003;24:853-9. 106. Minor P, Brown P. Diagnostic tests for antemortem screening of Creutzfeldt-Jakob disease. In: Turner ML, ed. Creutzfeldt-Jakob disease: Managing the risk of transmission by blood, plasma, and tissues. Bethesda MD: AABB Press, 2006:119-48.

107. Bellon A, Seyfert-Brandt W, Lang W, et al. Improved conformationdependent immunoassay: Suitability for human prion detection with enhanced sensitivity. J Gen Virol 2003;84:1921-5. 108. Korth C, Stierli B, Streit P, et al. Prion (PrPSc)-specific epitope defined by a monoclonal antibody. Nature 1997;390:74-7. 109. Lane A, Stanley CJ, Dealer S, Wilson SM. Polymeric ligands with specificity for aggregated prion protein (abstract). Clin Chem 2003;49:1774-5. 110. Grosset A, Moskowitz K, Nelsen C, et al. Rapid presymptomatic detection of PrP via conformationally responsive palindromic PrP peptides. Peptides 2005; 26:2193-200. 111. Nishida N, Harris DA, Vilette D, et al. Successful transmission of three mouse adapted scrapie strains to murine neuroblastoma cell lines over-expressing wild-type mouse prion protein. J Virol 2000;74:320-5. 112. Minor P, Newham J, Jones N, et al. Standards for the assay of Creutzfeldt-Jakob disease specimens. J Gen Virol 2004;85:1777-84. 113. Minor PD. Technical aspects of the development and validation of tests for variant Creutzfeldt-Jakob disease in blood transfusion. Vox Sang 2004;86:164-70. 114. Minor PD. The challenge for the public health system. Contrib Microbiol 2004;11:208-15. 115. Blajchman MA, Goldman M, Webert KE, et al. Proceedings of a consensus conference: The screening of blood donors for variant CJD. Transfus Med Rev 2004;18:73-92. 116. McCullough J, Anderson D, Brookie D, et al. Consensus conference on vCJD screening of blood donors: Report of the panel. Transfusion 2004;44:675-83. 117. Duncan RE, Delatycki MB, Collins SJ, et al. Ethical considerations in presymptomatic testing for variant CJD. J Med Ethics 2005;31:625-30. 118. Kleinman S. New variant Creutzfeldt-Jakob disease and white cell reduction: Risk assessment and decision making in the absence of data. Transfusion 1999;39:920-4. 119. Murphy MF. New variant Creutzfeldt-Jakob disease (nvCJD): The risk of transmission by blood transfusion and the potential benefit of leukocyte-reduction of blood components. Transfus Med Rev 1999;13:75-83. 120. St. Romaine C, Hazlehurst G, Jewell AP. Leucodepletion for transmissible spongiform encephalopathies. Br J Biomed Sci 2004;61:48-54. 121. Sowemimo-Coker SO, Pesci S, Andrade F, et al. Pall leukotrap affinity prion-reduction filter removes exogenous infectious prions and endogenous infectivity from red cell concentrates. Vox Sang 2006;90:265-75. 122. Gregori L, Gurgel PV, Lathrop JT, et al. Reduction in infectivity of endogenous transmissible spongiform encephalopathies present in blood by adsorption to selective affinity resins. Lancet 2006;368:2226-30. 123. Taylor DM. Inactivation of TSE agents: Safety of blood and bloodderived products. Transfus Clin Biol 2003;10:23-5. 124. Foster PR. Removal of TSE agents from blood products. Vox Sang 2004;87(Suppl 2):7-10. 125. Stenland CJ, Lee DC, Brown P, et al. Partitioning of human and sheep forms of the pathogenic prion protein during the purification of therapeutic proteins from human plasma. Transfusion 2002;42:1497-500. 126. Reichl HE, Foster PR, Welch AG, et al. Studies on the removal of a bovine spongiform encephalopathy-derived agent by processes

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127.

128.

129.

130.

131.

132. 133.

134.

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used in the manufacture of human immunoglobulin. Vox Sang 2002;83:137-45. Foster PR, Griffin BD, Bienek C, et al. Distribution of a bovine spongiform encephalopathy-derived agent over ion-exchange chromatography used in the preparation of concentrates of fibrinogen and factor VIII. Vox Sang 2004;86:92-9. Berardi VA, Cardone F, Valanzano A, et al. Preparation of soluble infectious samples from scrapie-infected brain: A new tool to study the clearance of transmissible spongiform encephalopathy agents during plasma fractionation. Transfusion 2006;46:652-8. Caulfield T, Dossetor J, Boshkov L, et al. Notifying patients exposed to blood products associated with Creutzfeldt-Jakob disease: Integrating science, legal duties and ethical mandates. CMAJ 1997;157:1389-92. Galea G, Turner ML. Cell, tissue, and organ transplantation. In: Turner ML, ed. Creutzfeldt-Jakob disease: Managing the risk of transmission by blood, plasma, and tissues. Bethesda MD: AABB Press, 2006:215-43. Head MW, Farquhar CF, Mabbott N, Fraser JR. The transmissible spongiform encephalopathies: Pathogenic mechanisms and strategies for therapeutic intervention. Expert Opin Ther Targets 2001;5:568-85. Trevitt CR, Collinge J. A systematic review of prion therapeutics in experimental models. Brain 2006;129:2241-65. Krever H. Commission of inquiry on the blood system in Canada. Ottawa, Ontario, Canada: Health Canada, 1997. [Available at http://www.hc-sc.gc.ca/ahc-asc/activit/com.krever_e.html (accessed May 16, 2008). Wilson K, Graham I, Ricketts N, et al. Variant Creutzfeldt-Jakob disease and the Canadian blood system after the tainted blood tragedy. Soc Sci Med 2007;64:174-85.

135. Childress JF, Beauchamp TL. Principles of biomedical ethics. 5th ed. Oxford: Oxford University Press, 2001. 136. Sandler SG. Variant Creutzfeldt-Jakob disease—the availability of a screening test will be the beginning, not the end, of difficult transfusion-related issues. Curr Opin Haematol 2006;13:445-6. 137. Cleemput I, Leys M, Ramaekers D, Bonneux L. Balancing evidence and public opinion in health technology assessments: The case of leucoreduction. Int J Technol Assess Health Care 2006;22:403-7. 138. Turner ML. Prion reduction filters. Lancet 2006;368:2190-1. 139. Evatt B, Austin H, Barnhart E, et al. Surveillance for CreutzfeldtJakob disease among persons with hemophilia. Transfusion 1998;38:817-20. 140. McClelland B, Contreras M. Appropriateness and safety of blood transfusion. Br Med J 2005;330:104-5. 141. Murphy M. Strategies for reducing the exposure to donor blood. Clin Med 2005;5:337-40. 142. Hart J, Leier B, Nahirniak S. Informed consent for blood transfusion: Should the possibility of prion risk be included? Transfus Med Rev 2004;18:177-83. 143. Boixiere A, Hergon E, Moutel G, et al. [Legal obligation to inform the patient on the theoretical risk of CJD transmission by blood]. Transfus Clin Biol 2004;11:101-5. 144. Farrugia A, Ironside JW, Giangrande P. Variant Creutzfeldt-Jakob disease transmission by plasma products: Assessing and communicating risk in an era of scientific uncertainty. Vox Sang 2005;89:186-92.

51

Pathogen Inactivation Bjarte G. Solheim1 & Jerard Seghatchian2 1 2

Professor Emeritus, Institute of Immunology, Rikshospitalet University Hospital and University of Oslo, Oslo, Norway Consultant, Blood Components Technology and Haemostasis/Thrombosis Consultancy, London, United Kingdom

Transfusion-transmitted infections (TTIs) involving the infectious agents described in Chapters 46-50 have been significantly reduced in industrialized countries during the last two decades by careful donor selection and extensive laboratory testing. However, blood transfusions represent an ideal portal of entry for infectious agents and often contain trace amounts of endogenous bacteria (mostly from the gut or from presymptomatic infections) and/or exogenous bacteria (from the skin). With the volumes generally collected during a donation, trace contaminations are a problem. This is particularly true for platelet products (stored at 22ºC) and in the transfusion of immunocompromised patients. Important interventions to reduce the risk for trace contaminations are careful donor selection, enhanced cleansing of the venipuncture site, use of diversion pouches for the first 15 to 20 mL of a blood collection,1 and the testing of platelet preparations for the presence of bacteria. Risks for TTIs are escalated by emerging infections combined with increased international air travel.2 This has been demonstrated by the mosquito-spread viruses, West Nile virus in the United States and the chikungunya virus in the Indian Ocean region. For the former a nucleic acid amplification test (NAT) has been developed, while this is not the case for chikungunya virus, which spread to Italy in 2007 and posed a risk in the Mediterranean area. On the Île de Réunion in the Indian Ocean in 2006, more than 25% of the inhabitants were infected. As a temporary measure in order to ensure a safe blood supply, components were supplied from France [except platelets, which were prepared locally by apheresis and subjected to pathogen inactivation (PI)].3 Programs for careful donor selection and extensive laboratory testing pose both organizational and economic challenges in most developing countries. In addition, the epidemiology is less favorable than in most developed countries, the use of whole blood is still dominating, and safe fractionated plasma proteins

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

are mostly unavailable. Therefore, TTIs are a serious problem, causing thousands of infections a year particularly with hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and malaria. Robust and affordable PI technologies for whole blood and blood components would be of great value in these countries. In developed countries, the risk for bacterial and protozoan infections and emerging infectious agents are the main drive for PI. Several approaches have been explored for PI. Except for solvent/detergent (SD) treatment, all are based on PI of single plasma units from recovered or apheresis plasma. Whether applied under good manufacturing practice (GMP) conditions in blood centers or on a larger scale outside blood centers, inprocess control of single plasma units is not performed. Thus, exceptionally high loads of a pathogen could slip through; NAT should be performed in spite of PI.2 Pathogen inactivation of pooled fractionated plasma proteins has virtually eliminated the risk of TTIs, without compromising the quality of the products significantly. After addition of stabilizers, some proteins such as albumin have been pasteurized with excellent pathogen safety records. However, it took almost 60 years before attention was drawn to the fact that the addition of caprylic acid and N-acetyl-DL-tryptophan as stabilizers during pasteurization impairs drug binding of pharmaceutical grade albumin.4 In this chapter the much more challenging PI of blood components are discussed. Solvent/detergent treatment, which is successfully applied for biopharmaceutical plasma products, dissolves cell membranes; hence, it is highly effective also against intracellular pathogens but not applicable for cellular blood components. PI methods that target nucleic acids have been the prime choice for PI of cellular components, but their efficiency against intracellular microorganisms depends on the ability to penetrate cell membranes. PI of blood components should inactivate or remove all types of infectious agents, without inducing neoantigens or reducing the function or life span of a blood component. PI should not result in residual toxic substances or involve a risk greater than any TTI associated with the original blood component.5 Because

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toxicity may first be revealed after large-scale clinical use, Phase IV postmarketing studies and hemovigilance programs are important.

Plasma Pathogen Inactivation Solvent/Detergent Treatment SD treatment is the most extensively used and best validated PI technology: 50 to 60 million doses of SD-treated pooled fractionated plasma proteins have been given without the reported transmission of enveloped viruses. However, the method does not inactivate nonenveloped viruses, and nanofiltration or heat treatment has been introduced as a second PI step for many fractionated plasma proteins. SD treatment removes infectivity by disrupting the membranes of enveloped viruses; bacteria, protozoa, and cells are also affected, but so far this has not been extensively validated.2 The most frequently used technique is the combination of 1% tri-(N-butyl)-phosphate (TNBP) and 1% polyoxyethylene-p-t-octylphenol (Triton X-100) for 4 hours at 30 to 31ºC.6 TNBP acts as an organic solvent and removes lipids

from the membranes; it is used alone in some protocols for PI. Triton X-100 is a non-ionic detergent that disrupts lipid bilayers for easier extraction of lipids; it also stabilizes TNBP.6 SD treatment was first licensed for clotting factors in 1985 and has been in use for the treatment of plasma since 1991.7,8 The inactivation rate of enveloped viruses is fast and the safety of the method very high (Table 51-1).7,8 Inactivation is below virus detection levels (⬎6 log) within 15 minutes,9 except for vaccinia virus, which shows a relative resistance, but is inactivated with TNBP and Triton X-100 according to the protocol mentioned above.10 So far, all tested enveloped viruses, including the recently emerging ones, are inactivated. For plasma no second PI step is available, but immune antibodies in a plasma pool can protect against disease with hepatitis A virus (HAV) and parvovirus B19.11,12 In addition, NAT screening for HAV and parvovirus B19 ensures the safety of SD-treated plasma, as an essential regulatory requirement. A major advantage of SD treatment is that the inactivation reagents in general do not affect proteins and protein activity in plasma. One important exception is reduction of the α2-antiplasmin activity by 70% to 80% during SD treatment. This seems to

Table 51-1. Inactivation Agents, Susceptible Pathogens, and Modified Targets Inactivation Agent

Susceptible Organism

Solvent/Detergent

● ●

Methylene blue plus light

● ●

S-59 plus ultraviolet A light

● ● ● ● ● ●

Riboflavin plus light

● ● ● ● ● ●

S-303

● ● ● ● ● ●

PEN 110

● ● ● ● ● ●

802

Target/Modification

Enveloped viruses Intracellular viruses

Lipid envelope solubilization

Enveloped viruses Some nonenveloped viruses

Free radical Guanidine

Enveloped viruses Intracellular viruses Some nonenveloped viruses Bacteria not including spores Protozoa Leukocytes

Pyrimidine adducts and cross-links

Enveloped viruses Intracellular viruses Some nonenveloped viruses Bacteria (spores unknown) Protozoa Leukocytes

Guanidine oxidation and possible adducts to thymine and adenin

Enveloped viruses Intracellular viruses Some nonenveloped viruses Bacteria (spores unknown) Protozoa Leukocytes

Nucleic acid adducts

Enveloped viruses Intracellular viruses Some nonenveloped viruses Bacteria (spores unknown) Protozoa Leukocytes

Guanine adducts

Chapter 51: Pathogen Inactivation

be caused by Triton X-100 because a reduction of only about 10% is observed with Triton X-45, which in preliminary virus inactivation validation is as effective as Triton X-100.13 Triton X-45 and X-100 differ in molecular mass, the former having only five ethylene oxide groups, the latter nine or 10 groups. This difference in molecular mass suggests a differential impact of the two non-ionic detergents on the conformation (and activity) of α2-antiplasmin.13 After removal with oil extraction and hydrophobic interaction chromatography, TNBP and Triton X-100 are either undetectable or present as trace contaminations far below toxicity levels.6 SD treatment is the only current technology for PI of pooled plasma. This allows for the production of a standardized biopharmaceutical product with extensive in-process control and without the significant normal variation in plasma protein concentrations observed in single plasma units. Pooling also dilutes and in some instances neutralizes antibodies and allergens, and allows the production of ABO-independent universal plasma (see later). There is a risk that emerging TTIs would be increased by pooling. However, SD plasma pools (60-380 L) are 10 to 100 times smaller than those used in the production of PI fractionated plasma proteins. Recently, a very gentle method for SD treatment of single plasma units and cryoprecipitate at blood centers was published.14,15 SD-treated pooled Fresh Frozen Plasma (SDPP) was introduced in Europe in 1991, with Octaplas (Octapharma AG, Lachen, Switzerland) as the leading product. Octaplas is a licensed biopharmaceutical product in 29 countries worldwide (including Canada and Mexico in North America). A total of over 6 million units of SDPP have been transfused so far. In the United States, SDPP (PLAS⫹SD, Vitex, Watertown, MA) was licensed in the second half of the 1990s but later withdrawn from the market.2 After SD treatment, these SDPP products are normally frozen in 200-mL plastic bags; however, the South African Bioplasma FDP is lyophilized. Nine studies covering all indications for plasma (including six European prospective randomized trials with SDPP) have investigated the clinical efficacy and tolerance, with Octaplas as the bestdocumented product.16,17 Although the SD treatment process has been the same for all SDPP products, there have been some differences in the quality of plasma and manufacturing processes.16,17 Apheresis plasma is preferred, followed by recovered plasma optimally frozen within few hours after collection. Plasma from whole blood stored for 15 hours before separation and freezing has a reduced content of coagulation factors and inhibitors.18 The usage pattern of such plasma and some differences in production processes could account for the thromboembolism and compositional differences observed with PLAS⫹SD but not with Octaplas.19-21 All SDPP products have low levels of α2-antiplasmin. Except for protein S in the lower normal range and the reduction of α2antiplasmin, Octaplas has a concentration of coagulation factors and inhibitors and clinical effect similar to apheresis FFP (frozen within 24 hours after collection).17,22 SD treatment of single plasma units (1% TNBP ⫹ 1% Triton X-45 or only 2% TNBP) has been reported to have minimal effect on coagulation factors

and inhibitors, including protein S and α2-antiplasmin.15 This promising method, however, needs further validation. α2-antiplasmin is an acute-phase serine protease inhibitor of plasmin that is synthesized in the liver.23 This explains why most clinical observations show that low α2-antiplasmin levels in SDPP are insignificant except in patients with liver deficiency or fibrinolysis.17 In these patients, antifibrinolytic treatment or administration of the serine protease inhibitor aprotinin has to be considered.2,24 In an enhanced version of Octaplas α2-antiplasmin levels are now reduced by only 30% to 40% (T-E Svae, Octapharma, personal communication). Low levels of protein S have been associated with thromboembolism observed in liver transplantation with PLAS⫹SD,17 and a retrospective study in Europe indicated possible thromboembolism after repeated plasma exchanges with Octaplas in patients with thrombotic thrombocytopenic purpura (TTP).25 However, in a large study, the same authors subsequently observed no problems with venous thromboembolism and concluded that there was no difference in the number of exchanges needed when cryosupernatant and Octaplas were compared. In addition, they reported that allergic/urticarial and citrate reactions were more common with cryosupernatant.26 Adverse events are less common with SDPP than with ordinary FFP. Particularly important is that there have been no reports of transfusion-related acute lung injury (TRALI) after transfusion of over 5 million units of SDPP in Europe.27,28 Hemovigilance data indicate that febrile, allergic, or anaphylactic reactions are reduced by 70% to 80% with SDPP.21 These observations are best explained by dilution/neutralization that results from pooling and removal of all cellular components during the SD treatment process. An improved version of SDPP is ABO-universal plasma that eliminates the need for ABO group-specific plasma.2 It is based on the principle that anti-A and anti-B can be neutralized by soluble A and B antigens in plasma. This was first exploited during World War II, when the Allied Forces used randomly pooled plasma. Such a lyophilized product was licensed for emergency needs in several European countries until the 1970s, but represented a high risk of transfusion-transmitted hepatitis.2 Since 1996, a lyophilized ABO-universal SDPP, Bioplasma FDP (National Bioproducts Institute, Pinetown, South Africa), has been prepared from lowtitered plasma pools in South Africa.29 More than 370,000 units have been transfused so far, with an excellent record of safety and efficacy.2 In Europe an ABO-independent universal SDPP has been developed by Octapharma by proportionally pooling A, B, and AB plasma. The product (tentatively named Uniplas) is not yet licensed, but has been used successfully in clinical trials in cardiac surgery and liver resection in Europe.30-32

Photosensitizers Methylene Blue Light Treatment Methylene blue (MB) is a positively charged phenothiazine dye with high affinity for negatively charged compounds, such as guanine, proteins, and some lipids. The virus inactivating activity of illuminated phenothiazines has been recognized since the

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early 1930s.33 Evidence suggests that MB interacts with nucleic acids through intercalative binding and produces singlet oxygen-mediated damage, primarily to nucleic acid guanosine residues, upon illumination with visible light.34 MB light treatment is effective against enveloped and some nonenveloped viruses (Table 51-1). MB does not effectively permeate plasma membranes; thus intracellular pathogens and leukocytes may not be fully inactivated. Therefore, the original protocol developed by Institute Springe introduced a freeze-thaw step to destroy residual leukocytes. Grifols (Barcelona, Spain) employs leukocyte filtration of blood, which also removes platelets and to some degree cellular fragments and microparticles, and brings residual leukocyte counts in plasma well below 1 ⫻ 106.35 However, only plasma membrane filters remove all cellular components, and reduce intracellular HIV infectivity to detection limits after MB light treatment of plasma. Binding of MB to membrane proteins may affect red cell viability, while binding to plasma proteins reduces coagulation factor activity in plasma, cryoprecipitate, and cryosupernatant.37 Toxicity of MB is low. Intravenous doses of 1 to 5 mg/kg have been administered in clinical situations without the report of serious adverse effects; a unit of MB-treated plasma would represent 0.0012 mg/kg for an adult, and could be reduced to 0.00012 mg/kg with absorption devices.2 No neoantigenicity caused by various intercalated proteins remaining after MB treatment have been reported. However, on theoretical basis, long-term side effects cannot be ruled out completely because of the binding of MB to proteins.21 MB-treated plasma is the second most frequently used pathogen-inactivated FFP, and more than 4 million units have been used clinically in European countries, including Germany, Belgium, France, Greece, Italy, Spain, Switzerland, and the UK. The method is applied on single plasma units. The original Springe protocol was factory based, and in use for more than a decade in Germany and Switzerland.38 Grifols has introduced a similar factory-based method, but with leukocyte filtration instead of a freeze-thaw step. This method is still in use in Spain. Macopharma (Tourcoing, France) has modified MB treatment of plasma for use in blood centers, and improved the method. The firm has introduced the Theraflex MB-Plasma system, with a 0.65-micron plasma filter and a removal device for MB that eliminates more than 90% MB and its photoproducts, without any significant additional loss of coagulation factors.2 The system is granted Conformité Européenne (CE) mark and used to prepare PI plasma in house at a number of blood centers in Europe. In Germany the Paul Ehrlich Institute (Frankfurt, Germany) has granted authorization for manufacture and sale of plasma treated with the Theraflex MB-Plasma system. In spite of extensive use, there have been no full reports of large randomized trials with MB-treated plasma using relevant endpoints such as blood loss or exposure to other blood components.39 In one small study, cardiac surgery patients were transfused with either MB-treated plasma or SDPP. The former gave better replacement of protein S and α2-antiplasmin, but no difference in blood loss.40 A 5-year observational study in Greece

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with the Theraflex MB-Plasma system reported good safety and a reduced number of adverse events.41 As with all other PI methods, MB treatment reduces the potency of some active elements of plasma. Fibrinogen, Factor V, and Factor VIII activity are decreased by approximately 30%; the effect on other coagulation factors and inhibitors is smaller.2,41 Significant prolongation of fibrin polymerization time has been reported; the prolongation is, however, eliminated after admixture of 60% Octaplas.42 Thrombin generation capacity is impaired in MB-treated plasma.43 Overall, satisfactory clinical results and efficacy have been reported with MB-treated plasma. However, it seems to be less effective than FFP as replacement fluid in plasma exchange for TTP, in spite of normal levels of von Willebrand factor cleaving enzyme (ADAMTS13) in MB-treated plasma.2,44

Psoralen Ultraviolet Light Treatment Furocoumarins, which include psoralens, are active compounds isolated from plants; they have been known as photosensitizers since ancient times. Amotosalen hydrochloric acid (also known as S-59) is a synthetic psoralen (Fig 51-1) specially selected by Cerus (Concord, CA) for PI of blood components because it crosses plasma membranes efficiently and demonstrates excellent protection against pathogens.6 S-59 is activated by ultraviolet A (UVA) light. Because hemoglobin absorbs UVA light, S-59 is not applicable for PI of red cells.

H H HOC C CH2OH OH HOCH O

NH2

O

CH2

O

O

H3 C

N

H3 C

N

S-59

N

N

NH

Riboflavin

O

CH3

N⫹

S

O

N S

N

CH

Flexible bond

Methylene blue

O NH

O

N

N

CH3 R1 R2

Thiazole orange

N N—[R5-N⫹(R6,R7)]nR8⫹Xn⫺

S-303 R3 R4

Inactine

Figure 51-1. Structures and general formula for some pathogen inactivating agents. The formula for Thiazole orange is used with permission from Skripchenko et al.45

Chapter 51: Pathogen Inactivation

S-59 targets the helical region of single- or double-stranded DNA or RNA, and intercalates almost instantly. Upon exposure to UVA light, S-59 binds covalently to pyrimidine, and with continued UVA light exposure a second bond is generated cross-linking double-stranded structures, while single-stranded structures are cross-linked in loops. S-59 photodegrades into well-characterized molecules that are rapidly excreted.6 In addition to targeting nucleic acids, S-59 also binds to lipids and proteins. Thus, about 15% of the initially added S-59 remains bound to plasma and platelets even after removal of S-59 and its photoproducts with a compound absorbing device (CAD). Most S-59 is associated with lipids, but 1% to 2% is associated with proteins.2 Psoralen UVA light treatment with S-59 inactivates a broad spectrum of enveloped viruses, bacteria, protozoa, and residual leukocytes, but its effect on nonenveloped viruses is more variable (Table 51-1).6,46 The INTERCEPT Blood System device developed by Cerus uses S-59, UVA illumination, and a CAD for the removal of residual S-59 and metabolites. In Europe the system was granted CE mark for PI of plasma in 2006, and is supposed to be implemented under GMP conditions in blood centers. In the United States it is approved only for clinical trials by the FDA. Preclinical studies have demonstrated high levels of safety.47,48 Fibrinogen, Factor V, Factor VII, Factor VIII, and Factor X are reduced by 17% to 30%, while coagulation factor inhibitor activity is less affected.46 Four randomized studies indicate adequate clinical effect, including support of plasma exchange for TTP48-51; however, more than 60% of the patients with congenital coagulation deficiencies experienced urticaria.49 Although no neoantigenicity or toxicologically relevant effects have been observed with INTERCEPT-treated plasma, Phase IV postmarketing studies could still reveal such effects resulting from some binding of S-59 to lipids and proteins.

Riboflavin Light Treatment In spite of phototoxic targeting of nucleic acids, vitamin B2 (riboflavin) is generally recognized as safe by the US FDA. This safety is supported by extensive experience with phototherapy treatment of neonatal jaundice (see Chapter 27). Riboflavin and its photoproducts are present in a wide range of foods and natural products in common use. In addition, riboflavin, its photoproducts, and its catabolites are detectable in normal blood, which suggests that their presence may be ubiquitous.52 However, further research is required because the level of photoproducts is considerable higher in riboflavin-treated blood components. Both oxygen-dependent (formation of free radicals) and oxygen-independent (electron transfer) processes are involved in the phototoxic PI effect of riboflavin, which damages guanine and may form adducts of thymine and adenine.53 Riboflavin light treatment has proven effective against a range of pathogens, including bacteria, enveloped viruses, protozoa, leukocytes, and some nonenveloped viruses (Table 51-1).52 Navigant Biotechnologies (Lakewood, CO) has developed the Mirasol system for riboflavin treatment of blood components.

Because riboflavin can be photoactivated by visible as well as UV light, the Mirasol system is being adapted for PI for all blood components, including red cells and whole blood. The system is similar to INTERCEPT, except that removal of reagents may not be necessary, and is not currently performed. Mirasol has been adapted for PI of platelets and plasma, while treatment of red cells and whole blood still is under development. Extensive PI of selected viruses (including parvovirus), bacteria, and protozoa in addition to leukocyte inactivation has been reported in platelet concentrates.52 Similar results have been obtained with Mirasol treatment of FFP (RP Goodrich, Navigant Biotechnologies, personal communication). Compared with the INTERCEPT system, the Mirasol system for PI of plasma reduces the time plasma is stored in liquid state, and early studies indicate that plasma protein activity is only marginally affected.52 Preclinical studies with Mirasol-treated plasma are promising, and no neoantigenicity has been observed. Pending the successful completion of remaining field verification and clinical studies in Europe, the Mirasol system for PI of plasma is expected to receive a CE mark (RP Goodrich, Navigant Biotechnologies, personal communication).

Platelet Pathogen Inactivation Psoralen UV Light Treatment The INTERCEPT Blood Systems device for PI of platelets is similar to the one for plasma, and was granted CE mark in Europe in 2002; the FDA so far has approved it only for clinical trials. During the rather complex operation of PI with INTERCEPT, platelets come in contact with many surfaces that may induce surface changes and loss of granule contents. Studies have demonstrated inactivation of a broad spectrum of viruses, bacteria, protozoa, and leukocytes, including clinically relevant nonenveloped viruses such as parvovirus B19 (Table 51-1).6 Typical experiments have been reported by Lin et al.54 INTERCEPT treatment also inactivates mitochondrial DNA and inhibits cytokine synthesis, while platelet metabolic functions seem retained.55 Invitro INTERCEPT-treated platelets demonstrate increased CD61 microparticle formation, higher metabolic rate, accelerated metabolic changes, and reduced agonist-induced aggregation responses. Spontaneous platelet activation measured by fluorescent-activated cell sorter showed increased expression of CD62P and CD42b after INTERCEPT treatment; in addition, phosphadidyl-serine (PS) on the platelet surface (a hallmark of apoptosis) is significantly increased.56-58 These changes are accelerated when the storage time is increased to 7 days. To what degree the formation of apoptotic cells, as measured by PS exposure, is of clinical significance remains to be established. In-vitro data show cytokine accumulation also in INTERCEPT-treated platelet concentrates.56 A European study including 166 thrombocytopenic patients transfused with INTERCEPT-treated buffy-coat platelets showed comparable platelet count increments and adverse reactions to untreated platelets.59 However, in a larger US study (n ⫽ 645)

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assessing apheresis platelets, the 1-hour posttransfusion corrected count increment and average number of days to next platelet transfusion were slightly reduced, while the number of platelet transfusions was increased compared to untreated platelets.60 In these studies storage time was limited to 5 days. Many facilities have increased storage time to 7 days without additional clinical trials in spite of the in-vitro study results. After CE marking in 2002 the system has been used for the production of more than 50,000 doses of PI platelets for routine clinical use in 14 European countries, and is reported to have replaced bacterial testing, gamma irradiation, and the use of cytomegalovirus-negative platelets. In addition there has been a report of a reduction in allergic nonhemolytic transfusion reactions with INTERCEPT-treated platelets.2 Although no neoantigenicity or toxicologically relevant effects have so far been observed after clinical use of INTERCEPT-treated platelets, Phase IV postmarketing studies could reveal such effects resulting from some binding of S-59 to platelet lipids and proteins.

Riboflavin-Treated Platelets The Mirasol system for PI of platelets is similar to the one for plasma. It is less complex than the INTERCEPT system in that removal of reagents is not performed; thus it may be more gentle to the platelets. Nevertheless, this PI process also leads to some platelet activation as expressed by increased metabolic activity and P-selectin expression.53 However, both in-vitro activity and mitochondria function seem not to be affected.61,62 Extensive PI of selected viruses (including parvovirus B19), bacteria, and protozoa and inactivation of leukocytes has been reported.53 Preclinical studies have demonstrated that in-vitro and in-vivo capabilities of Mirasol-treated platelets are similar to INTERCEPT-treated platelets.53,61,63 After a Phase III study was completed in Europe in 2007, the Mirasol system for platelet PI was granted CE marking for application under GMP conditions in blood centers.

Thionine Light Treatment The phenothiazine dye thionine and UV light combined with strong agitation is effective in the PI of platelets, and development of a protocol is being pursued by Blood Centers of the German Red Cross and MacoPharma (H Mohr, Institute Springe, Germany, personal communication). However, strong agitation could pose problems with irreversible activation of platelets.

Red Cell Pathogen Inactivation PI systems for red cells and whole blood have been explored with some negative results but also some promising developments.

Alkylating Agents FRALE Compounds Frangible anchor linker effector (FRALE) compounds are alkylating agents that specifically inactivate DNA/RNA via

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covalent adducts to the nucleic acid. The compounds consist of a nucleic acid-binding ligand that serves as an anchor and promotes nucleic acid specificity, an alkylating agent that functions as an effector, and a chain that joins the two moieties.6,64 Cerus has developed the Helinx system for PI of red cells with S-303 (Fig 51-1), which is a compound with an acridine-based anchor. S-303 is designed so that the alkyl chain is cleaved upon contact with blood at a rate slower than the rate of binding and alkylation to nucleic acids. This does not inhibit PI, but promotes the breakdown of residual unreacted compound into S-300, which does not further interact with nucleic acids.6,64 Acridine nitrogen mustards have been shown to alkylate proteins in addition to nucleic acids.65 Helinx treatment is performed at room temperature for 12 hours followed by incubation with a CAD in the final storage container for 8 hours in order to remove residual S-303 and S-300.6 Helinx PI has been shown to inactivate viruses, bacteria, protozoa, and leukocytes efficiently (Table 51-1), and extensive toxicology studies indicated that the process was safe.66 However, the literature with regard to the technology is sparse and mostly based on abstracts.6 PI with FRALE compounds has been applied to red cells, and the only available protocol is Helinx with S-303. After satisfactory results with in-vitro studies and genotoxicity tests, successful Phase I and II trials were performed, followed by Phase III trials in cardiac surgery and sickle cell/thalassemia patients. S-303 PI and conventional red cells were equivalent with regard to supporting the transfusion needs of cardiac surgery patients.67 Nevertheless, the Phase III trial was put on hold because some patients in each group developed antibodies reactive with S-303treated red cells, but no hemolysis.67 Neoantigen formation may have been caused by alkylation of proteins by acridine nitrogen mustards.65 Some patients even demonstrated antibodies before transfusion with S-303-treated red cells. These antibodies could have been induced by S-303-like agents such as the local anesthetic quinacrine, which also can alkylate proteins. Experiments to modify the Helinx system by adding quenchers has so far eliminated immunoreactivity of S-303 PI red cells,68 but has not totally eliminated the formation of modified groups when red cells are tested with sensitive assays after treatment with S-303.2

Aziridine Compounds Aziridine compounds, such as ethylenimine, have been used to inactivate vaccines for the past 30 years. The method inactivates both enveloped and nonenveloped viruses, including members of the Parvoviridae family, which is often difficult to accomplish.69 By forming oligomers of ethylenimine the selectivity of aziridines for nucleic acids is improved. Inactines are small molecules with a cationic tail, conferring DNA binding and an effector group based on ethylenimine or azindine. PEN110 is a small, highly water-soluble cation with an ethylenimine oligomer (Fig 51-1), and selective affinity for nucleic acids,6 but can also alkylate proteins.70 It diffuses readily through cell membranes and, when forming ionic bounds with nucleic acids, the molecule is activated. The active form can then alkylate guanine. This

Chapter 51: Pathogen Inactivation

induces a break and results in a stop message. Vitex has developed the INACTINE system with PEN110 for PI of red cells. The treatment is performed at room temperature for 6 to 24 hours, followed by washing with unbuffered saline to levels below the limit of detection of PEN110.71 Extensive studies with selected enveloped and nonenveloped viruses, bacteria, protozoa, and leukocytes (Table 51-1) demonstrate the high PI efficiency of PEN110.6 Toxicology studies indicated that even if an individual human received multiple PI red cell components at once or over time, the dose would be far less than that required to have reproductive effects or induce genotoxicity.71 After preclinical tests, successful Phase I and II trials were performed, but Phase III trials were stopped because of antibody responses to PEN110treated red cells. In spite of protocol modifications the problem was not solved, and in June 2005 Vitex discontinued further investment in the development of PI with INACTINE.

Photosensitizers PI of red cells with photosensitizers poses problems because of light absorption by hemoglobin and the high number of red cells, which requires complex technical adjustments to secure uniform distribution of light for the treatment of Red Blood Cell (RBC) units (see Chapter 4). Several studies have been conducted with porphyrin and phenothiazine dyes, which are rigid planar aromatic compounds activated by visible light, while psoralens are not applicable because hemoglobin absorbs UV light. However, because of hemolysis following PI of red cells with these aromatic dyes, the methods have been abandoned. Some of the hemolysis upon illumination arises from dye bound to the red cell membrane, while other damage is caused by singlet oxygen produced by illumination of dye residing in the cytoplasma or supernatant, and reacting with membrane lipids or proteins.72 A new approach was opened by the discovery of flexible dyes that can act as photosensitizers when rigidly bound to substrate but cannot generate reactive oxygen species when in solution because the energy from absorbed light is dissipated as heat. Wagner and associates have pioneered the work with PI of red cells with two flexible dyes.45,72 Thiopyrylium (TP) is a flexible photosensitizer that intercalates with nucleic acids but also binds to red cells. Dipyramidole (DP) (vasodilatator, antioxidant, red cell band 3 ligand) acts as a competitive inhibitor of TP binding to red cells, and a protocol for PI of red cells was therefore designed with TP and DP. This method inactivated model viruses including intracellular HIV ⬎6 log, and six bacterial species ⬎5 log. Hemolysis increased within acceptable levels and most, but not all, red cell properties were retained after 42 days of storage.72 Thiazole orange (TO) (Fig 51-1) is another flexible nucleic acid intercalating dye, which shows less binding to red cells. Without addition of quenchers or competitive inhibitors, five tested viruses (including intracellular HIV) were inactivated ⬎5.4 log, and eight tested bacteria were inactivated from 2.3 to ⬎7 log. The PI red cells exhibited only slightly increased hemolysis, moderately elevated potassium efflux, and similar

levels of adenosine triphosphate compared to controls.45 However, much work remains to understand and improve the technique, perform toxicology studies, characterize in-vitro and in-vivo properties of TO PI red cells, and eventually scale up the technique to treat entire RBC units in order to perform clinical trials. Because of some binding of TO to red cells, this dye has an inherent risk of neoantigen formation with subsequent immunization after transfusion.

Riboflavin Light Treatment Preliminary studies show promising results with PI of red cells using the Mirasol system adapted for red cells. Pathogen activation of microorganisms and leukocytes is similar to Mirasol treatment of platelets and plasma, while hemolysis and in-vitro red cell properties are within acceptable limits (RP Goodrich, Navigant Biotechnologies, personal communication). The Mirasol system has now also been adapted for PI of whole blood, which afterwards can be separated into red cells, platelets, and plasma. Studies with this modified protocol include a full panel of in-vitro evaluations with the three separated blood components during storage, and radiolabeled red cell recovery and survival in healthy volunteers after 42 days of storage (RP Goodrich, Navigant Biotechnologies, personal communication). Because the chemistry employed by the Mirasol system does not involve covalent linking or alkylation and riboflavin is a natural constituent in human bodies, the risk that altered proteins in plasma or on platelet and red cell surfaces will induce antibody formation against neoantigens is small. Further studies with Mirasol PI of whole blood are, therefore, of great interest.

Emerging Technologies CryoFacets (Raleigh, NC) has under development an integrated system for PI, reduction of noninfectious complications, “fractionation of plasma,” and effective, gentle cell processing. PI of this integrated system is based on the following: ● Vacuum ultrasound removal of oxygen in plasma or suspending solutions in order to eliminate oxidative damage of plasma proteins or cell membranes ● UVC illumination ● Ozone treatment Plasma is first vacuum treated, then exposed to UVC light, and finally mixed with ozone. Following PI, plasma is separated into three fractions according to molecular weight: “above albumin,” “albumin,” and “below albumin.” Platelets are highly purified by separation with a specially developed counterflow centrifugal elutriation system; plasma, cell fragments, and bacteria are washed away. After isolation, platelets are mixed with an ozone solution in order to inactivate remaining bacteria. After a brief exposure to ozone the platelets are suspended in the “below albumin” fraction for storage. When platelets are needed the product is washed with degassed saline, which removes metabolic byproducts and cell fragments, and prepares the platelets for UVC exposure in order to inactivate viruses and possibly leukocytes. Because UVC-induced damage

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to the platelets is first observed after several days, this treatment is undertaken shortly before transfusion. Red cell treatment follows essentially the same process as that used for platelets. PI with the CryoFacets protocols is reported to be very efficient and, because of innovative technologies, processing time is low. However, considerable in-vitro and in-vivo characterization of the PI products will be required before applicability assessments in hospitals and clinical trials can be planned.

Prion Elimination None of the above-mentioned PI technologies eliminate prions. However, a significant reduction in infectivity occurs during plasma fractionation, resulting in relatively low risk for prion transmission with fractionated plasma proteins (see Chapter 50).73 For SDPP (Octaplas) a reduction of 2.5 log has been reported.73 Phenothiazines may react with prions,74 but there are no reports on a possible effect of MB treatment of plasma. Several companies are developing prion reduction filters for red cell components and interesting studies are in progress with ligand chromatography, which may reduce prion infectivity of plasma significantly (see Chapter 50). After introduction of such ligand chromatography in the production process of Octaplas an additional reduction of at least 3 log is achieved (T-E Svae, Octapharma, personal communication).

Summary SDPP is the most extensively used and documented PI plasma. An ABO-universal variant, introduced in 1996 in South Africa, eliminates the need for ABO group-specific plasma. Photochemical PI of plasma with MB light treatment has gained extensive use in Europe in spite of sparse published documentation; one protocol has gained CE mark. The protocol for psoralen light treatment of FFP gained CE mark in 2006, while the protocol for riboflavin light treatment of FFP is in advanced preclinical and clinical development. Photochemical PI of platelets with psoralen light treatment gained CE mark in 2002, and so far ⬎50,000 platelet units have been transfused in Europe. The protocol for riboflavin light treatment of platelets gained CE mark in 2007, and is introduced in clinical settings in Europe. Two PI methods for red cells employing alkylating agents reached Phase III clinical trials; however, formation of antibodies against neoantigens on red cells has halted further progress. Riboflavin light treatment of red cells has now been adapted for PI of whole blood, which subsequently could be separated into plasma, platelets, and red cells. It is the only protocol for whole blood; therefore, further development will be followed with particular interest. Clinical experience has shown reduced frequency (40%-80%) of allergic/immunologic (febrile nonhemolytic) adverse events with SDPP, MB-treated FFP (PI with the CE marked system), and psoralen light-treated platelets. This may be explained by the elimination of cellular components in the plasma and additional

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dilution/neutralization in SDPP. Leukocyte activation and cytokine production are blocked in the platelets. In addition, no cases of TRALI have been documented after extensive use of SDPP; this may be the result of dilution and possible neutralization of leukocyte-reactive antibodies by pooling of FFP. For biopharmaceutical SDPP, the reduction of 2.5 log in prion infectivity is enhanced by ligand chromatography. Cost-effectiveness of PI is rather low in most developed countries when compared to other strategies to reduce noninfectious risks of transfusion,75 but comparable to NAT of blood components. When only the effect of virus inactivation is considered, the cost of a quality-adjusted life year has been calculated to approach US$10 million for SDPP, and is assumed to be similar for PI of plasma by other methods.2 The high cost is due to very low residual risk for recognized viral infections. During outbreaks of emerging infections and in developing countries with high residual infection risk the cost-effectiveness is much better. When TRALI is taken into consideration, the cost for SDPP is reduced to US$40,000-170,000 per life year saved depending on patient age.2 Further reduction is obtained when the reduction in allergic/anaphylactic adverse events and the benefits of ABO-universal SDPP are also considered.21 For PI of platelets, no cost-effectiveness data have been published, but it has been suggested that the cost per life year saved in immunocompromised patients may be in the same range as for SD treated plasma, assuming that TRALI and allergic/ anaphylactic adverse events are taken into account.16 A possible introduction of riboflavin light treatment of whole blood may be expected to have considerable cost implications for all countries. A consensus conference76 recently concluded that cost-effectiveness studies should be carried out by public agencies before general introduction of a PI method and that an additional cost consequence analysis77 should be included. The conference also pointed out that: “Large adequately powered randomized clinical trials should be performed to evaluate and/or confirm the effectiveness of any new PI. Postlicensure Phase VI studies should be integrated with hemovigilance systems to enhance the ability to detect adverse events.”76

Disclaimer The authors have disclosed no conflicts of interest.

References 1. McDonald C, Roy A, Mahajan P, et al. Relative values of the interventions of diversion and improved donor-arm disinfection to reduce the bacterial risk from blood transfusion. Vox Sang 2004;86:178-82. 2. Solheim BG, Cid J, Osselaer JC. Pathogen reduction technologies. In: Lozano M, Contreras M, Blajchman M, eds. Global perspectives in transfusion medicine. Bethesda, MD: AABB Press, 2006:103-48. 3. Rasonglès P, Isola H, Kientz D, et al. Rapid implementation of photochemical pathogen inactivation (INTERCEPT) for preparation of platelet components during an epidemic of chikungunya virus (abstract). Vox Sang 2006;91(Suppl 3):32.

Chapter 51: Pathogen Inactivation

4. Olsen H, Andersen A, Nordbo A, et al. Pharmaceutical-grade albumin: Impaired drug-binding capacity in vitro. BMC Clin Pharmacol 2004;4:4. 5. Epstein JS, Vossal JG. FDA approach to evaluation of pathogen reduction technology. Transfusion 2003;43:1347-50. 6. Pelletier JPR, Transue S, Snyder EL. Pathogen inactivation techniques. Best Pract Res Clin Haematol 2006;19:205-42. 7. Horowitz B, Bonomo R, Prince AM, et al. Solvent/detergent-treated plasma. A virus-inactivated substitute for fresh frozen plasma. Blood 1992;79:826-33. 8. Hellstern P, Sachse H, Schwinn H, Oberefrank K. Manufacture and in-vitro characterization of a solvent/detergent-treated plasma. Vox Sang 1992;63:178-85. 9. Horowitz B, Lazo A, Grossberg H, et al. Virus inactivation by solvent/detergent treatment and the manufacture of SD-plasma. Vox Sang 1998;74(Suppl 1):203-6. 10. Roberts P. Resistance of vaccinia virus to inactivation by solvent/ detergent treatment of blood products. Biologicals 2000;28:29-32. 11. Solheim BG, Rollag H, Svennevig JL, et al. Viral safety of solvent/ detergent-treated plasma. Transfusion 2000;40:84-90. 12. Rollag H, Solheim BG, Svennevig JL. Viral safety of blood derivatives by immune neutralization. Vox Sang 1998;74(Suppl 1):213-17. 13. Burnouf T, Goubran HA, Radosevich M, et al. Impact of Triton X-100 on alpha2-antiplasmin (SERPINF2) activity in solvent/ detergent-treated plasma. Biologicals 2007;35:349-53. 14. Burnouf T, Goubran HA, Radosevich M, El-Ekiaby M. Preparation and viral inactivation of cryoprecipitate in blood banks in resourcelimited countries. ISBT Science Series 2007;2:121-8. 15. Burnouf T, Goubran HA, Radosevich M, et al. A process for solvent/ detergent treatment of plasma for transfusion at blood centers that use a disposable-bag system. Transfusion 2006;46:2100-8. 16. Solheim BG. Pathogen reduction of blood components. Transfus Apher Sci 2008; 39:75-82. 17. Hellstern P. Solvent/detergent-treated plasma: Composition, efficacy, and safety. Curr Opin Hematol 2004;11:346-50. 18. Runkel S, Haubelt H, Hitzler W, Hellstern P. The quality of plasma collected by automated apheresis and of recovered plasma from leukodepleted whole blood. Transfusion 2005;45:427-32. 19. Solheim BG, Hellstern P. Composition, efficacy, and safety of S/Dtreated plasma (letter). Transfusion 2003;43:1176-8. 20. Salge-Bartels U, Breitner-Ruddock S, Hunfeld A, et al. Are quality differences responsible for different adverse reactions reported for SD-plasma for USA and Europe? Transfus Med 2006;16:266-75. 21. Solheim BG, Seghatchian J. Update on pathogen reduction technology for therapeutic plasma: An overview. Transfus Apher Sci 2006;35:83-90. 22. Hellstern P. Fresh-frozen plasma, pathogen-reduced single donor plasma or biopharmaceutical plasma? Transfus Apher Sci 2008; 39:69-74. 23. Matsuda M, Wakabayashi K, Aoki N, Morioka Y. Alpha 2-plasmin inhibitor is among acute phase reactants. Thromb Res 1980;17:527-32. 24. Solheim BG, Bergan A, Brosstad F, et al. Fibrinolysis during liver transplantation is enhanced by using solvent/detergent virusinactivated plasma (ESDEP/Octaplas) (letter). Anesth Analg 2003;96:1230-1. 25. Yarranton H, Cohen H, Pavord SR, et al. Venous thromboembolism associated with the management of acute thrombotic thrombocytopenic purpura. Br J Haematol 2003;121:778-85.

26. Scully M, Longair I, Flynn M, et al. Cryosupernatant and solvent detergent fresh-frozen plasma (Octaplas) usage at a single centre in acute thrombotic thrombocytopenic pupura. Vox Sang 2007;93:153-8. 27. Solheim BG. Plasma induced TRALI is avoided with solvent/ detergent-treated plasma (abstract). Transfusion Alternatives in Transfusion Medicine 2005;7(Suppl):57. 28. Bux J. Transfusion-related acute lung injury (TRALI): A serious adverse event of blood transfusion. Vox Sang 2005;89:1-10. 29. Chapanduka ZC, Fernandez-Costa FJ, Rochat C, et al. Comparative safety and efficacy of Bioplasma FDP versus single-donor freshdried plasma in cardiopulmonary bypass patients (letter). S Afr Med J 2002;92:356-7. 30. Solheim BG. Universal pathogen-reduced plasma in elective openheart surgery and liver resection. Clin Med Res 2006;4:209-17. 31. Tollofsrud S, Noddeland H, Svennevig JL, et al. Universal fresh frozen plasma (Uniplas): A safe product in open-heart surgery. Intensive Care Med 2003;29:1736-43. 32. Solheim BG, Granov DA, Juralev VA, et al. Universal fresh-frozen plasma (Uniplas): An exploratory study in adult patients undergoing elective liver resection. Vox Sang 2005;89:19-26. 33. Clifton CE. Photodynamic action of certain dyes on the inactivation of staphylococcus bacteriophage. Proc Soc Exp Biol Med 1931;28:745-6. 34. Wagner SJ. Virus inactivation in blood components by photoactive phenothiazine dyes. Transfus Med Rev 2002;16:61-6. 35. Krailadsiri P, Seghatchian J, Macgregor I, et al. The effects of leukodepletion on the generation and removal of microvesicles and prion protein in blood components. Transfusion 2006;45:407-17. 36. Abe H, Yamada-Ohnishi Y, Hirayama J, et al. Elimination of both cell-free and cell-associated HIV infectivity in plasma by a filtration/methylene blue photoinactivation system. Transfusion 2000;40:1081-7. 37. Seghatchian J, Krailadsiri P. What’s happening? The quality of methylene blue treated FFP and cryo. Transfus Apher Sci 2001;25:227-31. 38. Mohr H, Lambrecht B, Seltz A. Photodynamic virus inactivation of blood components. Immunol Invest 1995;24:73-85. 39. Williamson LM, Cardigan R, Prowse CV. Methylene blue-treated fresh-frozen plasma: What is its contribution to blood safety? Transfusion 2003;43:1322-9. 40. Wieding JU, Rathgeber J, Zenker D. Prospective, randomized and controlled study on solvent/detergent versus methylene blue/light virus inactivated plasma (abstract). Transfusion 1999;39(Suppl):23S. 41. Politis C, Kavallierou L, Hantziara S, et al. Quality and safety of fresh-frozen plasma inactivated and leucoreduced with the Theraflex methylene blue system including the Blueflex filter: 5 years’ experience. Vox Sang 2007;92:319-26. 42. Pock K, Heger A, Janisch S, et al. Thrombin generation capacity is impaired in methylene-blue treated plasma compared to normal levels in single-donor fresh-frozen plasma, a licensed solvent/detergenttreated plasma (Octaplas) and a development product (Uniplas). Transfus Apher Sci 2007;37:223-31. 43. Depasse F, Sensebe L, Seghatchian J, et al. The influence of methylene blue light treatment and methylene blue removal fillter on fibrinogen activity states and fibrin polymerization indices. Transfus Apher Sci 2005;33:63-9. 44. Alvarez-Larran A, Del Rio J, Ramirez C, et al. Methylene blue-photoinactivated plasma vs. fresh-frozen plasma as replacement fluid for plasma exchange in thrombotic thrombocytopenic purpura. Vox Sang 2004;86:246-51.

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45. Skripchenko A, Wagner SJ, Thompson-Montgomery D, Awatefe H. Thiazole orange, a DNA-binding photosensitizer with flexible structure, can inactivate pathogens in red blood cell suspensions while maintaining red cell storage properties. Transfusion 2006;46:213-19. 46. Singh Y, Sawyer L, Pinkoski L, et al. Photochemical treatment of plasma with amotosalen and long-wavelength ultraviolet light inactivates pathogens while retaining coagulation function. Transfusion 2006;46:1168-77. 47. Ciaravino V, McCullough T, Cimino G, Sullivan T. Preclinical safety profile of plasma prepared using the INTERCEPT blood system. Vox Sang 2003;85:171-82. 48. de Alarcon P, Benjamin R, Dugdale M, et al. Fresh frozen plasma prepared with amotosalen HCl (S-59) photochemical pathogen inactivation: Transfusion of patients with congenital coagulation deficiencies. Transfusion 2005;45:1362-72. 49. Hambleton J, Wages D, Radu-Radulescu L, et al. Pharmacokinetic study of FFP photochemically treated with amotosalen (S-59) and UV light compared to FFP in healthy volunteers anticoagulated with warfarin. Transfusion 2002;42:1302-7. 50. Mintz PD, Bass NM, Petz LD, et al. Photochemically treated fresh frozen plasma for transfusion of patients with acquired coagulopathy of liver disease. Blood 2006;107:3753-60. 51. Mintz PD, Neff A, MacKenzie M, et al. A randomized, controlled phase III trial of therapeutic plasma exchange with fresh frozen plasma prepared with amotosalen and UV light compared to untreated fresh frozen plasma in thrombotic thrombocytopenic purpura. Transfusion 2006;46:1693-704. 52. Goodrich RP, Edrich RA, Li J, Seghatchian J. The Mirasol system for pathogen reduction of platelets and plasma: An overview of current status and future trends. Transfus Apher Sci 2006;35:5-17. 53. Kumar V, Lockerbie O, Keil SD, et al. Riboflavin and UV light based pathogen reduction: Extent and consequence of DNA damage at molecular level. Photochem Photobiol 2004;75:561-4. 54. Lin L, Cook DN, Wiesehahn GP, et al. Photochemical inactivation of viruses and bacteria in platelet concentrates by use of a novel psoralen and long-wavelength ultraviolet light. Transfusion 1997;37:423-35. 55. van Rhenen DJ, Vermeij J, Mayaudon V, et al. Functional characteristics of S-59 photochemically treated platelet concentrates derived from buffy coats. Vox Sang 2000;79:206-14. 56. Apelseth TO, Hervig TA, Wentzel-Larsen T, Bruserud Ø. Cytokine accumulation in photochemical treated and gamma-irradiated platelet concentrates during storage. Transfusion 2006;46:800-10. 57. Seghatchian J, de Sousa G. Pathogen-reduction systems for blood components: The current and future trends. Transfus Apher Sci 2006;35:189-96. 58. Apelseth TO, Bruserud Ø, Wentzel-Larsen T, et al. In vitro evaluation of metabolic changes and residual platelet responsiveness in photochemical treated and gamma-irradiated single donor platelet concentrates during long-term storage. Transfusion 2007;47:653-65. 59. van Rhenen DJ, Gulliksson H, Cazenave JP, et al. Transfusion of pooled buffy coat platelet components prepared with photochemical pathogen inactivation treatment: The euroSPRITE trial. Blood 2003;101:2426-33.

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60. McCullough J, Vesole DH, Benjamin RJ, et al. Therapeutic efficacy and safety of platelets treated with a photochemical process for pathogen inactivation: The SPRINT trial. Blood 2004;104:1534-41. 61. Perez-Pujol S, Tonda R, Lozano M, et al. Effects of a new pathogenreduction technology (Mirasol PRT) on functional aspects of platelet concentrates. Transfusion 2005;45:911-19. 62. Li J, Lockerbie O, de Korte D, et al. Evaluation of platelet mitochondria integrity after treatment with Mirasol pathogen reduction technology. Transfusion 2005;45:920-6. 63. AuBuchon JP, Herschel L, Roger J, et al. Efficacy of apheresis platelets treated with riboflavin and ultraviolet light for pathogen reduction. Transfusion 2005;45:1335-41. 64. Cook D, Wollowitz D, inventors. Method for inactivating pathogens in red cell compositions using quinacrine mustard. US patent 5691132. November 25, 1997. 65. Creech HJ, O’Connell AP. Immunochemistry of conjugates prepared from serum albumins and acridine nitrogen mustards (ICR mutagens). Cancer Res 1981;41:3844-51. 66. Corash L. Inactivation of viruses, bacteria, protozoa and leukocytes in platelet and red cell concentrates. Vox Sang 2000;78(Suppl 2):205-10. 67. Benjamin RJ, McCullough J, Mintz PD, et al. Therapeutic efficacy and safety of red blood cells treated with a chemical process (S-303) for pathogen inactivation: A Phase III clinical trial in cardiac surgery patients. Transfusion 2005;45:1739-49. 68. Stassinopoulos A, Castro GM, Schott MA. A modified S-303 pathogen inactivation process eliminates immunoreactivity of S-303 RBC detected in pivotal clinical trials (abstract). Haematologica 2005;90:774. 69. Preuss T, Kamstrup S, Kyvsgaard NC, et al. Comparison of two different methods for inactivation of viruses in serum. Clin Diagn Lab Immunol 1997;4:504-8. 70. Käsermann F, Wyss K, Kempf C. Virus inactivation and protein modifications by ethyleneimines. Antiviral Res 2001;52:33-41. 71. Chapman J, Moore K, Butterworth BE. Pathogen inactivation of RBCs: PEN110 reproductive toxicology studies. Transfusion 2003;43:1386-93. 72. Wagner SJ, Skripchenko A, Cincotta L, et al. Use of a flexible thiopyrulium photosensitizer and competitive inhibitor for pathogen reduction of viruses and bacteria with retention of red cell storage properties. Transfusion 2005;45:752-60. 73. Svae TE, Neisser-Svae A, Bailey A, et al. Prion safety of transfusion plasma and plasma-derivatives typically used for prophylactic treatment. Transfus Apher Sci 2008; 39:59-67. 74. Achour A. Phenothiazines and prion diseases: A potential mechanism of action towards oxidative stress. Int J Antimicrob Agents 2002;20:305-6. 75. Klein HG. Pathogen inactivation technology: Cleansing the blood supply. J Intern Med 2005;257:224-37. 76. Klein HG, Anderson D, Bernardi MJ, et al. Pathogen inactivation: Making decisions about new technologies—preliminary report of a consensus conference. Vox Sang 2007;93:179-82. 77. Mauskopf JA, Paul JE, Grant DM, et al. The role of cost-consequence analysis in health care decision-making. Pharmaeconomics 1998;13:277-88.

PART III

Noninfectious Hazards

52

Hemolytic Transfusion Reactions Robertson D. Davenport Associate Professor, Department of Pathology, The University of Michigan Medical School, Ann Arbor, Michigan, USA

A hemolytic transfusion reaction (HTR) is the accelerated clearance or lysis of transfused red cells because of immunologic incompatibility. It is distinguished from autoimmune hemolysis or nonimmune causes of shortened survival of transfused red cells. HTR may occur when antigen-positive red cells are transfused to a patient with a preexisting alloantibody, or when a recently transfused patient makes a new alloantibody. The great majority of HTRs are a result of Red Blood Cell (RBC) transfusion. However, HTRs may also result from transfusion of plasma-containing blood components, such as Fresh Frozen Plasma or Platelets, which contain red cell antibodies but very few, if any, red cells.1,2 Occasionally, HTRs may be caused by incompatibility between red cells from one donor and antibodycontaining plasma from a different donor transfused to the same recipient.3

Incidence The actual incidence of HTR is difficult to determine. Much of the data are derived from retrospective studies and are likely to suffer from underreporting. A prospective study of bedside transfusion errors over a 15-month period found that major errors occurred in 1.24% of transfused patients.4 Although only one of these errors resulted in a clinical reaction, none were reported— indicating that most errors in transfusion practice are not recognized at the time. In some cases, underlying medical conditions such as liver disease, sickle cell anemia, or bleeding may make a definite diagnosis difficult, particularly in delayed HTR (DHTR). The reported incidence of HTR depends to some degree on the recipient patient population, and is likely to be higher in academic medical centers with heavily transfused patient populations. Reports from blood centers have largely relied on surveys of transfusion services, which may result in underestimation. Improvement in antibody identification techniques over time, Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

as well as increased knowledge of immunohematology, have undoubtedly resulted in a progressive decrease in HTRs. Estimates of the incidence of HTR derived from both blood centers and transfusion services are summarized in Table 52-1. Some reports do not differentiate between acute and delayed reactions, or between intravascular and extravascular hemolysis. HTRs have not necessarily been differentiated from serologic reactions without hemolysis. Overall, there has been an apparent increase in the incidence of DHTR and a decreased in acute HTR (AHTR) with time. Factors contributing to a higher rate of delayed reactions include improved serologic detection, longer survival of transfusion patients, and increasing total number of red cell transfusions. Because most patients receive more than one RBC unit, estimates of the incidence of HTR per transfused patient range from 1:854 to 1:524, which is higher than the incidence per unit transfused.11,15 The population incidence rate of DHTR has been estimated at 1.69 events per 100,000 population per year.15 Table 52-1. Estimated Incidence of Hemolytic Transfusion Reactions (HTRs) Setting

Period

HTR Risk Estimate per Unit Transfused

Overall HTR (acute and delayed reactions not differentiated) 1981-1987 1:15,605 Blood center5 Oncology patients6 1974-1981 1:35,739 Acute HTR Tertiary care medical center7 Tertiary care medical center8 Reported transfusion errors*9

1964-1973 1974-1977 1990-1999

1:12,100 1:21,222 1:77,000

Delayed HTR Tertiary care medical center10 Tertiary care medical center†8 Tertiary care medical center11 Blood center12 Tertiary care medical center13 Tertiary care medical center14

1964-1973 1974-1977 1980-1992 1980-1981 1986-1987 1974-1978

1:11,652 1:4,015 1:5,405 1:6,875 1:9,094 1:2,339

*Clinical reactions in ABO transfusion errors. † Same institution as previous, improved serologic detection.

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Section IV: Part III

Relative value (not to scale)

Serum bilirubin Plasma hemoglobin Haptoglobin Urine hemoglobin Platelet count Fibrinogen

0

12 Time after transfusion (hours)

Since 1996, the Serious Hazards of Transfusion (SHOT) program in the United Kingdom has reported and tracked HTRs. In 2004, HTRs caused by ABO-incompatible RBC transfusions were reported in 19 of 2,607,410 units issued, for a rate of 1:137,000.16 The number of patients transfused or units transfused was not reported. However, the wastage rate was estimated to be less than 5%. The number of ABO-incompatible transfusions reported annually to SHOT officials since the program’s inception has varied from 13 to 36. The number of units issued was not reported for years prior to 2004. Data on HTRs from the Quebec Hemovigilance System covering 2000 to 2004 have been reported.17 Over a 5-year period, 47 ABO-incompatible transfusions, 55 AHTRs, and 91 DHTRs out of 7059 total transfusion reactions occurred. The incidence per RBC unit issued was 1:27,318 for ABO incompatibility, 1:14,901 for AHTR, and 1:9313 for DHTR. The most common cause of AHTR was ABO incompatibility, accounting for 30% of reactions. HTRs are classified as acute or delayed reactions, based on whether they occur within 24 hours or after 24 hours of the implicated transfusion. A more important distinction is between intravascular and extravascular hemolytic reactions. Intravascular hemolysis is characterized by hemoglobinemia and hemoglobinuria. In contrast, extravascular hemolysis lacks these dramatic signs, but is characterized by shortened survival of transfused red cells along with the accumulation of hemoglobin breakdown products. Generally, intravascular hemolysis is seen with AHTR, while extravascular hemolysis is usually seen in DHTR. This distinction is not absolute, however. Occasional acute reactions

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24

Figure 52-1. Time course of hemolytic and coagulation parameters in intravascular HTR. Adapted with permission from Duvall et al.18

result in extravascular hemolysis, and some intravascular hemolytic reactions are delayed. Accelerated clearance of incompatible red cells is an essential feature of HTRs. Transfusion may also stimulate the production of alloantibody without hemolysis. This phenomenon has been termed delayed serologic transfusion reaction (DSTR), and needs to be differentiated from delayed HTR.11,13 DSTR is clinically benign, but predisposes to later HTR. Earlier reports of hemolytic reactions have not necessarily differentiated clearly between serologic and true hemolytic reactions.

Signs and Symptoms There is a broad range of initial clinical presentations of HTR. The typical progression of intravascular and extravascular hemolysis is shown in Fig 52-1 and Fig 52-2. In all cases, there is an unexpected degree of anemia from the loss of transfused red blood cells. In some cases, particularly with extravascular hemolysis, this is the only clue to HTR. Some reactions, both immediate and delayed, are asymptomatic. In intravascular hemolysis, however, most symptomatic patients experience fever and/or chills. Nausea or vomiting, pain, dyspnea, and hypotension or tachycardia are also common initial symptoms. The reported pain may localize to the infusion site, back, flanks, chest, groin, or head. The cause of pain in HTR is unclear, but is most likely the result of direct stimulation of nociceptive nerves in perivascular

Chapter 52: Hemolytic Transfusion Reactions

Relative value (not to scale)

Hemoglobin

Figure 52-2. Time course of hemolytic parameters in extravascular hemolysis. Adapted from Cummins et al.19

Conjugated bilirubin Unconjugated bilirubin

0

7

14

Time after transfusion (days)

tissue by bradykinin generated from activation of the complement system.20 In extravascular hemolysis, fever and/or chills are the most commonly reported initial symptoms. Jaundice also may be an initial sign because elevation of serum bilirubin occurs with both intravascular and extravascular hemolysis. The degree of hyperbilirubinemia depends upon the patient’s liver function and rate of red cell destruction. Conjugated and unconjugated bilirubin fractions tend to follow a parallel course, peaking at the same time.19 Delta bilirubin, a minor albumin-bound fraction, persists past the peak of total bilirubin, and is a useful clue to previous hemolysis. Although HTR typically presents during or shortly after the offending transfusion, the time from transfusion to clinical presentation of DHTR is quite variable. Most delayed reactions present within 2 weeks after transfusion, but the initial presentation may be up to 6 weeks later, because of the time required for antibody production.

Complications Autoantibody is found in about 28% of patients concomitantly with alloantibody, although the reported range is 15% to 53%.21 A positive direct antiglobulin test (DAT) caused by IgG may persist for many months, and there may be evidence of complement deposition on autologous red cells, with a positive DAT caused by C3 persisting for up to 100 days.22 In most cases, there is no evidence of loss of autologous red cells; however, so-called “bystander hemolysis” can occur. It is not possible to distinguish between autoimmune hemolytic anemia (AIHA) and bystander hemolysis by serologic testing alone. Patients with sickle cell anemia can present a particular challenge. HTR can precipitate sickle crisis. Factors that contribute to the occurrence of sickle crisis include increased oxygen consumption resulting from fever, the relative loss of circulating hemoglobin A compared to hemoglobin S, and the release of vasoactive mediators causing reduction of local blood flow. HTRs may be particularly

severe in patients with sickle cell disease. In such reactions the degree of anemia may actually be greater than before transfusion, probably because of bystander hemolysis of autologous red cells. This phenomenon has been termed the sickle cell HTR syndrome.23 In addition to hemolysis, there is often suppression of erthropoiesis as indicated by a marked drop in the reticulocyte count. Demonstration of an increase in corrected reticulocyte count, increase in the absolute number of hemoglobin-Scontaining red cells, or decrease in the ratio of red cell hemoglobin to reticulocyte hemoglobin can indicate the occurrence of hyperhemolysis.23-25 Pain crisis in a sickle cell patient following transfusion should suggest the occurrence of sickle cell HTR syndrome. Further transfusion in this setting may exacerbate the anemia and even result in fatality. Serologic studies often do not provide an explanation for HTR in these patients, because the causative antibody can be at an undetectable level in the serum during the reaction. In addition, the presence of multiple alloantibodies may make the serologic diagnosis difficult. Hypotension occurs in some cases of intravascular HTR but is rare in extravascular reactions. Complement activation is likely to be the most important determining factor. The anaphylatoxins C3a, C4a, C5a, and C5a-des-arg are released during immune hemolysis. Additionally, consumption of C1-esterase inhibitor contributes to activation of the kinin pathway, leading to generation of bradykinin.26 The proinflammatory cytokines tumor necrosis factor (TNF) and interleukin (IL)-1 produced by phagocytes during HTR also may contribute to hypotension and shock. Impairment of renal function is seen in both intravascular and extravascular HTR, although it is more common in the former. The degree of renal function abnormality varies from an asymptomatic elevation of serum blood urea nitrogen (BUN) and creatinine to complete anuria. Both hypotension and intravascular coagulation contribute to renal impairment. Thrombus formation in renal arterioles caused by disseminated intravascular coagulation (DIC) may cause cortical infarcts. Free hemoglobin contributes to renal injury, causing so-called pigment nephropathy. Experimental evidence indicates that hemoglobin is toxic to renal tubular epithelium cells.27

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Section IV: Part III

Intravascular hemolysis modulates blood pressure and local blood flow through alterations in the metabolism of the potent physiologic vasodilator nitric oxide (NO). NO can combine with heme and thiol groups of hemoglobin.28 In oxyhemoglobin, NO causes reduction of ferrous iron (Fe2⫹) to form ferric methemoglobin [Hb(Fe3⫹)]. NO combines with deoxyhemoglobin to form Hb(Fe2⫹)NO, but does not cause reduction. Scavenging of NO by free hemoglobin through these pathways results in vasoconstriction and hypertension. NO can also combine with cysteine on the beta globin chain to form S-nitrosohemoglobin (SNO-Hb). This process is reversible, so SNO-Hb can act as a NO donor with resultant vasodilation. Free hemoglobin reacts with NO much more rapidly than does intraerythrocytic hemoglobin.29 The effects of intravascular hemolysis may be very similar to the infusion of stroma-free hemoglobin. Indeed, prominent hypertensive effects limited early trials of hemoglobin solutions as a blood substitute.30 Disseminated intravascular coagulation occurs in intravascular HTR, but is relatively rare. Rarer still is the occurrence of DIC in extravascular HTR. The production of proinflammatory cytokines is likely to be a major factor in DIC. It can be difficult to distinguish DIC from other causes of coagulopathy, particularly in massive transfusion or liver disease. Uncontrolled bleeding caused by DIC may be the initial manifestation of an acute HTR, particularly in the intraoperative setting. Unfortunately, if HTR is not recognized early in this situation, more incompatible blood may be transfused in an attempt to keep up with blood loss. In one report, nine of 35 patients experiencing HTR while under anesthesia received 4 to 6 additional units because of excessive bleeding.31 Fatality from HTR is usually associated with intravascular hemolysis, but severe extravascular reactions may also cause death.7,32 It is difficult to estimate the actual rate of mortality, but the best available data are derived from reports to regulatory agencies. A review of transfusion-associated deaths reported to the Food and Drug Administration (FDA) between 1976 and 1985 attributed 158 fatalities to acute hemolysis and 26 to delayed hemolysis.33 Assuming that 100 million RBC units were transfused in this time, the mortality rate for AHTR would be 1:630,000 and that for DHTR would be 1:3,850,000 per unit transfused. Similarly, assuming that 30 million patients were transfused in this time, the per patient mortality rate for AHTR would be 1:190,00, and for DHTR would be 1:1,150,000. An earlier review of transfusion fatalities reported to the FDA covering 1976 to 1978 estimated the death rate from HTR to be 1:587,000 per unit transfused.34 A recent update has extended the analysis of FDA fatality reports through 1995.35 Since 1990 the number of deaths reported to the FDA annually has increased, probably because of increased vigilance in reporting. The total number of deaths (151) from acute and delayed hemolysis reported between 1986 and 1995 was essentially unchanged from the prior 10 years. However, acute hemolysis accounted for a higher proportion of deaths (62%) during the first decade, than during the second decade (50%). This difference was caused by a higher incidence

814

of deaths from other causes including bacterial contamination and acute lung injury in the later report. The total number and proportion of deaths from delayed HTR was unchanged between the two reporting periods (26 or 10% vs 29 or 10%). An analysis of blood transfusion errors reported to the New York State Health Department, from January 1990 to October 1991, reported a death rate of 1:600,000 per unit transfused.36 When these data were extended to the period 1990 to 1999, the fatality rate from erroneous administration of RBC units was 1:1,800,000.9 It is not surprising that different studies result in discrepant estimates of mortality rate, because these figures are based on small numbers of events. The cumulative SHOT data from 1996 to 2004 includes 245 ABO-incompatible transfusions. Among these, death related to transfusion occurred in 7% of cases, major morbidity in 22%, and no ill effect in 63%.16,37 Mortality is dependent on the amount of incompatible red cells transfused. A review of 41 HTRs causing acute renal failure indicated that no deaths occurred among patients receiving less than 500 mL of incompatible blood; there was 25% mortality in the group receiving 500 to 1000 mL and 44% mortality among those receiving greater than 1000 mL of incompatible blood.31 However, transfusion of even small amounts of incompatible blood is not necessarily safe. At least 12 deaths have been reported to the FDA involving transfusion of less than 1 unit of blood.33

Causes of Hemolytic Transfusion Reactions Hemolytic transfusion reactions usually result from inadvertent administration of incompatible blood components, or the failure to detect a potential incompatibility. Rarely, incompatible blood components are deliberately transfused before ABO-incompatible marrow infusion or when compatible blood cannot be obtained, with an expectation of possible HTR. Among transfusion-related fatalities from acute hemolysis reported to the FDA during 1976 to 1985, 86% were the result of process errors.33 Among these errors, 10% occurred in phlebotomy and ordering departments, 33% occurred within the blood bank, and 57% occurred during transfusion administration. These frequencies are similar to reported transfusion errors in New York State, where 22% occurred in phlebotomy and ordering departments, 32% occurred within the blood bank, and 46% occurred during transfusion administration.36 More recent figures indicate that non-blood-bank errors account for 56%, blood bank errors account for 29%, and compound errors account for the remaining 15% of transfusion errors.9 In part, differences between the FDA and New York figures are accounted for by the inclusion of nonfatal errors in the latter. Serendipitously, a transfusion error may result in administration of compatible blood components, as occurred in a third of the reported New York State events. Misidentification of a pretransfusion sample has been termed “wrong blood in tube” (WBIT). A study covering 62 institutions

Chapter 52: Hemolytic Transfusion Reactions

in 10 countries performed by the Biomedical Excellence for Safer Transfusion Working Party of the International Society for Blood Transfusion has assessed the frequency of WBIT.38 On the basis of results from over 690,000 samples, it was determined mislabeling occurred in 1 in every 165 samples. Two countries with national patient identification systems, Sweden and Finland, had miscollection rates too low to quantify. Outside these nations, miscollected samples demonstrating WBIT occurred at a median rate of 1 in every 1986 samples. The apparent discrepancy between mislabeling and WBIT is because not all mislabeled tubes are miscollected. Data on the failure of pretransfusion tests to detect potential incompatibility are scant. This is more likely to be a factor in DHTR than in AHTR. A Mayo Clinic study suggested that at least five of 37 cases (14%) of delayed HTR could have been prevented by improving the sensitivity of red cell antibody screening procedures.8 The deliberate, physician-guided administration of incompatible blood components may occur in platelet transfusion, urgent transfusion required in patients with multiple alloantibodies or antibodies to high-incidence antigens, or in marrow transplantation. ABO-incompatible red cells have been successfully administered to patients before major ABO-incompatible marrow transplants in an effort to reduce the titer of isoagglutinins.39 These patients have predictable, but manageable, reactions. In a retrospective study of 35 patients receiving major ABO-incompatible peripheral blood hematopoietic progenitor cell transplants who were transfused with 1 ABO-incompatible RBC unit, 23 had no clinical reaction, and four had severe reactions necessitating discontinuation of the transfusion.40 Some patients who have received incompatible transfusions involving non-ABO antibodies were treated with high-dose intravenous immunoglobulin (IVIG) before transfusion and have not experienced HTR.41 High levels of anti-A or anti-B in platelet concentrates, particularly apheresis products, can cause acute hemolysis.42-45 There is considerable variability in the strength of the implicated antibody, but a high-titer isoagglutinin is usually present in donor plasma when hemolysis occurs. A study of apheresis group O platelets found that 26% had anti-A or anti-A,B titer of 64 or greater, a level that has been associated with HTR.46 A study of pooled whole-blood-derived platelets found 60% of group O pools had anti-A titer at least 64.47 Repeated platelet transfusions within a brief period can result in sufficient accumulation of anti-A to cause severe acute hemolysis.48 However, there is no clear consensus as to what constitutes a critical titer of ABO antibodies. At variance with reports of acute hemolysis, apheresis platelet concentrates are often transfused across ABO groups without adverse consequences. In one study 16 patients who received both ABO-compatible and -incompatible platelet concentrates were evaluated for evidence of hemolysis.49 There was no difference between preand posttransfusion hemoglobin levels in 24 paired transfusion episodes.

Diagnosis Diagnosis of HTR requires clinical suspicion, especially when the transfusion occurred days to weeks previously. The initial laboratory evaluation includes confirmation of the ABO group, a test for free hemoglobin, and a DAT performed on a postreaction blood specimen (Table 52-2). Visual inspection of the postreaction plasma can detect hemoglobin in the range of 20 to 50 mg/ dL, equivalent to the lysis of approximately 10 mL of red cells in an adult.50 It should be remembered that free serum hemoglobin also may be present in nonimmune hemolysis, red cell fragility syndromes, hemoglobinopathies, severe burns, polyagglutination, or infusion of hemoglobin-based oxygen-carrying solutions. A common cause of a false-positive test result for free hemoglobin is drawing a sample through an indwelling catheter using inappropriate technique. Percutaneous intravascular thromboectomy can cause marked hemoglobinemia.51 A false-negative test result for free hemoglobin may occur if too much time has been allowed to elapse before obtaining the postreaction specimen. Table 52-2. Laboratory Investigation of Hemolytic Transfusion Reactions First-tier investigation ● Posttransfusion serum hemoglobin (qualitative) ● Posttransfusion direct antiglobulin test ● Confirmation of posttransfusion ABO/Rh Second-tier investigation ● Repeat pretransfusion ABO/Rh ● Pre- and posttransfusion antibody screen ● Repeat special antigen typing ● Crossmatch with pre- and postreaction specimens Third-tier investigation ● Antibody identification panels on pre- and postreaction samples ● Enhanced antibody screening method: PEG, extended incubation, gel, enzymes ● Red cell eluate on pre- and postreaction samples ● Investigation of transfusion technique and blood storage conditions ● Check of the blood bag, tubing, and segments for hemolysis ● Enhanced crossmatches: PEG, enzymes ● Minor crossmatches of implicated units ● Antibody detection tests on donor units ● Tests for polyagglutination ● Hemoglobin electrophoresis ● Quantitative serum hemoglobin ● Serum haptoglobin ● Serum bilirubin (conjugated and unconjugated) ● Urine hemoglobin and hemosiderin ● Bacterial culture and Gram’s stain of blood bags ● Serum BUN and creatinine ● Peripheral blood smear ● Serial hemoglobin, hematocrit, and platelet count ● Blood coagulation studies (PT, aPTT, fibrinogen, FDP) ● DAT on donor units PEG ⫽ polyethylene glycol; BUN ⫽ blood urea nitrogen; PT ⫽ prothrombin time; aPTT ⫽ activated partial thromboplastin time; FDP ⫽ fibrin degradation product; DAT ⫽ direct antiglobulin test.

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A low level of free hemoglobin may be difficult to detect in an icteric specimen. The DAT may be positive because of the effects of drugs, autoimmune disease, or autoantibodies. If the postreaction DAT is positive, then a test should be performed on a stored prereaction sample. If the prereaction sample is also a positive, then the test is not valid for the purpose of detecting, or excluding, the presence of alloantibody-coated transfused cells and further testing is required. The DAT will be negative if transfused antigen-positive cells have been cleared from circulation. In addition, when small numbers of antibody-coated allogeneic red cells are present, a DAT performed by routine methods may not be sufficiently sensitive. In a study that compared the relative sensitivity of DAT performed with monospecific IgG antiglobulin technique, flow cytometry, and antibody elution, the DAT could detect 10% antibody-coated cells.52 Antibody could be detected in the eluate of the samples with as little as 1% antibody-coated red cells present, although in some cases it was not nearly as sensitive. Flow cytometry, however, was consistently the most sensitive method with a detection limit of approximately 1%. The hemoglobin and antiglobulin tests are useful for screening purposes. However, if either of these is positive, or if there is a strong clinical suspicion, then further testing must be performed. At this point, the implicated blood bags with their attached administration sets should be returned to the blood bank, and all units dispensed to the patient should be returned and quarantined. Units that were transfused within the past 24 hours should also be identified. The ABO grouping and Rh typing of both pre- and postreaction blood specimens should be repeated. Care should be taken to look for mixed-field agglutination. The antibody screen should be repeated on both specimens, and any special antigen typing of donor units should be repeated. Blood from saved segments or recovered bags and tubing of units transfused within 24 hours should be crossmatched against both the pre- and postreaction specimens. This testing should include a 37ºC reading and an indirect antiglobulin test. If a large number of units have been transfused in the last 24 hours, the blood bank medical director may elect to substitute reconfirmation of ABO grouping of donor units. Negative results in these investigations usually rule out HTR, except in unusual circumstances. Other causes of hemolysis or shortened red cell survival that should be considered in the differential diagnosis of HTR are listed in Table 52-3. Nonimmune hemolysis can have a similar clinical presentation to HTR. Lysis of red cells can be caused by overheating in a blood warmer, or by freezing. Hemolysis can also be caused by inadequate removal of glycerol from frozen red cells or by attempting to force blood through a filter or small-bore needle. The transfusion of outdated blood has been reported to cause hemoglobinuria and transient hemodynamic, pulmonary, and renal changes.53 Transfusion administered with hypotonic solutions or some drugs may also cause hemolysis.54 Intravenous dimethylsulfoxide infusion has been reported to mimic HTR.55 In general, patients tolerate the infusion of

816

hemolyzed blood remarkable well. Often the only sign of an adverse event is hemoglobinuria. However, deaths caused by the transfusion of hemolyzed blood have been reported.33 Some hematologic abnormalities, particularly autoimmune hemolytic anemia, can have presentations similar to HTR. Patients with congenital hemolytic anemias, such as glucose-6phosphate dehydrogenase deficiency, may manifest hemolysis after blood transfusion.56 Conversely, blood donated by individuals with glucose-6-phosphate dehydrogenase deficiency can cause hemoglobinemia and hyperbilirubinemia in transfusion recipients.57 Establishing the diagnosis of HTR may be particularly difficult in patients with liver disease, autoimmune hemolytic anemia, sickle cell anemia, or active bleeding. In chronic liver disease there is often a positive DAT, hyperbilirubinemia, and elevated lactate dehydrogenase (LDH). The clinical and laboratory presentation of AIHA may be identical to HTR. Concern has been raised that transfusion may aggravate hemolysis in

Table 52-3. Differential Diagnosis of Hemolytic Transfusion Reactions ● ● ● ● ●

●

●

● ●

● ● ● ● ●

Alloantibody-induced hemolysis Delayed serologic transfusion reaction Autoimmune hemolytic anemia Cold hemagglutinin disease Nonimmune hemolysis – Incompatible fluids – Improper storage – Malfunctioning blood warmer – Small needles, high hematocrit – Improper deglycerolization – Infusion pumps – Bacterial contamination – Mechanical thrombectomy Hemolytic anemia – G6PD deficiency – Congenital spherocytic anemia Hemoglobinopathies – Sickle cell disease – Sickle cell transfusion reaction syndrome Drug-induced hemolysis Microangiopathic hemolytic anemias – Thrombotic thrombocytopenic purpura – Hemolytic uremic syndrome – HELLP syndrome Bleeding Artificial heart valve dysfunction Paroxysmal nocturnal hemoglobinuria Polyaggultination Infections – Clostridium perfringens – Malaria – Babesiosis

G6PD ⫽ glucose-6-phosphate dehydrogenase; HELLP ⫽ hemolysis, elevated liver enzymes, and low platelet count.

Chapter 52: Hemolytic Transfusion Reactions

AIHA, although one published study has suggested that this is not usually the case even in the face of serologic incompatibility.58 Characteristically, in both bleeding and AIHA there is proportionate loss of both autologous and transfused red cells. One indication of HTR in these settings is the persistence of transfused red cells that lack the implicated antigen, but the absence of transfused cells bearing the antigen. Resorption of a hematoma can have manifestations very similar to extravascular HTR. Such patients have an unconjugated hyperbilirubinemia, elevated LDH, and depressed haptoglobin levels. In addition, the presence in the serum of fibrin degradation products (FDPs) from the hematoma may be confused with DIC. In these patients, as in bleeding patients, persistent circulating antigen-positive red cells and a negative posttransfusion DAT are evidence against the diagnosis of HTR. The serologic specificity of a red cell antibody is an indication of its clinical significance. However, there is not an absolute correlation between specificity and presence or absence of red cell destruction. The general clinical significance of many red cell antibody specificities is summarized in Table 52-4.

Pathophysiology The pathophysiologic mechanisms involved in HTR are not well understood. There are essentially three phases: antibody-antigen interaction, phagocytosis and inflammatory cell activation, and systemic response. Initially, there is a binding of antibody to red cell antigens, which can result in complement activation. Second, immunoglobulin- and complement-coated cells interact with phagocytes, resulting in clearance of red cells and activation of phagocytes. Third, the inflammatory mediators produced in the first two phases act on a variety of cell types, causing a clinical manifestations of HTR. The course of immune hemolysis is determined by antigen site density, immunoglobulin class of the alloantibody, and activation of complement. ABO antigens are present in high numbers on a red cell surface, approximately 5 ⫻ 105 per cell.50 In contrast, there are 103 to 104 antigens per cell in the Rh, Kell, Kidd, and Duffy systems. Complement fixation is facilitated by close proximity of antigens that allows bridging of IgG molecules by C1q. However, IgM antibodies can fix complement without a requirement for bridging between molecules. IgM antibodies are common in the ABO system, but relatively unusual as alloantibodies to other antigens. Activation of the classical pathway of complement proceeds from C1q binding through C3 activation. Cleavage of C3 results in C3a liberation into circulation and C3b deposition on the red cell membrane. Activated C3 may then cleave C5 with release of C5a. Assembly of the membrane attack complex then may proceed with resultant intravascular hemolysis. Factor I, also known as C3b inactivator, is the major regulator of C3b activity. Cleavage of membrane-bound C3b by factor I results in the generation of iC3b and release of the small peptide fragment C3c. This terminates the complement cascade

because iC3b is enzymatically inactive. iC3b is further degraded into C3dg and C3d by factor I and trypsin-like proteinases. Erythrophagocytosis results from interaction of immunoglobulin- and/or complement-coated red cells with phagocyte receptors. Cell-bound antibodies promote red cell clearance primarily through interaction of the Fc portion of IgG with specific receptors. Among the IgG receptors, FcγRI principally mediates red cell phagocytosis by monocytes.59 However, this receptor has a high affinity for monomeric IgG, and is blocked by normal serum concentrations of IgG.60 FcγRIII appears to be the most important IgG receptor on splenic macrophages in alloimmune and autoimmune red cell clearance, as well as in autoimmune thrombocytopenia.61-63 The principal complement receptor expressed by macrophages and monocytes, CR3, primarily recognizes iC3b. Receptors for C3a and C5a are present on a wide variety of cells including monocytes, macrophages, neutrophils, platelets, endothelium, and smooth muscle. The physiologic

Table 52-4. General Clinical Significance of Red Cell Antibodies Blood Group System

Generally Clinically Significant* Specificities

Generally Clinically Insignificant Specificities

ABO, H Lewis P I/i

All -Lea, -Lea⫹Leb -P, -P⫹P1⫹Pk (Tja) -I, -i

-A1 not reactive at 37ºC -Leb -P1, -Pk -IH, -IA, -IB, -iH, -IP1 not reactive at 37ºC

Rh Duffy MNSs Lutheran Kell Kidd Cartwright Diego Colton Dombrock Cromer Augustine Vel Lan Sid LW Gerbich Xg Scianna Chido/Rogers Indian Cost/York Knops/McCoy JMH Holly/Gregory (Dombrock) Bg (HLA)

All All All -Lub All All -Yta, -Ytb -Dia,- Dib, -Wra -Coa, -Co3 -Doa, -Dob -Cra, -Tca -Ata -Vel -Lan

-Inb

Possibly -Fy6 -M not reactive at 37ºC -Lua

-Cob

-Sda All All All All All -Ina -Csa, -Yka, -Ykb -Kna, -Knb -JMH -Gya, -Hy All

*Resulting in hemolytic transfusion reaction or decreased red cell survival.

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Section IV: Part III

effects of C3a and C5a include oxygen radical production, granule enzyme release, leukotriene production, NO production, and cytokine production. These low-molecular-weight peptides can also produce vasodilation and bronchoconstriction. Ligation of phagocyte receptors results in cellular activation and production of inflammatory response factors. An experimental model of ABO incompatibility has suggested that monocytes are the leukocyte subpopulation most directly involved in AHTR.64 Incompatible red cells induce a reduction in CD14 and increase in CD44 expression on monocytes in whole blood. After 24 hours incubation with incompatible red cells, monocytes showed particularly high levels of CD44. These data demonstrate that monocyte activation is critical in the development of intravascular HTR. Immune hemolysis stimulates the production of a variety of cytokines that are crucial to the initiation, maintenance, and ultimate resolution of HTR (Table 52-5). ABO incompatibility with intravascular HTR strongly stimulates production of TNF-α and the chemokines CXCL8 (IL-8) and CCL2 (MCP-1).65-68 TNF-α is an early response, appearing in plasma within 2 hours, and has potent proinflammatory effects, including pyrogenic activity, leukocyte activation, stimulation of procoagulant activity, and expression of a large number of gene products related to the inflammatory response. TNF-α produced in blood during ABO incompatibility will stimulate endothelial cells to express leukocyte adhesion molecules, chemotactic cytokines, and

Table 52-5. Cytokines Involved in Immune Hemolysis Terminology ●

Proinflammatory cytokines: – Interleukin-1 (IL-1β) – Tumor necrosis factor (TNF-α)

Biologic Activities ● ● ● ● ●

● ●

– Interleukin-6 (IL-6)

● ● ● ●

●

Chemokines: – CXLC8

● ● ● ●

– CCL5

● ● ● ●

●

Anti-inflammatory cytokines: – Interleukin-1 receptor antagonist (IL-1ra, IRAP)

818

●

Fever Hypotension, shock, death (synergy) Mobilization of leukocytes from marrow Activation of T cells and B cells Induction of cytokines (IL-1β, IL-6, CXCL8, TNF-α, CCL5) Induction of adhesion molecules Induction of procoagulants Fever Acute phase protein response B-cell antibody production T-cell activation Chemotaxis of neutrophils Chemotaxis of lymphocytes Neutrophil activation Basophil histamine release Chemotaxis of monocytes Induction of respiratory burst Induction of adhesion molecules Induction of IL-1β Competitive inhibition of IL-1 type I and II receptors

procoagulant activity.69 CXCL8 and CCL2 produced in blood during ABO incompatibility appear later than TNF-α and reach very high levels. CXCL8 primarily activates neutrophils to undergo the respiratory burst, release granule contents and alter surface adhesion molecules.70 CCL2 is primarily a chemotactic and activating factor for monocytes.71 There is also evidence for the production of cytokines in IgG-mediated extravascular hemolysis.72,73 There appear to be two categories of cytokine responses in this setting: those produced at high levels (greater than 1 ng/mL by 24 hours), and others produced at lower level (in the range of 100 pg/mL).72 Low-level cytokine responses include IL-1β, IL-6, and TNF-α. CXCL8 is a high-level response with a time course similar to that of ABO incompatibility. In contrast to the setting of ABO incompatibility, TNF-α is produced in a delayed fashion in response to IgG-coated red cells, achieving a level of less than 100 pg/mL. However, cell-associated TNF-α can be demonstrated by immunocytochemical staining in monocytes engaged in erythrophagocytosis. While the in-vitro models employed in these studies are not directly comparable, these findings do suggest a possible reason for the clinical differences between intravascular and extravascular HTRs. In the former case, TNF-α is released into systemic circulation where it can have diverse effects on many cell types; in the latter case, TNF-α effects may be confined to the site of erythrophagocytosis, primarily the spleen. Both IL-1β and IL6 produced by monocytes in response to IgG-coated red cells increase progressively over 24 hours to levels approximating 100 pg/mL. Because IL-1β and IL-6 are B-cell growth and differentiation factors, the production of these two cytokines promotes the production of red cell allo- and autoantibodies that are often associated with DHTRs. IgG-mediated hemolysis also results in the production of the IL-1β inhibitor IL-1ra.74 Significant levels of IL-1ra appear in a parallel fashion to IL-1β. Immunocytochemical staining has demonstrated strong reactivity for IL-1ra in monocytes engaged in erythrophagocytosis. Northern blot analysis of mononuclear cell RNA shows that IL-1β gene expression precedes that of IL1ra in response to IgG-coated red cells. However, neutralizing antibodies to IL-1β do not suppress either IL-1ra or IL-1β gene expression in this setting. Therefore, it appears that IL-1ra production is a primary response to the IgG-coated red cell stimulus, rather than an autocrine phenomenon induced by initial IL-1β production. Treatment of mononuclear cells with the steroid dexamethasone inhibits IL-1ra production in response to IgG-coated red cells. These data suggest the possibility that the clinical variability of DHTR, and some of the clinical differences from intravascular HTR, may be accounted for, in part, by the relative balance of IL-1β and IL-1ra production. Labile blood pressure is a feature of severe HTR, particularly with intravascular hemolysis. Both complement activation products such as C5a and cytokines such as IL-1β and TNF-α can contribute to hypotension. The common pathway of these mediators is the production of NO by endothelial cells. NO, in

Chapter 52: Hemolytic Transfusion Reactions

turn, causes relaxation of vascular smooth muscle. Hypotension and deposition of thrombi in arterioles, which impair cortical blood flow, are the major factors that contribute to renal failure. In addition, there may be direct effects of inflammatory mediators on the kidneys. There are several mechanisms by which HTR results in intravascular coagulation. TNF-α produced during immune hemolysis can induce tissue factor expression by endothelial cells.69 Tissue factor is an initiator of the extrinsic pathway that functions as a cofactor for Factors VII and VIIa to accelerate the activation of Factors IX and X. TNF-α and IL-1β, acting on endothelial cells, will also decrease the cell surface expression of thrombomodulin. Thrombomodulin is normally present on endothelial cells, and binds thrombin to activate the coagulation inhibitor protein C. Intravascular hemolysis, as in ABO incompatibility, will also induce procoagulant activity in blood leukocytes, largely because of tissue factor expression.74 This cellular procoagulant is partly inhibited by blocking antibodies to tissue factor and partly dependent of the multifunctional adhesion protein CD11b. Knowledge of the pathophysiology of HTR has been limited by the lack of good animal models. However, a recently developed model using transgenic mice expressing human glycophorin A holds promise.75 Administration of IgM and IgG anti-GPA results in intravascular or extravascular hemolysis, respectively. Dependence on active complement and Fcγ receptors has been shown.

summarized in Table 52-6. The severity of HTR is directly related to volume and rate of infusion of incompatible blood. Thus, early recognition, stopping transfusion, and preventing the transfusion of additional incompatible units is the first essential step of treatment. Initial attention must be paid to cardiovascular support. If hypotension is present, fluid resuscitation and pressor support should be considered. Care should be taken to avoid fluid overload, however, especially in patients with impaired cardiac or renal function. Pulmonary artery catheterization is useful in selected patients to guide resuscitation. Because intravascular hemolysis is an expected consequence of the infusion of ABO-incompatible marrow, some guidance can be obtained from published reports. Isoagglutinin titer clearly influences the clinical response to ABO-incompatible marrow infusion. In general, antibody titers below 64 are associated with mild or no reactions while high titers such as 1024 are associated with significant clinical reactions. The volume of incompatible red cells infused with the marrow also determines the magnitude of the response. One protocol reported to be successful in patients receiving major ABO-incompatible transplants involved preparatory hydration with 5% dextrose in onehalf normal saline with 30 to 40 mEq sodium bicarbonate/L and 15 mEq potassium chloride/L at a rate of 3000 mL/m2/day, and 100 mL/m2. Mannitol 20% was given 1 hour before marrow infusion.76 During marrow infusion, the infusion rate of fluids was increased to 4500 to 6000 mL/m2/day and additional mannitol was given at a rate of 30 mL/m2/hour for the next 12 hours. The rationale for this protocol was to maintain a high rate of urine output and prevent precipitation of hemoglobin in the renal tubules. All these patients had a preinfusion antibody titer no greater than 32. None experienced a clinical reaction to the infusion of approximately 120 to 160 mL of incompatible red cells. The deliberate transfusion of ABO-incompatible red cells before transplantation has been employed to reduce

Therapy Patients who have minimal symptoms are best managed by careful observation. However, early vigorous intervention in severe reactions saves lives. Therapeutic options in HTR are Table 52-6. Therapeutic Options in Hemolytic Transfusion Reactions Therapeutic Intervention

Indication

Hydration

● ●

Alkalinization of urine

● ●

Diuresis

●

Typical Dose

Prevent renal impairment Maintain urine output ⬎100 mL/hour

●

Normal saline and/or 5% dextrose 200 mL/m2/hour

Prevent renal impairment Maintain urine pH ⬎7.5

●

NaH2CO3 40 to 70 mEq in 1 liter 5% dextrose

Prevent renal impairment

●

Mannitol 20% 100 mL/m2* Furosemide 40 to 80 mg

●

Vasodilation

●

Increase renal blood flow

●

Dopamine 1 to 5 µg/kg/minute

Anticoagulation

●

Treat intravascular coagulation

●

Heparin 5 to 10 units/kg/hour, 0.15 to 0.25 unit per mL

Red cell exchange transfusion

●

Decrease load of incompatible red cells

●

Exchange of one estimated red cell mass

Plasma or platelet transfusion

●

Treat hemorrhagic complications of disseminated intravascular coagulation

●

Platelets: 1 unit Platelets/10 kg (max. 6 units) or 1 unit Apheresis Platelets

●

Plasma: 10 mL/kg Fresh Frozen Plasma

●

400 mg/kg

Intravenous immunoglobulin

●

Prevent extravascular hemolysis

†

*Ensure adequate renal function to prevent fluid overload from increased intravascular volume. † Investigational. Not standard therapy.

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Section IV: Part III

isoagglutinin titers. In one such protocol, 1 incompatible RBC unit was given over 8 hours on each of 2 days immediately before transplant.39 These patients were monitored in an intensive care unit and hydrated with normal saline and 5% dextrose (1:1 ratio) at a rate of 3000 mL/m2/day. Sodium bicarbonate was administered to maintain the urine pH above 7.0. Of the 12 patients reported in this series, isoagglutinin titers before transfusion ranged from 32 to 1024. One patient developed renal failure requiring hemodialysis for 17 days, but this resolved. In another series of deliberate administration of ABO-incompatible red cells, 35 patients received 50 to 150 mL of incompatible red cells before marrow transplantation.40 All patients received prednisolone 250 mg, dimethindene 4 mg, and ranitidine 150 mg 30 minutes before the transfusion. No clinical reaction was observed in 23 patients (66%), while severe reactions (chest pain, headache or agues, and/or high changes of heart rate, blood pressure, and/ or oxygen saturation) occurred in 4 patients (11%). The highest pretransfusion titer in this series was 64. Because the severity and course of HTR is dictated by the load of incompatible red cells in circulation, exchange transfusion with antigen-negative blood may be considered. Although it is not appropriate to expose a patient to added risk of transfusion-related infectious disease if the hemolytic process is well tolerated, with a severe reaction to ABO incompatibility exchange transfusion might greatly reduce the chance of morbidity or death. Early treatment of hypotension and DIC are the most important interventions to limit the extent of possible renal impairment. Maintenance of urine output with intravenous fluids and diuretics, such as mannitol or furosemide, early in the course of the reaction has been used successfully. However, if oliguria in the face of normovolemia is present, fluid loading is contraindicated. The use of vasopressor agents with direct vasodilatory effects on the renal vascular bed, such as low-dose dopamine (1-5 µg/kg/min) has been suggested, but is controversial.77,78 The prevention and treatment of DIC is also controversial. Heparin has been advocated by some authors as a treatment for DIC.79 In addition, heparin may have a direct anticomplement effect, which limits intravascular hemolysis and the sequelae of complement activation.80 An obvious drawback of heparin therapy, especially in the intraoperative or postoperative patient, is the potential for hemorrhage. Therefore, heparin should be reserved for patients with clear evidence of intravascular coagulation (thrombocytopenia, hypofibrinogenemia, presence of FDPs and D-dimers). The use of Fresh Frozen Plasma or platelet concentrates in DIC is also controversial, and transfusion of these components should be limited to those patients with active hemorrhage. Most extravascular HTRs are not life-threatening and require no acute treatment. However, some patients with extravascular HTR may benefit from intravenous immunoglobulin (IVIG) infusion. A single dose of IVIG, 400 mg/kg infused within 24 hours of transfusion, has been used successfully to prevent transfusion reactions in alloimmunized patients for whom compatible

820

blood was not obtainable.41,81 Five patients so treated did not experience transfusion reactions and had sustained increases in hematocrit. IVIG, 1 g/kg or 400 mg/kg, has also been used in the treatment of sickle cell HTR syndrome.82,83 The selection of blood components for a hemorrhaging patient undergoing HTR is a critical decision. The first consideration is that no patient should be allowed to suffer a fatal hemorrhage while a search for serologically compatible blood is undertaken. Second, red cells lacking known clinically significant antigens to which the patient currently has an antibody should be obtained, if at all possible. For instance, one should not reflexively issue group O-negative red cells to a patient known to have anti-e. When the specificity of the antibody causing the reaction is not known, the results of serologic tests performed up to that point in time must be considered, and clinical judgment exercised. Although the focus of attention in most HTRs is on red cells, care should be taken to avoid transfusion of type-incompatible plasma or platelets that may aggravate hemolysis, especially when ABO incompatibility is a possible cause. Undue haste in both serologic evaluation and decision making must be avoided, because human errors are often committed under pressure. Future therapies that have not yet been subjected to clinical trials might be directed against inflammatory mediators produced during HTRs. HTRs have a similar clinical presentation to the systemic inflammatory response syndrome (SIRS) (eg, fever, tachycardia, tachypnea or hypoxemia, and leukocytosis).84 Cytokine dysregulation is central to the pathophysiology of SIRS, in which elevated levels of TNF-α IL-1, and IL-6 are associated with mortality.85,86 These considerations suggest that TNF-α or IL-1 blockade may be beneficial in the treatment of HTR. Presently, there are three TNF-α blocking agents marketed in the United States; etanercept (Enbrel, Amgen, Thousand Oaks, CA), a soluble p75 TNF-α receptor fusion protein to the Fc portion of IgG; infliximab (Remicade, Centocor, Malvern, PA), a chimeric (mouse/human) TNF-α antibody; and adalimumab (Humira, Abbott Laboratories, Abbott Park, IL), a fully human monoclonal antibody. Anakinra (Kineret, Amgen) is recombinant human IL1ra, which is also approved by the FDA. Unfortunately, clinical trials of these agents in SIRS have been rather disappointing, with most trials not showing improved outcome. Inhibitors of complement show promise in the treatment of HTR. Human recombinant soluble form of complement receptor 1 (sCR1) has been shown to inhibit complement-mediated red cell destruction in vitro and in a mouse model of hemolysis.87 It should be noted that there are no data at present regarding the safety or effectiveness of drugs that inhibit complement or cytokines in HTR.

Prevention Much of the activity in blood banks is directed toward the prevention of HTRs. Proper performance of donor unit typing, pretransfusion testing, antibody identification, and crossmatching are critical and are covered in Chapter 5.

Chapter 52: Hemolytic Transfusion Reactions

Because human errors are the most common cause of severe HTR, administrative systems designed to analyze errors and prevent future recurrence are the most important protective measure. An event reporting system specifically designed for transfusion services, MERS-TM, has been developed.88 It allows for the recognition and analysis of errors, determination of patterns of errors, and monitoring for changes in frequency after corrective action is implemented. In one application of MERSTM, high severity events with the potential for patient harm were discovered to account for 241 (5%) of the 4670 events over a 47-month period.89 Proper identification of the transfusion recipient and pretransfusion blood specimen is the single most important aspect of the prevention of HTR. Every transfusion service must establish and enforce the procedures to be followed in its institution. At a minimum, these procedures should include permanent and unique identification of each patient using a permanent identification method such as a wristband, confirmation of the proper labeling of blood specimens by comparison to the wristband, and confirmation of the patient identification before starting the transfusion. Deviation from institutional policies on patient and specimen identification should be taken very seriously. Because proper identification of the transfusion recipient is crucial to preventing HTRs, barrier systems that are intended to physically prevent the transfusion of blood without correct identification of the patient have been devised. One such system uses a plastic lock that is preset to a three-letter code at the time of blood component issuance by the blood bank. Before the unit can be administered, the identical code must be entered to unlock the system. Use of this system in one hospital over the course of 1 year detected two misidentified pretransfusion blood samples, and prevented one attempt to transfuse blood to the wrong patient.90 Use of a special wristband for identification of transfusion recipients may prevent some errors. Such systems generally have a unique identifier on the wristband that is only used by the blood bank. A report of the use of one system over a period of 17 years found that potentially ABO-incompatible transfusions were avoided in five of 411,705 samples typed.91 A report of the use of another identification system found that of 2198 cases, two potentially ABO-incompatible transfusions were avoided.92 An alternative system uses bar codes on patient wristbands, blood sample tubes, blood component bags, and nurses’ identification badges; and point-of-care reading devices to verify identity.93 Radio frequency identification devices also have great promise for reduction error. These tools require investment in information technology, but can be integrated with other systems, such as medication administration, for overall enhancement of patient safety. However, sole reliance on such a device to prevent incorrect administration of blood may undermine other more important steps in proper patient identification. Confirmation of the recipient’s ABO type by point-of-care testing before transfusion is a possible strategy for avoiding AHTR. In principle, seven out of eight ABO-incompatible transfusions could be thus avoided. However, bedside testing is subject

to analytic and interpretive errors. At one institution performing bedside ABO confirmation, 13 ABO-incompatible RBC transfusions occurred in 8 years.94 Of these, an error in bedside ABO testing occurred in seven cases. User inexperience is the most important factor in bedside testing errors, although there are device factors as well.95 In situations where patient identification error has caused HTR, immediate consideration should be given to the possibility that another patient has been involved in the misidentification, and may, too, be at risk of receiving incompatible blood. This is especially likely if there are two patients with similar names or if two samples are received simultaneously from the same patient care location. Identification of such errors can prevent a second hemolytic reaction. The selection of donor units lacking antigens corresponding to alloantibodies is essential for the prevention of HTR, but whether phenotype matching of donors and recipients is an appropriate strategy for the prevention of alloimmunization and HTR in the nonimmunized patient is controversial. Arguments in favor of this practice have been put forth in the setting of sickle cell disease where, because of ethnic gene pool diversity, there is often a mismatch between the donor and recipient phenotypes. Examination of alloimmunization rates among children in one urban area indicated that children with non-European ethnic origins had a 42.9% incidence of alloimmunization compared to 17.6% in patients of European ancestry.96 This was not simply caused by variation in transfusion rates, because the patients of European ethnicity received more transfusions than the others. In another study that compared sickle cell patients to those with other forms of chronic anemia, 30% of the patients with sickle cell anemia were alloimmunized, in contrast to 5% in the group of patients with other forms of anemia.97 Of the 32 alloimmunized patients with sickle cell anemia, 17 had multiple antibodies and 14 had DHTRs. Although many institutions have not performed phenotype matching for sickle cell patients in the past, it is becoming commonly accepted practice.98,99 Phenotype matching may possibly prevent some HTRs in other multitransfused patient populations. In a retrospective study of patients with at lest one red cell antibody who received subsequent transfusions, 21% of 653 patients produced additional antibodies.100 Patients with hematologic disease or malignancy, warm-reacting autoantibodies, antibodies to high- or low-frequency antigens, and those receiving prophylactic antigen matching were excluded. Of those who formed additional antibodies, 57% did so after one transfusion episode of a median of 2 RBC units. The authors predicted that matching for Rh, Jka, and Fya could have prevented the formation of 83% of new antibodies. However, whether prevention of serologic reactions will prevent hemolytic reactions is not clear. In this study, 1% to 10% of potential donors would have been available for 3% of patients and more than 10% of potential donors would have been available for 61% of patients, had phenotype matching been performed. However, the frequency of suitable donors may be different in other locations.

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Section IV: Part III

Hemolytic transfusion reactions could be avoided entirely if the responsible antigens on the red cell surface could be removed or camouflaged. Experimental work has suggested that treating red cells with polyethylene glycol (PEG) might be an effective means of camouflaging antigens.101 PEG modification appears to work by creating a sphere of hydration around the red cell that effectively excludes IgG or IgM from coming into contact with antigenic structures on the membrane surface. PEG-treated red cells have properties of size, shape, intracellular ion content, and oxygen binding that are identical to untreated red cells. However, PEG-treated red cells have a low shear viscosity compared to normal red cells. This also may be advantageous in sickle cell disease in which increased blood viscosity within capillaries can result in occlusive crises. The effectiveness of PEG modification is dependent on the molecular weight and branching characteristics of PEG molecules and the chemistry of covalent attachment.102 Use of a dichlorotriazine derivative of 5-kD PEG results in complete inhibition of direct agglutination by anti-D. However, such cells are still agglutinated by anti-D in the indirect antiglobulin test. A and B epitopes are partially, but not completely, masked. In contrast, red cells coated with branched chain 10-kD PEG after treatment with succinimidyl-propionate-modified 20-kD PEG are not agglutinated by anti-A, anti-B, and anti-D. Treatment of red cells with maleimidophenyl-PEG 5 kD and 20 kD can inhibit agglutination by Rh and ABO antibodies.103 However, preliminary data from human trials with PEG-treated red cells have shown that antibodies to the modified red cells resulting in accelerated clearance occur in some recipients, which may significantly limit the usefulness of this technique (G. Garratty, personal communication). Group A and group B red cells can be converted to group O cells by enzymatic cleavage of terminal determinant saccharides with α-N-acetylgalactosaminidase or α-D-galactosidase.104-106 The use of such technology for large-scale conversion of red cells raises the possibility that acute HTRs fromABO incompatibility may be completely avoidable in the future. However, there are issues with regard to completeness of antigen removal and the possibility of exposure of neoantigens by enzymatic treatment. Treatment of red cells with α-N-acetylgalactosaminidase results in rapid loss of A epitopes binding Dolichos biflorus lectin.104 Inhibition of complement-mediated hemolysis is somewhat slower. However, the epitopes of A antigen that react with human source anti-A are relatively resistant to enzymatic degradation. Additionally, there are differences in the enzymatic sensitivity of A epitopes on the red cell membrane. Glycosphingolipids with short oligosaccharide chains display the greatest resistance to enzymatic treatment. Work on enzyme-converted group O (ECO) red cells from group B red cells has advanced to the stage of the clinical trials.107,108 An initial trial of a 2-unit transfusion of ECO RBCs to group O subjects demonstrated good 24-hour posttransfusion survival (95%), with a half-life of 29.5 days.107 There was no clinical or laboratory evidence of hemolysis. A subsequent study

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with larger volume transfusions had similar results. Subjects who received a second transfusion did not show evidence of alloimmunization or increase in anti-B titer.108 In another study, occasional serologic incompatibility with enzyme-converted red cells was seen, as well as transient positive DAT and increases in antiB titer after transfusion. However, no adverse effects of whole unit transfusion were seen, and normal posttransfusion red cell survival was demonstrated in the face of incompatibility.109 A screening technique has recently been used to discover several novel exoglycosidases with high specific activity for the removal of A and B determinants at neutral pH.110 Complete conversion of whole RBC units from group A and group B to group O, as indicated by no reactivity with licensed typing reagents, was achieved with low enzyme concentrations. Although this technique has not yet been subjected to clinical trials, it is promising as a feasible method of ECO production on a clinical scale.

Summary Although the prevention of HTRs continues to be a major focus in transfusion medicine, advances in serology and transfusion service practices have significantly reduced their incidence. Simultaneously, advances in the understanding of the pathophysiology of HTRs have given us insights to help guide the management of patients undergoing reactions. It is conceivable that technological advances in red cell modification and oxygen-carrying solutions will significantly change red cell transfusion practice in the future and virtually eliminate the occurrence of HTR. Until such time, however, HTRs will remain a major adverse consequence of blood transfusion.

Disclaimer The author has disclosed no conflicts of interest.

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Chapter 52: Hemolytic Transfusion Reactions

7. Pineda AA, Brzica SM Jr, Taswell HF. Hemolytic transfusion reaction. Recent experience in a large blood bank. Mayo Clin Proc 1978;53:378-90. 8. Moore SB, Taswell HF, Pineda AA, Sonnenberg CL. Delayed hemolytic transfusion reactions. Evidence of the need for an improved pretransfusion compatibility test. Am J Clin Pathol 1980;74:94-7. 9. Linden JV, Wagner K, Voytovich AE, Sheehan J. Transfusion errors in New York State: An analysis of 10 years’ experience. Transfusion 2000;40:1207-13. 10. Pineda AA, Taswell HF, Brzica SM Jr. Transfusion reaction. An immunologic hazard of blood transfusion. Transfusion 1978;18:1-7. 11. Vamvakas EC, Pineda AA, Reisner R, et al. The differentiation of delayed hemolytic and delayed serologic transfusion reactions: Incidence and predictors of hemolysis. Transfusion 1995;35:26-32. 12. Davis KG, Richard LA. Delayed haemolytic transfusion reactions. Review of three cases. Med J Aust 1982;1:335-7. 13. Ness PM, Shirey RS, Thoman SK, Buck SA. The differentiation of delayed serologic and delayed hemolytic transfusion reactions: Incidence, long-term serologic findings, and clinical significance. Transfusion 1990;30:688-93. 14. Croucher BEE. Differential diagnosis of delayed transfusion reactions. In: Bell CA, ed. A seminar on laboratory management of hemolysis. Washington, DC: AABB, 1979:151-60. 15. Vamvakas EC, Pineda AA, Moore SB. Incidence of delayed hemolytic transfusion reactions (letter). Vox Sang 1995;69:86. 16. The Serious Hazards of Transfusion Steering Group. SHOT Annual Report 2004. Manchester, UK: SHOT Office, 2004. [Available at http://www.shotuk.org/SHOTREPORT2004.pdf.] 17. Robillard P, Karl Itaj N, Chapdelaine A. Five-year trends in the incidence of serious adverse transfusion reactions in the Quebec Hemovigilance System (abstract). Transfusion 2006;46(Suppl):86A. 18. Duvall CP, Alter HJ, Rath CE. Hemoglobin catabolism following a hemolytic transfusion reaction in a patient with sickle cell anemia. Transfusion 1974;14:382-7. 19. Cummins D, Ferrier A, Murphy F. Bilirubinuria, conjugated hyperbilirubinaemia and delta bilirubinaemia following acute haemolysis. Ann Clin Biochem 1997;34:109-10. 20. Kindgen-Milles D, Klement W, Arndt JO. The nociceptive systems of skin, paravascular tissue and hand veins of humans and their sensitivity to bradykinin. Neurosci Lett 1994;181:39-42. 21. Ahrens N, Pruss A, Kahne A, et al. Coexistence of autoantibodies and alloantibodies to red blood cells due to blood transfusion. Transfusion 2007;47:813-16. 22. Salama A, Mueller-Eckhardt C. Delayed hemolytic transfusion reactions. Evidence for complement activation involving allogeneic and autologous red cells. Transfusion 1984;24:188-93. 23. Petz LD, Calhoun L, Shulman IA, et al. The sickle cell hemolytic transfusion reaction syndrome. Transfusion 1997;37:382-92. 24. King KE, Shirey RS, Lankiewicz MW, et al. Delayed hemolytic transfusion reactions in sickle cell disease: Simultaneous destruction of recipients’ red cells. Transfusion 1997;37:376-81. 25. Ballas SK, Marcolina MJ. Hyperhemolysis during the evolution of uncomplicated acute painful episodes in patients with sickle cell anemia. Transfusion 2006;46:105-10. 26. Joseph K, Kaplan AP, Frederick WA. Formation of bradykinin: A major contributor to the innate inflammatory response. Adv Immunol 2005;86:159-208. 27. Chan WL, Tang NL, Yim CC, et al. New features of renal lesion induced by stroma free hemoglobin. Toxicol Pathol 2000;28:635-42.

28. Patel RP. Biochemical aspects of the reaction of hemoglobin and NO: Implications for Hb-based blood substitutes. Free Radic Biol Med 2000;28:1518-25. 29. Vaughn MW, Huang KT, Kuo L, Liao JC. Erythrocytes possess an intrinsic barrier to nitric oxide consumption. J Biol Chem 2000;275:2342-8. 30. Gould SA, Moss GS. Clinical development of human polymerized hemoglobin as a blood substitute. World J Surg 1996;20:1200-7. 31. Bluemle LW Jr. Hemolytic transfusion reactions causing acute renal failure. Serologic and clinical considerations. Postgrad Med 1965;38:484-9. 32. Schorn TF, Knospe WH. Fatal delayed hemolytic transfusion reaction without previous blood transfusion. Ann Intern Med 1989;110:241-2. 33. Sazama K. Reports of 355 transfusion-associated deaths: 1976 through 1985. Transfusion 1990;30:583-90. 34. Honig CL, Bove JR. Transfusion-associated fatalities: Review of Bureau of Biologics reports 1976-1978. Transfusion 1980;20:653-61. 35. Sazama K. Death from transfusion: A 20-year review. Presented at the annual meeting of the North Carolina Association of Blood Banks, Charlotte, NC, September, 2000. 36. Linden JV, Paul B, Dressler KP. A report of 104 transfusion errors in New York state. Transfusion 1992;32:601-6. 37. The Serious Hazards of Transfusion Steering Group. Additional Cumulative Data 1996-2003. Manchester, UK: SHOT Office, 2003. [Available at http://www.shotuk.org/additional_cumulative.htm.] 38. Dzik W, Murphy M, Andreu G, et al. An international study of the performance of sample collection from patients. Vox Sang 2003;85:40-7. 39. Nussbaumer W, Schwaighofer H, Gratwohl A, et al. Transfusion of donor-type red cells as a single preparative treatment for bone marrow transplants with major ABO incompatibility. Transfusion 1995;35:592-5. 40. Scholl S, Klink A, Mugge L-O, et al. Safety and impact of donor-type red blood cell transfusion before allogeneic peripheral blood progenitor cell transplantation with major ABO mismatch. Transfusion 2005;45:1676-83. 41. Kohan AI, Niborski RC, Rey JA, et al. High-dose intravenous immunoglobulin in non-ABO transfusion incompatibility. Vox Sang 1994;67:195-8. 42. McManigal S, Sins KL. Intravascular hemolysis secondary to ABO incompatible platelet produces. Am J Clin Pathol 1999;111:202-6. 43. Conway LT, Scott EP. Acute hemolytic transfusion reaction due to ABO incompatible plasma in a platelet apheresis concentrate (letter). Transfusion 1984;24:413. 44. Larsson LG, Welsh VJ, Ladd DJ. Acute intravascular hemolysis secondary to out-of-group platelet transfusion. Transfusion 2000;40:902-6. 45. Harris SB, Josephson CD, Kost CB, Hillyer CD. Nonfatal intravascular hemolysis in a pediatric patient after transfusion of a platelet unit with high-titer anti-A. Transfusion 2007;47:1412-17. 46. Josephson CD, Mullis NC, Van Demark C, Hillyer CD. Significant numbers of apheresis-derived group O platelet units have “high-titer” antiA/A,B: Implications for transfusion policy. Transfusion 2004;44:805-8. 47. Cooling LW, Butch S, Downs T, Davenport R. Isoagglutinin titers in pooled group O platelets are comparable to apheresis platelets (abstract). Transfusion 2007;47(Suppl):78A. 48. Sadani DT, Urbaniak SJ, Bruce M, Tighe JE. Repeat ABO-incompatible platelet transfusions leading to haemolytic transfusion reaction. Transfus Med 2006;16:375-9.

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49. Mair B, Benson K. Evaluation of changes in hemoglobin levels associated with ABO incompatible plasma in apheresis platelets. Transfusion 1998;38:51-5. 50. Mollison PL, Engelfriet CP, Contreras PM. Blood Transfusion in Clinical Medicine. 9th ed. Oxford: Blackwell Scientific Publications, 1993:487. 51. Mair DC, Eastlund T, Rosen G, et al. Hemolysis during percutaneous mechanical thrombectomy can mimic a hemolytic transfusion reaction. Transfusion 2005;45:1291-4. 52. Alvarez A, Rives S, Montoto S, et al. Relative sensitivity of direct antiglobulin test, antibody elution and flow cytometry in the serologic diagnosis of immune hemolytic transfusion reactions. Haematologica 2000;85:186-8. 53. Gossinger H, Laggner A, Druml W, et al. Hemodynamic, pulmonary, and renal reactions to inadvertent transfusion of outdated blood. Crit Care Med 1986;14:70-1. 54. Whitelaw JP. Hemolysis caused by half-physiologic-strength saline (letter). Transfusion 1990;30:78. 55. Samoszuk M, Reid ME, Toy PT. Intravenous dimethylsulfoxide therapy causes severe hemolysis mimicking a hemolytic transfusion reaction (letter). Transfusion 1983;23:405. 56. Mimouni F, Shohat S, Reisner SH. G6PD-deficient donor blood as a cause of hemolysis in two preterm infants. Isr J Med Sci 1986;22:120-2. 57. Shalev O, Manny N, Sharon R. Posttransfusional hemolysis in recipients of glucose-6-phosphate dehydrogenase-deficient erythrocytes. Vox Sang 1993;64:94-8. 58. Salama A, Berghofer H, Mueller-Eckhardt C. Red blood cell transfusion in warm-type autoimmune haemolytic anaemia. Lancet 1992;340:1515-7. 59. Ruegg SJ, Jungi TW. Antibody-mediated erythrolysis and erythrophagocytosis by human monocytes, macrophages and activated macrophages. Evidence for distinction between involvement of highaffinity and low-affinity receptors for IgG by using different erythroid target cells. Immunology 1988;63:513-20. 60. Leslie RGQ. Immunoglobulin and soluble immune complex binding to phagocyte Fc receptors. Biochem Soc Trans 1984;12:743-6. 61. Davenport RD, Kunkel SL. IgG receptor roles in red cell binding to monocytes and macrophages (abstract). Transfusion 1994;34(Suppl):79S. 62. Unkeless JC. Function and heterogeneity of human Fc receptors for immunoglobulin G. J Clin Invest 1989;83:355-61. 63. Clarkson SB, Kimberly RP, Valinsky JE, et al. Blockade of clearance of immune complexes by anti-Fc gamma receptor monoclonal antibody. J Exp Med 1986;164:474-89. 64. Udani M, Rao N, Telen MJ. Leukocyte phenotypic changes in an in vitro model of ABO hemolytic transfusion reaction. Transfusion 1997;37:904-9. 65. Butler J, Parker D, Pillai R, et al. Systemic release of neutrophil elastase and tumour necrosis factor alpha following ABO incompatible blood transfusion. Br J Haematol 1991;79:525-6. 66. Davenport RD, Strieter RM, Kunkel SL. Red cell ABO incompatibility and production of tumour necrosis factor-alpha. Br J Haematol 1991;78:540-4. 67. Davenport RD, Strieter RM, Standiford TJ, Kunkel SL. Interleukin-8 production in red blood cell incompatibility. Blood 1990;76:2439-42. 68. Davenport RD, Burdick MD, Strieter RM, Kunkel SL. Monocyte chemoattractant protein production in red cell incompatibility. Transfusion 1994;34:16-19.

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69. Davenport RD, Burdick M, Kunkel SL. Endothelial cell activation in hemolytic transfusion reactions (abstract). Transfusion 1992;32(Suppl):53S. 70. Matsushima K, Oppenheim JJ. Interleukin 8 and MCAF: Novel inflammatory cytokines inducible by IL 1 and TNF. Cytokine 1989;1:2-13. 71. Jiang Y, Beller DI, Frendl G, Graves DT. Monocyte chemoattractant protein-1 regulates adhesion molecule expression and cytokine production in human monocytes. J Immunol 1992; 148:2423-8. 72. Davenport RD, Burdick M, Moore SA, Kunkel SL. Cytokine production in IgG mediated red cell incompatibility. Transfusion 1993;33:19-24. 73. Hoffman M. Antibody-coated erythrocytes induce secretion of tumor necrosis factor by human monocytes: A mechanism for the production of fever by incompatible transfusions. Vox Sang 1991;60:184-7. 74. Davenport RD, Burdick MD, Strieter RM, Kunkel SL. In vitro production of interleukin-1 receptor antagonist in IgG mediated red cell incompatibility. Transfusion 1994;34:297-303. 75. Schirmer DA, Song SC, Baliff JP, et al. Mouse models of IgG- and IgM-mediated hemolysis. Blood 2007;109:3099-107. 76. Slavc I, Urban Ch, Schwinger W, et al. ABO-incompatible bone marrow transplantation: Prevention of hemolysis by alkaline hydration with mannitol diuresis in conjunction with red cell reduced buffy coat bone marrow. Wein Klin Wochenschr 1992;104:93-6. 77. Lauschke A, Teichgraber U, Frei U, Eckardt K. ‘Low-dose’ dopamine worsens renal perfusion in patients with acute renal failure. Kidney Int 2006;69:1669-74. 78. Bellomo R. Has renal-dose dopamine finally been relegated to join the long list of medical myths? Crit Care Resusc 2001;3:7-10. 79. Rock RC, Bove JR, Nemerson Y. Heparin treatment of intravascular coagulation accompanying hemolytic transfusion reactions. Transfusion 1969;9:57-61. 80. Gray JM, Oberman HA, Beck ML. Delay in the onset of immune hemolysis in vivo apparently due to heparinization. Transfusion 1973;13:422-4. 81. Win N, Madan B, Gale R, Matthey F. Intravenous immunoglobulin given to lymphoma patients with recurrent haemolytic transfusion reactions after transfusion of compatible blood. Hematology 2005;10:375-8. 82. Talano J, Hillery C, Gottschall J, et al. Delayed hemolytic transfusion reaction/hyperhemolysis syndrome in children with sickle cell disease. Pediatrics 2003;111:e661-5. 83. Win N, Doughty H, Telfer P, et al. Hyperhemolytic transfusion reaction in sickle cell disease. Transfusion 2001;41:323-8. 84. American College of Chest Physicians/Society of Critical Care Medicine. Consensus conference: Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Crit Care Med 1992; 20:864-74. 85. Terregino CA, Lopez BL, Karras DJ, et al. Endogenous mediators in emergency department patients with presumed sepsis: Are levels associated with progression to severe sepsis and death? Ann Emerg Med 2000;35:26-34. 86. Jean-Baptiste E. Cellular mechanisms in sepsis. J Intensive Care Med 2007;22:63-72. 87. Yazdanbakhsh K, Kang S, Tamasauskas D, et al. Complement receptor 1 inhibitors for prevention of immune-mediated red cell destruction: Potential use in transfusion therapy. Blood 2003;101:5046-52. 88. Callum J, Kaplan H, Merkley L, et al. Reporting of near-miss events for transfusion medicine: Improving transfusion safety. Transfusion 2001;41:1204-11.

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89. Callum J, Merkley L, Coovadia A, et al. Experience with the medical event reporting system for transfusion medicine (MERS-TM) at three hospitals. Transfus Apher Sci 2004;31:133-43. 90. Mercurilali F, Inghilleri F, Colotti MT, et al. One-year use of the Bloodloc system in an orthopedic institute. Tranfus Clin Biol 1994;1:227-30. 91. Figueroa P, Ziman A, Wheeler C, et al. Nearly two decades using the Check-Type to prevent ABO incompatible transfusions: One institution’s experience. Am J Clin Pathol 2006;126:1-5. 92. Lau FY, Wong R, Chui CH, et al. Improvement in transfusion safety using a specially designed transfusion wristband. Transfus Med 2000;10:121-4. 93. Sandler S, Langeberg A, Dohnalek L. Bar code technology improves positive patient identification and transfusion safety. Dev Biol 2005;120:19-24. 94. Ahrens N, Pruss A, Kiesewetter H, Salama A. Failure of bedside ABO testing is still the most common cause of incorrect blood transfusion in the barcode era. Transfus Apher Sci 2005;33:25-9. 95. Migeot V, Ingrand I, Salmi L, Ingrand P. Reliability of bedside ABO testing before transfusion. Transfusion 2002;42:1348-55. 96. Luban NL. Variability in rates of alloimmunization in different groups of children with sickle cell disease: Effect of ethnic background. Am J Pediatr Hematol Oncol 1989;11:314-19. 97. Vichinsky EP, Earles A, Johnson RA, et al. Alloimmunization in sickle cell anemia and transfusion of racially unmatched blood. N Engl J Med 1990;322:1617-21. 98. Afenyi-Annan A, Brecher M. Pre-transfusion phenotype matching for sickle cell disease patients. Transfusion 2004;44:619-20. 99. Osby M, Shulman I. Phenotype matching of donor red blood cell units for nonalloimmunized sickle cell disease patients: A survey of 1182 North American laboratories. Arch Pathol Lab Med 2005;129:190-3. 100. Schonewille H, van de Watering L, Brand A. Additional red blood cell alloantibodies after blood transfusions in a nonhematologic

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alloimmunized patient cohort: Is it time to take precautionary measures? Transfusion 2006;46:630-5. Scott MD, Bradley AJ, Murad KL. Camouflaged blood cells: Lowtechnology bioengineering for transfusion medicine? Transfus Med Rev 2000;14:53-63. Fisher TC, Armstrong JK, Meiselman HJ, et al. Second generation poly(ethylene glycol) surface coatings for red blood cells (abstract). Transfusion 2000;40(Suppl):119S. Nacharaju P, Boctor F, Manjula B, Acharya S. Surface decoration of red blood cells with maleimidophenyl-polyethylene glycol facilitated by thiolation with iminothiolane: An approach to mask A, B, and D antigens to generate universal red blood cells. Transfusion 2005;45:374-83. Hoskins LC, Larson G, Naff GB. Blood group A immunodeterminants on human red cells differ in biologic activity and sensitivity to alpha-N-acetylgalactosaminidase. Transfusion 1995;35:813-21. Hobbs L, Mitra M, Phillips R, et al. Deantigenation of human type B erythrocytes with Glycine max alpha-D-galactosidase. Biomed Pharmacother 1995;49:244-50. Zhu A, Leng L, Monahan C, et al. Characterization of recombinant alpha-galactosidase for use in seroconversion from blood group B to O of human erythrocytes. Arch Biochem Biophys 1996;327:324-9. Lenny LL, Hurst R, Goldstein J, Galbraith RA. Transfusions to group O subjects of 2 units of red cells enzymatically converted from group B to group O. Transfusion 1994;34:209-14. Lenny LL, Hurst R, Zhu A, et al. Multiple-unit and second transfusions of red cells enzymatically converted from group B to group O: Report on the end of phase I trials. Transfusion 1995;35:899-902. Kruskall M, AuBuchon J, Anthony K, et al. Transfusion to blood group A and O patients of group B RBCs that have been enzymatically converted to group O. Transfusion 2000;40:1290-8. Liu QP, Sulzenbacher G, Yuan H, et al. Bacterial glycosidases for the production of universal red blood cells. Nat Biotech 2007;25:454-64.

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53

Febrile, Allergic, and Nonimmune Transfusion Reactions Gregory J. Pomper Assistant Professor of Pathology, Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, North Carolina, USA

This chapter reviews a variety of acute, nonhemolytic, and noninfectious transfusion reactions, the most common of which are febrile, nonhemolytic transfusion reactions (FNHTRs) and allergic reactions. Other acute, nonhemolytic reactions are reported less frequently and include transfusion-related acute lung injury (TRALI) and anaphylactic or anaphylactoid reactions. Additional acute adverse effects can occur in massive transfusion because of the large volume of blood components transfused over a short period. The complications of massive transfusion include dilutional coagulopathy, hypothermia, citrate toxicity, and electrolyte disturbances among others. Some patients cannot tolerate the acute increase in intravascular blood volume caused by transfusion and experience the complications of transfusion-associated circulatory overload (TACO). Acute reactions can be caused by the toxicity of chemicals that leach into blood components from blood storage containers or filters or by chemicals added to improve storage conditions, such as dimethyl sulfoxide (DMSO). Other reactions are caused by endogenous mediators generated in the blood during filtration, processing, or storage, such as bradykinin-mediated hypotensive reactions. It is important that these complications of transfusion be recognized by patient care teams and blood bank personnel, and that appropriate treatments and preventive measures be instituted for patient safety and well-being.

Febrile Nonhemolytic Transfusion Reactions Description An FNHTR is commonly defined as an increase in body temperature of 1ºC or more that occurs during or within several hours of transfusion and is unrelated to hemolysis, sepsis, or other known causes of fever. The use of a 1ºC increase in body

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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temperature as a threshold for defining an FNHTR avoids undue concern over small fluctuations in body temperature unrelated to transfusion that do not justify discontinuation of transfusion and follow-up investigation. Many FNHTRs begin with the patient feeling uneasy and experiencing chills. In mild reactions, the signs and symptoms do not progress. Chills with or without an increase in body temperature can be classified as an FNHTR if other possible causes of chills are unlikely and the time course of the reaction correlates with the transfusion. In the most severe reactions, patients may experience rigors (severe shaking chills) or a fever elevation of 2ºC or more over baseline. Although signs and symptoms usually are limited to chills and fever, some patients may also rarely experience more severe symptoms such as headache, nausea, and/or vomiting. The fever of FNHTR usually persists no more than 8 to 12 hours after the start of transfusion. If fever persists 18 to 24 hours or longer, it is unlikely to be transfusion related. Generally, FNHTRs are self-limited and have no sequelae. However, elderly patients, patients with compromised cardiovascular status, or critically ill patients are at risk of cardiorespiratory complications associated with FNHTR. Because fever increases oxygen demand and consumption an estimated 13% for every 1ºC over 37ºC and shivering increases oxygen demand approximately 300%, FNHTRs can aggravate preexisting cardiac, pulmonary, and cerebrovascular insufficiency. Therefore, prompt recognition and antipyretic management of FNHTRs can be very beneficial. An FNHTR almost always is associated with transfusion of cellular blood components, such as red cells, platelets, and granulocyte preparations and less commonly with noncellular components, such as plasma and cryoprecipitate. The incidence of FNHTR varies widely and median rates have been reported as higher for platelets (4.6%) than for red cells (0.33%).1 The reaction risk of blood components, however, varies according to numerous factors, such as method of preparation of the blood component (eg, leukocyte reduction, storage time, medications, patient and donor characteristics, monitoring practices, and many others). These factors vary among different geographic regions and medical centers. In addition, rates based on reactions

Chapter 53: Febrile, Allergic, and Nonimmune Transfusion Reactions

reported to blood banks are lower than those based on systematic surveillance of responses to all transfusions. The cause of higher FNHTR reaction rates with platelets is not entirely known. Pools of whole-blood-derived concentrates have been reported to cause a higher rate of FNHTR than apheresis platelets.2,3 Platelet concentrates prepared from platelet-rich plasma are commonly used in the United States. These plateletrich plasma concentrates are reported to cause a higher rate of FNHTR than are platelet concentrates prepared from the buffycoat technique. Longer platelet storage times are also associated with higher rates of FNHTR.4-6 Reactions also are more frequent among certain recipients, such as multitransfused patients or multiparous women who have developed leukocyte or platelet alloantibodies.

Etiology An FNHTR appears to be part of the systemic inflammatory response syndrome (SIRS) provoked in transfusion recipients by the immune challenge of transfusing foreign cells or infusing soluble inflammatory mediators present in stored blood components. The term systemic inflammatory response syndrome was coined to describe the constellation of observed body responses to various insults, such as infection, trauma, burns, and ischemia. It is defined as the presence of two or more of the following: body temperature more than 38ºC or less than 36ºC; heart rate more than 90 beats/minute; tachypnea (respiratory rate ⬎20 breaths/minute or PaCO2 less than 32 mm Hg); white cell count more than 12,000/µL or less than 4 ⫻ 109/L, or more than 10% immature neutrophils (band forms). Although a mild FNHTR may not completely fulfill these criteria, FNHTR is nevertheless an inflammatory response. Exogenous pyrogens such as lipopolysaccharide (LPS) and pyrogenic cytokines initiate a series of responses leading to hyperthermia. These responses include rapid muscle contractions that cause shivering, rigors, and an increase in heat generation. Heat conservation is achieved through cutaneous vasoconstriction, which also contributes to the sensation of a chill. Perceived chills lead to behavioral changes that can further increase body temperature. For example, the patient may cover up, and the result is inhibition of heat dissipation. An FNHTR appears to have two possible underlying causes: 1) the more “classical” pathway of infused antigens, such as leukocytes, that stimulate the in-vivo generation of cytokines in the recipient and 2) the infusion of pyrogenic cytokines or other inflammatory response mediators (eg, activated complement proteins, LPS, or neutrophil-priming lipids) that accumulate in the plasma portion of cellular blood components during storage.7,8 A third cause of fever, infusion of blood components contaminated with bacteria or bacterial products, will produce a febrile response but is not usually categorized as a FNHTR but rather as a bacterial septic reaction, if recognized. The common pathway by which these different stimuli induce posttransfusion fever has been attributed to an increase in circulating pyrogenic cytokines in the recipient, such as interleukin-1β (IL-1β),

IL-6, and tumor necrosis factor α (TNF-α). Pyrogenic cytokines induce fever by mediating upregulation of the thermostatic set point for body temperature in the thermoregulatory center of the hypothalamus. This mechanism is supported by the association of febrile reactions with a specific cytokine polymorphism IL1RN*2.2 genotype.9 Recently, research has lead investigators to a more complex model of fever generation, building on a model where cytokines principally induce fever to one where central nervous system stimulation by prostaglandin E2 (PGE2) is pivotal. Alternative hypotheses have resulted from the finding that a febrile response to LPS occurs even with blockade of either IL-1 or TNF-α and that the presence of circulating cytokines lags behind the development of fever.10 Research has found that LPS-induced C5a production via complement activation results in rapid peripheral PGE2 production.11 In addition, LPS binds to toll-like receptor 4 and induces cytokine production, leading to a two-phase rapid and delayed febrile response. Hyperthermic stimuli compete with hypothermic stimuli to achieve a central thermal balance point that may elevate or decrease based upon physiologic stimuli.12 Central to the febrile response is the presence of EP3 prostaglandin receptors that bind PGE2 in the hypothalamus (Fig 53-1).13 These newer models of the fever response provide possible explanations for why FNHTRs continue to occur despite prestorage leukocyte reduction that minimizes cytokine accumulation in storage. Clinical evidence supports the hypothesis that febrile reactions can be caused by noncytokine hyperthermic stimuli present in cellular transfusion products.14 The active field of febrile response research shows promise for the development of more targeted anitpyretic medications that may eventually lead to the extinction of FNHTRs.

Alloimmunization to Leukocytes or Platelets Transfusion recipients at greatest risk of an FNHTR are those with leukocyte or platelet antibodies who receive transfusions with blood components containing large numbers of passenger leukocytes or platelets.15,16 Less frequently, donor antibodies to leukocytes, present in the plasma portion of blood components, are associated with FNHTRs. The implicated antibodies most often have HLA specificity, although they also may be platelet- or granulocyte-specific. A minimum of approximately 1 ⫻ 107 leukocytes per unit of Red Blood Cells (RBCs) appears necessary to cause an FNHTR, although this number varies among individuals.17,18 The role of donor leukocytes in FNHTR is supported by the finding that decreasing the leukocyte content of blood components below this threshold reduces the incidence of FNHTRs. A variety of mechanisms are possible by which antibody-leukocyte or antibody-platelet interactions cause fever. For example, donor monocytes may be activated and secrete pyrogenic cytokines when recipient antibodies bind to them. An alternative explanation is that immune complex formation between recipient antibodies and donor leukocytes or platelets leads to generation of activated complement components such as C5a, which stimulate the production of PGE2.

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COOH

PVH HO OH

O

Blo od v

esse l

PGE2

EP3R MnPO

DMH/DHA

RPa Spinal cord Activation of brown adipose tissue Skin vasoconstriction

Figure 53-1. A model for neuronal action of the EP3 receptors (EP3Rs) in the fever response after systemic immune challenge. In response to lipopolysaccharides or cytokines, prostaglandin E2 (PGE2) is generated by macrophages of the liver and lung and endothelial cells lining venules in the brain. PGE2 is transported or diffuses across the blood-brain barrier and acts on EP3Rs in the median preoptic area (MnPO). These are hypothesized to be GABAergic

neurons that inhibit thermogenic systems in the hypothalamic paraventricular nucleus (PVH), the dorsomedial nucleus/dorsal area (DMH/DHA), and the raphe pallidus nucleus in the medulla (RPa). These thermogenic nuclei activate sympathetic preganglionic neurons in the spinal cord, resulting in cutaneous vasoconstriction and activation of brown adipose tissue that ultimately raise body temperature. Used with permission from Lazarus et al.13

Storage-Generated Cytokines Antibodies to leukocytes or platelets do not appear to account for all FNHTRs, particularly those caused by platelet transfusions. For example, some patients with no history of transfusion or pregnancy experience an FNHTR to their first transfusion of platelets.2 It is unlikely that these reactions are mediated by recipient leukocyte or platelet antibodies because these recipients have no previous exposure to foreign cells. In addition, the rate of FNHTRs to platelet transfusion increases with increasing blood bank storage time of the transfused platelet concentrate.4-6 This indicates that time-dependent change occurs in the platelet concentrate during storage that has a role in stimulating an FNHTR in some patients. Furthermore, febrile reactions still occur with the use of prestorage leukocyte reduction.19,20 In some cases, this is the result of inappropriate filter use or filter failure. However, this observation also supports the possibility that a substance or substances in the plasma portion of blood components not removed by filtration may be responsible for mediating at least some FNHTRs. The discovery that proinflammatory cytokines accumulate in the plasma portion of platelet concentrates may account for many of these findings. A variety of leukocyte-derived, proinflammatory cytokines, including IL-1β, IL-6, IL-8, TNF-α, macrophage inflammatory protein 1α (MIP-1α), and growth-related oncogene α (GRO-α), are generated and accumulate in the plasma portion of platelet concentrates during storage.8,21-23 Extracellular levels of these cytokines generally increase with increasing component storage time and are roughly proportional to the passenger leukocyte content of the blood component bag. Prestorage or earlystorage leukocyte reduction (within 1 to 2 days of collection) prevents or greatly reduces generation of these cytokines. Because they have pyrogenic activity, many of these cytokines (if present in high enough concentration) can induce febrile responses in

transfusion recipients. Elevated levels of IL-1β, IL-6, and TNF-α in the plasma portion of platelet concentrates correlate positively with the occurrence of an FNHTR. Some studies have shown that IL-6 levels in the plasma portion of platelet concentrates correlate best with the occurrence of FNHTR. In one study, chills, fever, or both occurred more frequently after infusion of the plasma portion of the platelet concentrates than after infusion of the cellular portion containing platelets and leukocytes.21 The plasma portions that caused an FNHTR contained higher levels of IL-1β and IL-6 than did those that did not cause chills or fever. These data support the role of the plasma portion of platelet concentrates as a source of inflammatory mediators and as a possible stimulus of FNHTR in some transfusion recipients. Although levels of a variety of proinflammatory cytokines in the plasma portion of platelet concentrates correlate with the occurrence of FNHTRs, it is unknown which, if any, of these actually mediate FNHTRs. Other inflammatory mediators, such as C5a and biologically active lipids, accumulate in platelet concentrates during storage and also may be implicated.24,25 Platelet-derived cytokines, such as CD40L (CD154), CCL5 (RANTES), transforming growth factor β1 (TGF-β1), CXCL4 (platelet factor 4; PF4), CXCL8 (IL-8), and MIP-1α are present in the plasma portion of platelet concentrates and apheresis platelets. All platelet-derived cytokines accumulate during platelet storage and CD40L has been associated with clinical FNHTRs.26 These cytokines are not known to be directly pyrogenic, but they stimulate the synthesis of proinflammatory mediator, IL-1β, IL-6, IL-8, and TNF-α. Because RANTES can activate basophils and mast cells and stimulate histamine release, it may play a role in mediating some allergic reactions (see later, Allergic Reactions).27 RANTES data are conflicting as to the extent of accumulation during storage; however, it seems that some increase in levels does occur.28

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Some proinflammatory cytokines, such as IL-1β and IL-8, have been detected in the supernatant portion of stored RBCs, although at much lower levels than in platelet concentrates.29 Because the cold storage temperature of RBC units, 1-6ºC, has an inhibitory effect on cellular metabolism, the capacity of passenger leukocytes in RBC units to synthesize and secrete cytokines is less than those in platelet concentrates. As a result, the levels of cytokines in RBC units appear to be too low to mediate significant physiologic reactions. The stimulus for cytokine generation during storage of blood components remains unknown. Measurements of cytokine messenger RNA levels and total cytokine levels (intracellular plus extracellular) in platelet concentrates indicate that accumulation of leukocyte-derived cytokines is caused in part by new synthesis and secretion. The stimulus for synthesis and secretion of leukocyte-derived cytokines may be, for example, contact activation of monocytes after these cells interact with the plastic of the storage containers or tubing.30 Other possibilities include the direct stimulatory effects of C5a on PGE2 or the stimulatory effects of activated complement components on monocytes or other leukocytes in the blood component bag. The presence of plateletderived cytokines (such as CD40L, RANTES, and TGF-β1) in the plasma portion of platelet concentrates likely results from their release from preexisting stores, because the biosynthetic activity of platelets is limited.

Bacterial Contamination of Blood Components An FNHTR may result from infusion of a blood component contaminated with bacteria or bacterial products such as LPS. Unless a Gram’s stain and bacterial cultures are performed, mild septic transfusion reactions characterized by only fever and chills are likely to be classified clinically as FNHTR (see Chapter 49).31,32 Transfusion reactions caused by contaminating bacteria, whether mild or severe, are manifestations of the SIRS, described earlier. Proinflammatory cytokines (such as IL-1β, IL-6, IL-8, and TNF-α) are implicated in the pathogenesis of SIRS associated with sepsis.33 If bacterial contamination of blood components has occurred, the greatest source of cytokines is likely the transfusion recipient’s cells stimulated by the infused bacteria or bacterial products. However, cytokine production by leukocytes in the component bag during storage stimulated by bacteria or bacterial products also may contribute to the reaction.

Diagnosis As a routine part of the transfusion procedure, the vital signs of transfusion recipients (pulse, temperature, and respiratory rate) should be measured immediately before transfusion and at intervals during and soon after transfusion. The patient should be watched closely, particularly in the first 30 to 60 minutes of transfusion, for the onset of chills, shivering, or rigors, which often precede a fever. A transfusion reaction is a possibility if chills, fever (1ºC or more over pretransfusion temperature), or both develop any time during transfusion or up to several hours after the transfusion

has ended. A febrile response to transfusion, however, is not specific for an FNHTR. For example, a fever may be the early manifestation of a more serious acute hemolytic or septic transfusion reaction. When a patient has a febrile reaction to transfusion, an evaluation to rule out hemolysis and possibly bacterial contamination should be undertaken promptly. Nursing staff should stop the transfusion immediately and notify the physician caring for the patient. They should verify that the identity of the transfusion recipient, based on the patient’s identification bracelet and verbal confirmation with the recipient, if possible, matches that of the intended recipient, as indicated on the blood component tag. All containers and transfusion sets should be sent to the laboratory along with a posttransfusion blood specimen and a report that summarizes the clinical reaction. The clinical team should also have verbal communication with the blood bank staff to ensure that the postreaction blood specimen, component bag, and infusion set are received by the blood bank as soon as possible. Investigation of a febrile reaction in the blood bank generally begins with a recheck of the records for clerical errors. The posttransfusion serum must be visually evaluated for hemolysis and should be compared with the pretransfusion serum. A direct antiglobulin test must be performed on the posttransfusion blood specimen and ideally on a pretransfusion specimen for comparison. The ABO grouping of the patient sample and the donor unit should be repeated. When suggested by the preliminary serologic results, the crossmatch may be repeated for RBC transfusions to confirm patient-donor compatibility. The results of these tests confirm or exclude a hemolytic transfusion reaction as the basis for the fever. When a septic reaction is highly suspected, for example if the patient arrives with a high fever (2ºC or more) or accompanying hypotension, the bag contents should be examined by means of a Gram’s stain and culture for bacterial contamination. Blood cultures also should be obtained from the transfusion recipient’s posttransfusion blood specimen to correlate bacteremia with the same organism that may be detected in the blood component bag. Most blood banks do not test for HLA-specific, platelet-specific, or granulocyte-specific antibodies in the recipient’s serum as possible causes of an FNHTR. Identification of these antibodies and pyrogenic cytokines is reserved for specialized laboratories and does not play a role in the immediate evaluation of most reactions. The patient is examined by the primary team and the blood bank physician to determine whether associated symptoms or circumstances can explain the fever, such as drug reactions, sepsis, or other inflammatory conditions unrelated to transfusion. The time course of the development and resolution of fever should be examined in relation to the transfusion. In cases in which the transfusion recipient has a fever at the start of transfusion or is experiencing intermittent spiking fevers, a posttransfusion increase in body temperature can be difficult to interpret. In such cases possible FNHTR may be the most definitive diagnosis that can be made. An FNHTR is a diagnosis of exclusion, arrived at by means of eliminating the possibility of immune hemolysis, bacterial contamination of the blood component, TRALI, or other causes of fever unrelated to transfusion.

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Treatment The transfusion should be stopped immediately. The intravenous line should be kept open with normal saline solution to provide ready access for the possible infusion of crystalloid and intravenous medication in case the fever is a sign of a more serious hemolytic or septic reaction. Most patients, however, should be reassured that febrile transfusion reactions usually are harmless and that the fever typically responds to antipyretic therapy. The antipyretic agent of choice is acetaminophen (adults, 325 to 650 mg orally; children, 10 to 15 mg/kg orally or rectally). Aspirin and nonsteroidal antiinflammatory drugs are contraindicated in the treatment of many transfusion recipients, such as those receiving platelets. Unless the patient has signs of an allergic reaction, such as urticaria, erythema, or pruritus, antihistamines are not indicated in the management of FNHTR. However, it is not unusual for physicians to prescribe both an antipyretic and an antihistamine in combination for mild reactions. Patients occasionally develop rigors (severe shaking chills) after a transfusion and meperidine had been a mainstay therapy for many years. Because shivering can increase oxygen demand significantly, it is important to control the shaking chills, particularly for patients with cardiac or respiratory insufficiency. When administered to adults at doses of 25 to 50 mg intravenously, meperidine remains a very effective treatment for rigors. Meperidine is effective in rapidly arresting rigors through mechanisms not clearly understood. Unfortunately, meperidine has fallen out of favor with some hospitals because of unacceptable central nervous system toxicities and other downsides. Use of meperidine is generally contraindicated in the care of patients with renal failure because of accumulation of the proconvulsant metabolite normeperidine. Use of meperidine also is contraindicated in the care of patients who have taken monoamine oxidase inhibitors within the previous 14 days because of the risk of serotonin syndrome (excess serotonin activity). Toxicities secondary to metabolite accumulation, short half-life, and higher equianalgesic dose compared to morphine has decreased interest and formulary availability for this opioid.34 On the basis of anecdotal evidence, morphine may be slightly less efficacious for the treatment of rigors, but its safety profile is more acceptable when given as a one-time dose of 2 to 4 mg intravenously. After symptoms of an acute febrile reaction have been treated and the patient has been stabilized, any unused portion of the blood component should be returned to the blood bank and not transfused, even if blood bank testing rapidly rules out hemolysis. This is because no quick test is currently available that is sensitive enough to also rule out with confidence bacterial contamination of the blood component. The use of Gram’s stain helps detect heavily contaminated units (with detection limits not less than 106 organisms per mL), and lower levels of contamination may be missed. If the febrile reaction is caused by bacterial contamination of the component bag, restarting transfusion of the same component can cause a severe and even fatal septic transfusion reaction as more bacteria or bacterial products are infused. For this reason, a new blood component unit should

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be used if transfusion is still needed after the patient’s condition has been stabilized. Restarting transfusion with the same platelet preparation that caused an FNHTR may be considered as a last resort in the treatment of patients who repeatedly have mild and uncomplicated febrile responses to platelet transfusions despite prophylactic measures.35 In this situation, it may be necessary to cautiously complete the transfusion with the same component, because the patient is likely to react to other platelet preparations. The patient should have no other symptoms and the fever should be less than 1.5ºC over the pretransfusion baseline. A septic reaction caused by bacterial contamination of a unit is generally the major concern in this setting. Hemolysis of either donor or recipient red cells usually is not significant because of the small amount of red cells and plasma in platelet preparations. The transfusion should generally not be restarted for at least 30 minutes as a precaution to allow other possible signs or symptoms of a serious reaction to develop. High transfusion-related fevers, such as a 2ºC increment or more, are more likely to be associated with septic reactions and should preclude restarting the transfusion. However, lesser fevers do not rule out bacterial contamination of the blood component. If the transfusion is restarted, the patient should be made as comfortable as possible with appropriate antipyretic therapy, as described earlier. The transfusion should proceed slowly and the patient observed closely for further signs of a reaction or further temperature elevation throughout the transfusion, which should be stopped if symptoms recur. Restarting transfusion of a blood component that has caused an FNHTR should not be routine.

Prevention Premedication Premedication with acetaminophen but not diphenhydramine should be considered for patients with a history of FNHTR. Patients who have no history of FNHTR do not need premedication. Despite a number of studies showing no benefit to premedication in preventing transfusion reactions, the practice remains commonplace in many institutions.36 Premedication with the glucocorticoid hydrocortisone sodium succinate (adults, 100 mg intravenously) may be useful in the care of reactionprone patients when an antipyretic agent alone is ineffective. Glucocorticoids have antiinflammatory effects that may help prevent or reduce the severity of FNHTRs. For example, they inhibit the enzyme phospholipase A2, thereby blocking production of arachidonic acid and its metabolites such as PGE2, a key mediator of fever. Glucocorticoids also inhibit synthesis of pyrogenic cytokines, such as IL-1 and IL-6. A variety of glucocorticoids other than hydrocortisone are available. However, hydrocortisone has the advantage of being a short-acting glucocorticoid (biologic half-life, 8 to 12 hours), and it induces a shorter period of immunosuppression than do many other glucocorticoid preparations. Because glucocorticoids generally act through changes in gene expression, hydrocortisone should be administered at least 4 to 6 hours before transfusion so that its antiinflammatory action has time to take effect.

Chapter 53: Febrile, Allergic, and Nonimmune Transfusion Reactions

Rate of Infusion Slowing the speed of infusion of a blood component can possibly prevent or decrease the severity of FNHTR. The rate of increase in body temperature in FNHTR caused by leukocyte alloimmunization appears to be directly related to the rate of infusion of leukocytes in the blood components.18 A slower rate of infusion is of theoretic advantage in decreasing the severity of reactions caused by bacterial contamination or storage-generated cytokines. Slower infusion avoids a sudden bolus of bacterial toxins or cytokines that may provoke an immediate and possibly massive inflammatory response. Leukocyte Reduction The prophylactic transfusion of leukocyte-reduced components in the treatment of patients receiving repeated transfusions is effective in avoiding alloimmunization to leukocytes, which is one of the major causes of FNHTR. Leukocyte-reduced blood components ideally should be transfused to such patients beginning with the first transfusion. Leukocyte reduction is effective in the care of patients already alloimmunized to leukocytes, because FNHTRs in these patients are directly related to the number and rate of infusion of passenger leukocytes. The threshold number of white cells associated with the development of an FNHTR generally ranges from 0.25 ⫻ 109 to 2.5 ⫻ 109.18 The removal of approximately 90% of leukocytes (1010), which usually leaves less than 5 ⫻ 108 white cells per RBC unit, is sufficient to prevent most FNHTRs.17,37 For that reason, leukocyte reduction for the purpose of preventing FNHTRs often is defined as decreasing the passenger leukocytes to less than 5 ⫻ 108 per transfusion. Leukocyte reduction of blood components can be performed either at the time of component preparation (prestorage leukocyte reduction) or immediately before transfusion (poststorage leukocyte reduction). Poststorage leukocyte reduction by means of filtration can be performed in the blood bank before distribution of the component for transfusion or during administration of blood components. The latter often is called bedside leukocyte reduction. Leukocyte-reduced RBC units have in the past been prepared by various poststorage techniques, including simple centrifugation with buffy-coat removal, saline washing, and deglycerolization of frozen RBCs.38,39 Saline-washed and frozen deglycerolized RBCs are rendered leukocyte reduced because approximately 1 to 2 log10 leukocytes are removed by repeated centrifugation and washing steps on automated cell washers. Filtration of RBCs units through microaggregate filters designed to remove microaggregate debris more than 40 microns in diameter after an extra centrifugation step or after centrifugation and cooling (spin, cool, filter) also has been shown to reduce leukocytes in RBC units sufficiently to reduce the incidence of FNHTRs.40-43 High-efficiency leukocyte reduction filters for red cells and platelets have been developed that are capable of removing both microaggregate debris and nonaggregated leukocytes.44 These leukocyte reduction filters can remove 3 or more log (99.9% or more) leukocytes, thereby decreasing the leukocyte content to approximately 1 ⫻ 106/unit or less. Despite their efficacy in

leukocyte reduction, the use of bedside leukocyte reduction filters has had variable and sometimes disappointing results in reducing the incidence of FNHTRs to platelet concentrates.3 This may be the result of causes of FNHTR other than leukocyte antibodies in the transfusion recipient. For example, storagegenerated, extracellular cytokines in the component bag that either are not removed or are inadequately removed by means of poststorage filtration are now believed to mediate some reactions. As a result, the practice of prestorage leukocyte reduction is increasingly replacing poststorage leukocyte reduction. Prestorage leukocyte reduction not only removes leukocytes but also removes leukocytes before they have a chance to release cytokines that can accumulate extracellularly in blood component bags during storage. Prestorage leukocyte reduction has also yielded conflicting results on the efficacy of leukocyte reduction to mitigate FNHTRs.19,20,45 Prestorage leukocyte reduction of platelet concentrates or RBC units is achieved by use of blood component containers with in-line leukocyte reduction filters in a closed system between the primary collection bag and satellite containers. Prestorage leukocyte-reduced platelets also can be prepared with some automated apheresis instruments equipped with centrifugation chambers designed to minimize leukocyte collection (so-called process leukocyte reduction). Because some data indicate that only approximately 15% of patients who experience an FNHTR will have a similar reaction to the next transfusion, some blood banks provide a leukocyte-reduced component (either prestorage or bedside leukocyte reduced) only when a patient has had two or more documented febrile reactions.46 This practice is cost-effective but has the disadvantage of subjecting some patients to two uncomfortable reactions before a preventive measure is taken. Poststorage plasma removal from platelet preparations by saline washing techniques is an alternative to prestorage leukocyte reduction for eliminating extracellular cytokines.14,22 Prestorage leukocyte reduction by means of filtration is a more efficient and cost-effective way to eliminate extracellular leukocyte-derived cytokines while reducing passenger leukocytes. Moreover, in evaluations of plasma removal from platelet concentrates to reduce the risk of FNHTR, this technique still is associated with FNHTR in a relatively large percentage of recipients. Neither leukocyte reduction nor poststorage plasma removal has been effective in eliminating all FNHTRs to platelet transfusions.

Allergic Reactions Description An allergic reaction can be classified as an immediate hypersensitivity response consisting of transient localized or generalized urticaria, erythema, and pruritus. More serious allergic reactions can be complicated by hypotension and angioedema of the face and larynx. Allergic reactions can be categorized as those that

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have only cutaneous manifestations and usually are mild, resolving soon after administration of antihistamines. If other organ systems—cardiovascular, respiratory, or gastrointestinal—are involved beyond mild hypotension, particularly if the reaction is serious enough to necessitate treatment beyond antihistamines, the reaction would be considered anaphylactic or anaphylactoid (see later). Allergic and anaphylactic reactions, however, are part of a continuum. Allergic reactions occur during or soon after transfusion of plasma-containing blood components. Atopic individuals—those with other known allergies—appear at greater risk of allergic reactions. A large retrospective review of reported transfusion reactions noted that 17% of all reactions in a 9-year period were allergic and 1% of reactions were very severe.47 Other papers report allergic reaction rates of approximately 0.1% for red cells and 0.5% for platelet transfusions.19,45

Etiology Allergic reactions are mediated by recipient immunoglobulin E (IgE) or non-IgE antibodies to proteins or other allergenic soluble substances in the donor plasma. The result of the hypersensitivity reaction is secretion of histamine from mast cells and basophils, which mediates cutaneous reactions by increasing vascular permeability. Although the source of histamine in allergic reactions is believed in many cases to be the transfusion recipient’s mast cells and basophils, it has been hypothesized that histamine generated by leukocytes in stored cellular blood components may play a role. Several studies have shown that histamine accumulates in the plasma portion of platelet concentrates and RBC units with increasing storage time. However, histamine is not synthesized during storage but rather it leaks into the extracelluar plasma.48,49 These data are consistent with the observation that allergic transfusion reactions also are more common with increasing storage time of blood components.50 Several of the chemokines that accumulate in the plasma portion of platelet concentrates during blood bank storage, such as IL-8, RANTES, and MIP-1α, can recruit and activate basophils and stimulate histamine release. Therefore, it is theoretically possible that the infusion of storagegenerated donor cytokines during transfusion may contribute to the onset of allergic reactions among transfusion recipients. Consistent with this hypothesis, the platelet-derived cytokine, RANTES, is present at higher levels in platelet concentrates that cause allergic reactions.51 Allergic (and anaphylactic) reactions have been reported after infusion of antibodies in donor plasma, such as penicillin antibody infused into recipients receiving penicillin or related antibiotics, and after infusion of drugs in donor plasma, such as penicillin infused into recipients already sensitized to penicillin.

hemolysis usually are unrevealing. Isolated, mild urticarial reactions not accompanied by other signs and symptoms necessitate minimal diagnostic evaluation. If the reaction is severe, has atypical manifestations, or is accompanied by fever (uncharacteristic of allergic reactions), a more elaborate laboratory evaluation to rule out a hemolytic or septic transfusion reaction is indicated. In the diagnosis of an allergic reaction as transfusion-related, it is important to rule out, if possible, urticarial drug reactions that may be circumstantially attributed to transfusions. Careful attention to the timing of onset of urticaria relative to the transfusion may help avoid this confusion. Administration of medications should generally be discouraged in the peritransfusion period to avoid such confusion. Even mild allergic reactions should be reported to the blood bank. Monitoring allergic reactions and correlating reactions with any newly implemented changes in blood component collection, processing, storage, or filtration are important in detecting new and unexpected causes of reactions. In the care of patients with repeated allergic reactions, notification of the blood bank allows the blood bank medical director to consult on measures to manage or prevent such reactions in the future.

Treatment The patient can be treated with a first-generation, H1-blocking antihistamine (adults, 25 to 50 mg diphenhydramine intravenously or orally). If the sedating side effects of first-generation antihistamines must be avoided, newer, less-sedating antihistamines are available for oral administration (adults, cetirizine 10 mg orally, loratadine 10 mg orally, or fexofenadine 60 mg orally); however, parenteral antihistamines are preferred in the management of acute reactions because of their more rapid bioavailability. An H2 blocker, such as cimetidine (adults, 300 mg intravenously) or ranitidine (adults, 50 mg intravenously) may be added to the H1 blocker to speed resolution of the reaction. Combining H1 and H2 antagonists has given better results in treating patients with allergic reactions in nontransfusion settings than has use of an H1 antagonist alone.52,53 For reactions characterized by only localized urticaria, such as a few hives, the transfusion can be temporarily discontinued while an antihistamine is administered. The transfusion can be resumed in approximately 30 minutes if the urticaria has cleared and if no further symptoms occur. For patients with generalized urticaria or a more serious allergic reaction accompanied by facial or laryngeal edema or hypotension, the transfusion should be discontinued and the infusion set with any untransfused blood returned to the blood bank. If laryngeal edema causes breathing difficulties or if hypotension is severe, epinephrine [adult dose, 0.2 to 0.5 mL of 1:1000 solution (0.2 to 0.5 mg) subcutaneously] can be administered.

Diagnosis Urticaria is readily diagnosed clinically by the presence of the cutaneous wheal-and-flare reaction. Because allergic symptoms usually are mild and are not characteristic of hemolytic transfusion reactions, serologic blood bank investigations to rule out

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Prevention Transfusion recipients often are given routine premedication with an antihistamine such as diphenhydramine in an effort to prevent or reduce the severity of allergic transfusion reactions,

Chapter 53: Febrile, Allergic, and Nonimmune Transfusion Reactions

even when they have had no previous reactions. The value of this approach is uncertain, because few patients have allergic reactions. At least two randomized double-blind placebo-controlled studies of premedication using diphenhydramine and acetaminophen have failed to show a benefit of premedication to reduce reactions.54,55 When premedication is restricted to patients who have had two or more previous allergic reactions, overall reaction rates do not increase. Accordingly, premedication with an antihistamine should probably be reserved for recipients who have had a previous allergic reaction. For patients with repeated reactions not eliminated by premedication with an H1 blocker alone, a combination of H1 and H2 blockers has been shown more effective.52,53 Should premedication not prevent repeated allergic transfusion reactions, another option is to reduce the plasma content of transfused blood components. This can be achieved in RBC and platelet preparations with automated saline “washing.”56,57 However, washing or plasma removal steps generally should be reserved for patients with two or more serious allergic reactions (eg, those that include angioedema or hypotension) that are not prevented with premedication with both H1 and H2 blockers, because cell washing is time-consuming and can delay transfusion. Patients with two or more allergic reactions can undergo testing for IgA deficiency, because this is a reported cause of both allergic and anaphylactic reactions.

Anaphylactic and Anaphylactoid Reactions

binding of allergen to cell-bound IgE results in cross-linking of IgE and Fc receptors. This cross-linking activates the mast cells and basophils to secrete preformed mediators, such as histamine, as well as newly synthesized mediators, such as leukotrienes, prostaglandins, cytokines, and platelet-activating factor (PAF) (Fig 53-2).60 PAF induces downstream production of nitric oxide (NO) through inducible and possibly constitutively expressed NO synthase.61 As a potent vasodilator, NO is believed to be the principal compound causing hypotensive and cardiovascular collapse during anaphylaxis, although the exact mechanism remains under debate. Anaphylactoid reactions are clinically identical to anaphylactic reactions but occur by mechanisms that do not involve IgE. For example, immune complexes involving antibodies other than IgE may result in complement fixation and generation of

(A)

Proteoglycans Histamine

Etiology Anaphylactic reactions occur when an allergen present in plasma is transfused to a patient who through previous sensitization has an IgE directed against that allergen.59 Immunoglobulin E is bound by means of Fc receptors to mast cells and basophils. The

heparin, chondroitin sulphate

Proteases

Cytokines IL-4, IL-5, IL-6, IL-8, IL-13, GM-CSF, TNF-α, fibroblast growth factor, stem cell factor

tryptase, carboxypeptidase, chymase, cathepsin G, elastase, plasminogen activator, renin, matrix metalloprotease 9

Lipid mediators

Description Anaphylactic reactions are serious and potentially life-threatening immediate hypersensitivity reactions to allergens in the plasma of transfused blood components.58 These reactions can have a rapid onset beginning as early as seconds to minutes after the start of the transfusion, and can occur with small transfused volumes. Anaphylactic reactions are differentiated from other allergic (urticarial) transfusion reactions by their systemic nature and severity. These reactions generally affect multiple organ systems, as evidenced by cutaneous, respiratory, cardiovascular, and gastrointestinal effects. The symptom complex often includes the rapid onset of laryngeal edema and bronchospasm with stridor, wheezing, coughing, and respiratory distress. Other symptoms include generalized urticaria, erythema, tachycardia, hypotension, nausea, vomiting, diarrhea, and cramping abdominal or pelvic pain. Severe reactions can proceed rapidly to shock, syncope, respiratory failure, and death. Fatal anaphylactic reactions are less common than are fatal hemolytic or septic reactions.

PRODUCTS OF MAST CELL ACTIVATION

Other enzymes

(B)

β-hexosaminidase, β-glucuronidase, arylsulphatase

prostaglandin D2, leukotriene C4, platelet activating factor

PRODUCTS OF BASOPHIL ACTIVATION Histamine

Proteoglycans heparin chondroitin sulphate

Proteases tryptase, elastase cathepsin G

Basic proteins basogranulin, 2D7 antigen, eosinophil major basic protein, eosinophil cationic protein, eosinophil-derived neurotoxin

Other enzymes

Cytokines IL-4, IL-8, IL-13, MIP-1␣, IgE-dependent histamine releasing factor

Lipid mediators leukotriene C4, platelet activating factor β-hexosaminidase, β-glucuronidase, eosinophil peroxidase

Figure 53-2. (A) Mast cell with its activation products. (B) Basophil with its activation products. Note that currently only two products of mast cell activation (histamine and total tryptase) and one product of basophil activation (histamine) can be measured in clinical laboratories as markers of acute anaphylaxis events. Used with permission from Simons FE.60 IL ⫽ interleukin; GM-CSF ⫽ granulocytemacrophage colony-stimulating factor; TNF-α ⫽ tumor necrosis factor alpha; MIP ⫽ macrophage inflammatory protein.

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the anaphylatoxins C3a, C4a, and C5a, which activate basophils and mast cells. Some cytokines secreted by monocytes as part of the inflammatory cascade initiated by non-IgE immune complex formation also can directly activate basophils and mast cells and initiate anaphylactoid reactions. Moreover, IgG4 subclass antibodies can bind to Fc receptors of mast cells and basophils and, in a manner analogous to that of IgE, mediate cellular activation and degranulation after binding of allergen. The term anaphylactoid is sometimes used to describe mild or clinically atypical anaphylactic reactions. However, anaphylactoid is better used to differentiate the mechanism of the reaction, not its clinical severity or presentation. The best documented anaphylactoid reactions have resulted from the transfusion of donor plasma containing IgA to IgAdeficient recipients who have produced a class-specific IgG anti-IgA that reacts with all IgA subclasses. Less commonly, patients with normal total IgA levels have a subclass-specific IgA deficiency and may make an anti-IgA of restricted specificity. Although IgA deficiency is relatively common (approximately 1 case among 700 persons), anaphylactoid reactions occur only among some IgA-deficient transfusion recipients, because not all make anti-IgA. Anaphylactic or anaphylactoid reactions have been documented among patients with deficiencies of other plasma proteins, such as complement, von Willebrand factor, and haptoglobin.62,63 In an analogous manner, these patients produce an antibody to the missing factor that reacts with transfused, plasma-containing blood components. In most anaphylactic or anaphylactoid reactions, however, the allergen is never identified, nor is evidence obtained to differentiate anaphylactic from anaphylactoid mechanisms.

Diagnosis Anaphylactic and anaphylactoid reactions are diagnosed from clinical signs and symptoms.64 The cutaneous signs and symptoms and the often rapid onset of the reaction help differentiate anaphylactic reactions from acute hemolytic and septic transfusion reactions. Serum β-tryptase levels may be measured to confirm an anaphylactic reaction, because it is a marker for mast cell degranulation. However, no laboratory measurement is available in time to meaningfully affect recognition and management of an anaphylactic reaction. Recipient IgA levels should be measured in a pretransfusion blood specimen to determine if the recipient is IgA-deficient. Although the results of tests for IgA deficiency do not affect diagnosis or management of the reaction at hand, it is important for avoiding future reactions.58 Testing should be performed on a specimen drawn before transfusion, because IgA deficiency can be masked by any IgA provided by the transfusion. Recipient anti-IgA also can be measured, especially for rare cases in which the anti-IgA is subtype-specific and total IgA levels are within the reference range. Although IgA is the most commonly known allergen in anaphylactoid reactions, in most anaphylactic and anaphylactoid reactions, the offending allergen is not IgA and is never identified.

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Treatment Anaphylactic and anaphylactoid reactions are managed identically.64 Severe reactions are true medical emergencies and should be managed by experienced critical care staff, if possible. The patient should be placed in an intensive care unit as soon as it is practical without jeopardizing emergency care. Once anaphylaxis is evident clinically, 1:1000 epinephrine solution (1 mg/mL) should be administered subcutaneously in a dose of 0.2 to 0.5 mL for adults (0.01 mL/kg of body weight for children). The dose may be repeated every 15 to 30 minutes as needed. Intravenous crystalloid or colloid solution should be administered as needed to support the patient’s blood pressure. For example, 500 mL to 1 L of normal saline solution can be administered in the first 15 to 30 minutes. Further infusion should be titrated to blood pressure. If the systolic blood pressure is less than 60 mm Hg, intravenous epinephrine in a dose of 1 to 5 mL of a 1:10,000 solution (0.1 mg/mL) for adults and 0.1 mL/kg for children, is administered over 2 to 5 minutes by means of intravenous push. An epinephrine drip (1 to 4 µg/minute) may be started, and administration of other pressors, such as dopamine, can be considered. Blood pressure, pulse, and urine output should be monitored. It may be necessary to monitor the effectiveness of fluid replacement and pressor infusion through measurement of central venous pressure. Respiratory distress is managed with supplemental oxygen. The patient’s upper airway may have to be secured with endotracheal intubation if obstruction from laryngeal edema is imminent. Stridor is a sign of laryngeal edema. Endotracheal intubation and mechanical assistance with ventilation are indicated if the PaCO2 increases to more than 65 mm Hg. When intubation is difficult or impossible because of laryngeal obstruction, cricothyrotomy or tracheostomy is an option. Wheezing caused by obstruction of small bronchi and bronchioles by increased mucus production and smooth muscle contraction can be managed with nebulized albuterol or metaproterenol and intravenous aminophylline. Urticaria, angioedema, or gastrointestinal distress is managed with an antihistamine (adults, 50 mg diphenhydramine intravenously; children, 1 to 2 mg/kg intravenously). H2-blocking antihistamines may be added as an adjunct to H1 blockers. Glucocorticoids, such as hydrocortisone, 200 mg given intravenously every 6 hours, are also administered because they reduce late-phase inflammatory responses. Glucocorticoids, however, are not expected to be of benefit in the initial management of anaphylaxis because of their delayed onset of action.

Prevention Patients with IgA deficiency who have already had an anaphylactic reaction or who are known to have anti-IgA should receive transfusion of RBC and platelet preparations that have been saline-washed with an automated cell washer.56,58 If plasma transfusion is necessary, only IgA-deficient donors should be used. Patients who have anaphylactic reactions to any other known plasma allergen also should be treated with transfusion

Chapter 53: Febrile, Allergic, and Nonimmune Transfusion Reactions

of saline-washed RBCs or platelet preparations. Because anaphylactic reactions can be induced by very small amounts of allergen, washing must be extensive. Washing and saline replacement by means of automated cell washers have been shown generally successful in removing IgA sufficiently to prevent recurrences of anaphylactoid reactions. If a patient has had one anaphylactic reaction of unknown causation, the next transfusion need not necessarily be performed with washed RBCs or platelets, because the reaction might have been donor-specific. The next transfusion may be administered slowly with vigilance after premedication with both H1 and H2 blockers and a glucocorticoid. The patient care team should be prepared to respond to an anaphylactic reaction. The patient ideally should be in a critical care unit with monitoring at the time of transfusion and with a critical care physician and nurses in attendance. Some blood banks with the capability of automated cell washing, nevertheless, may choose to provide saline-washed RBCs and platelet concentrates for future transfusions after a single anaphylactic reaction as a precautionary measure, particularly if the patient is not expected to receive many more transfusions.

Complications of Massive and Rapid Transfusion Massive transfusion is defined as the replacement of one blood volume within a 24-hour period. For adults of average size, this is roughly equivalent to 10 units of RBCs with any accompanying crystalloid, colloid, platelet, or plasma infusions. The possible complications include citrate toxicity, electrolyte imbalance (hyperkalemia from transfusion of older RBCs, hypokalemia from citrate toxicity), circulatory overload, and hypothermia. Recipients of massive transfusions are at increased risk of hemolytic transfusion reactions (including ABO incompatibility), FNHTR, and allergic reactions because of the number of units they receive. Reactions can be more severe with massive transfusion because rapid infusion means the implicated unit often has been completely administered before the onset of symptoms. The large number of units transfused in a short time complicates the investigation of transfusion reactions, because each transfused component must be investigated. The lethal triad of severe trauma consists of hypothermia, acidosis, and coagulopathy. “Damage control” resuscitation methods have been developed to directly address the coagulopathy of trauma. The coagulopathy of trauma develops because of severe injury and is already present when a patient presents for emergency medical care. Trauma coagulopathy is not caused by the resuscitation efforts of emergency medical interventions as traditionally understood.65 Recent advances in trauma research have shown that early application of damage control resuscitation can greatly improve survival through an application of a 1:1 plasma:red cell ratio from patient presentation. In well-coordinated trauma centers, massive transfusion protocols are under investigation that provide 6 units of plasma, 6 units of RBCs,

6 whole-blood-derived platelet concentrates (or 1 apheresis platelet), and 10 units of cryoprecipitate as a part of a predefined effort to treat the coagulopathy of trauma.65-67 Aggressive damage control transfusion has been associated with a number of risks including hyperkalemia, and when a walking blood donor program is employed, increased infectious disease transmission.68,69 Despite these risks, retrospective studies have shown that the application of early, aggressive transfusion support improves overall survival in this severely injured population that would otherwise have very poor prognosis. Rapid transfusion can also occur during therapeutic apheresis and red cell exchange apheresis (erythrocytapheresis). During apheresis procedures, as many as 10 to 20 units of Fresh Frozen Plasma or 4 to 8 units of RBCs can be transfused over 1.5 to 2 hours. Although any acute transfusion reaction can occur during apheresis-associated transfusion, citrate toxicity in particular is a common but usually mild complication.

Citrate Toxicity Rapid blood transfusion can cause a transient decrease in the level of ionized calcium because of the calcium-chelating properties of the citrate anticoagulant in stored blood components.70 The clinical presentation of citrate-induced symptoms is also termed citrate toxicity. Citrate toxicity can occur whenever large volumes of plasma that contain citrate are transfused, such as during massive transfusion, plasma exchange, or other apheresis procedures.71,72 Citrate infusion can induce hypocalcemia, hypomagnesemia, and other electrolyte imbalances, and these imbalances are associated with clinical symptoms. Apheresis procedures can produce a unique clinical paradox of urinary calcium excretion in the setting of hypocalcemia.72,73 Hypocalcemia is a recognized complication of liver transplantation, in which large amounts of plasma are transfused. However, the precise mechanism of hypocalcemia is not well understood and may not be caused entirely by citrated plasma.74,75 Citrate ordinarily is rapidly metabolized to bicarbonate in mitochondria-rich tissue, such as liver, skeletal muscle, and kidney.70 In the routine transfusion of blood components, patients with normal liver function usually tolerate the citrate infusion without significant complications. However, patients with liver or renal failure or parathyroid dysfunction are at greater risk of citrate toxicity when they receive rapid transfusions of plasma or plasma-containing blood components. Citrate anticoagulates blood by binding divalent cations such as calcium, thus hypocalcemia is a primary symptom. Other divalent cations such as magnesium and zinc can also be bound by citrate, but the contribution of hypomagnesemia to clinical symptoms is less pronounced.76 During apheresis, citrate is administered as acid-citrate-dextrose formula A (ACD-A) in constant proportion to the whole-blood flow rate. Healthy plateletpheresis donors receive relatively large doses of citrate, and many experience mild symptoms of the citrate effect, but the symptoms usually do not progress because of the short duration of the procedure. Donors of peripheral blood stem cells (PBSCs),

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however, receive smaller doses of citrate per unit of time but usually experience more severe citrate toxicity because of the longer duration of the procedure. Paresthesia caused by transient hypocalcemia is common in apheresis. It typically occurs after the initial infusion of the priming solution (if citrate is used) or later as apheresis progresses. Apheresis practitioners should be aware that peripheral paresthesia caused by hypocalcemia can be masked in patients with a preexisting neuropathy as a result of chemotherapy (vincristine) or as part of a neurologic condition. Citrate toxicity is recognized clinically because of the signs and symptoms of hypocalcemia. It can be confirmed by measuring the plasma ionized calcium level in the patient. Symptoms of hypocalcemia include peripheral and perioral paresthesia (Chvostek and Trousseau signs can occasionally be elucidated), muscle spasm, cramping, nausea, vomiting, cardiac arrhythmia, bradycardia, hypotension, and if severe, tetany. An electrocardiogram (ECG) can show prolongation of the QT interval with hypocalcemia, but the relation is not linear with the ionized calcium level, and ECG findings are an unreliable guide to calcium therapy. Mild citrate toxicity during transfusion or apheresis is managed or prevented in part by means of slowing the rate of transfusion or reinfusion. When slowing the infusion rate is impossible or ineffective and the patient has signs and symptoms of hypocalcemia, calcium supplementation is indicated. The best guide to determining a need for calcium supplementation is measurement of the patient’s ionized calcium levels, if results can be obtained rapidly. Calcium replacement during apheresis should generally be given when a patient has symptoms, when the patient’s clinical condition may exacerbate citrate effects, or when prolonged large-volume leukapheresis is expected to cause citrate toxicity. Infusion of calcium itself, however, is associated with development of ventricular arrhythmia and even cardiac arrest. Therefore, intravenous calcium replacement for the management or prophylaxis of apheresis-induced citrate toxicity should be administered only by experienced apheresis staff. Under no circumstances should calcium be added directly to a unit of blood, because it causes clots to form in the bag. Citrate toxicity during apheresis is related to the citrate concentration of the reinfused blood or colloid solution, the infusion rate, the blood volume of the patient, and the total time over which the citrate is infused.77 It is difficult to establish a definitive safe rate of citrate infusion because of the large number of variables involved. However, citrate dosages of up to 1 mg/kg/ minute given during plateletpheresis usually are well tolerated. A safe rate of calcium replacement for controlling citrate toxicity during PBSC apheresis is 0.5 to 0.6 mg of calcium ion for every 1.0 mL of infused ACD-A.78,79 These dosages have been successful for prophylaxis against citrate toxicity during large-volume leukapheresis. To avoid excessive volume during PBSC apheresis, administration of a concentrated calcium solution (calcium chloride or calcium gluconate) is appropriate. Care must be taken to coordinate the calcium infusion with whole blood flow during the apheresis procedure to avoid the potential for catheter

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thrombosis. Calcium administration should be halted soon after interruptions in whole blood flow.

Electrolyte Disorders Because of inhibition of sodium-potassium-adenosine triphosphatase in red cell membranes by the cold storage temperature of RBC units, extracellular potassium accumulates with increasing blood bank storage times. Extracellular potassium increases at the rate of approximately 1 mEq/day during the first 3 weeks of RBC storage in citrate-phosphate-dextrose-adenine1 (CPDA-1).80 Potassium levels in additive solution-1 (Adsol) units are markedly higher on Day 7 of storage (17 mmol/L) than on Day 0 (1.6 mmol/L). The increase is even greater by Day 42 (46 mmol/L).81 Hyperkalemia resulting from massive transfusion of older RBC units with an elevated amount of extracellular potassium can cause significant cardiac complications and possibly death in some patients.68 Acidosis can contribute to hyperkalemia and severely injured patients presenting with a potassium level greater than 4 mmol/L are at increased risk. Other patients at risk of hyperkalemic complications are neonates and those with renal failure. The diagnosis of hyperkalemia is made by means of measurement of potassium in the serum and observation of ECG changes, which include peaked T waves, prolongation of the PR interval, and ventricular arrhythmia.82 In neonatal transfusions, hyperkalemia can be avoided by use of fresh RBC units (less than 7 days old) or older units that have been saline-washed to remove the extracellular fraction containing the potassium.83,84 However, transfusion of older RBC units does not place neonates at risk of hyperkalemia if small-volume transfusions (10 to 15 mL/kg) are given slowly.85 Hypokalemia can also develop during massive transfusion or large volume apheresis.72 As the anticoagulant citrate in blood components is metabolized to bicarbonate, the blood can become alkalotic, producing hypokalemia. The degree of hypokalemia may be sufficient to necessitate infusion of potassium if symptoms develop. However, the use of newer RBC additive solutions such as Adsol has helped to decrease the effect of hypokalemia. RBC units are plasma-reduced before the addition of additive solution, which itself contains no additional citrate. Therefore, most of the citrated plasma is removed from additive solution RBC units during production. Animal studies have shown fewer physiologic aberrations during massive transfusion with Adsol RBCs than with CPDA-1 units.86 The complications of hypokalemia, therefore, are more likely when large numbers of units of plasma rather than RBC units are transfused.

Hypothermia Hypothermia, defined as a core body temperature of less than 35ºC, may be caused by rapid infusion of large quantities of cold (1ºC to 10ºC) blood or RBC units. Hypothermia during massive transfusion has been shown to induce cardiac arrhythmia and arrest.87 Even smaller quantities of cold blood can be cardiotoxic if transfused into central venous lines, because the newly infused cold blood can reach the heart before sufficient warming has

Chapter 53: Febrile, Allergic, and Nonimmune Transfusion Reactions

occurred. Data published in the early 1960s showed that massive transfusion at a rate of approximately 1 unit every 5 to 10 minutes was sufficient to lower the temperature of an esophageal probe behind the right atrium to nearly 30ºC.87 The resulting decrease in sinoatrial node temperature was associated with the development of ventricular fibrillation. For most routine transfusions given at a standard rate of administration, blood does not have to be warmed.88 The patient may experience minor chills, but this is easily remedied by warming the patient, as with extra blankets. Transfusion of cold RBC units through central venous lines, however, should be avoided. Indications for warming blood include rapid transfusion, which are generally considered to be more than 50 mL/kg/hour for an adult and more than 15 mL/kg per hour for a child, and exchange transfusion for infants. Because blood warming during certain massive transfusions sometimes delays infusion and impedes resuscitation, it is not always practical. Warming blood for transfusion in the treatment of patients with cold agglutinin disease has a theoretic basis but is debatable because supportive outcome data are lacking. If blood has to be warmed, an approved warming device should be used and the temperature must be kept below a point where hemolysis occurs.88 Infusion of thermally injured cells can induce disseminated intravascular coagulation and shock. Heating blood with a device other than an approved bloodwarming device, such as a commercial microwave oven, is unacceptable. Blood that has been warmed but not used should not be reissued for another patient because of the increased risk of bacterial proliferation at warmer temperatures. The maximum flow rate that can be achieved with commercially available blood warmers is 850 mL/minute; however, most can provide a rate of only 150 mL/minute. Most recent research has focused on comparisons between commercial warming devices and the comparison studies usually evaluate warming capability in the rapid transfusion setting.89 Most of the available federally approved blood warming devices safely warm blood and other intravenous fluids across a range of flow rates. However, while little recent data exist correlating the clinical benefits of warming blood, many emergency centers and trauma services use such devices routinely without incident. An alternative to using mechanical blood warmers that circumvents such flow limitations is rapid admixture with warm or hot saline solution immediately before transfusion.88,90 This technique immediately warms a unit of RBCs, yet does not cause significant hemolysis. However, it necessitates that warmed saline solution be available at all times in trauma care and requires attention to technique to avoid the direct infusion of hot saline solution into the patient.

Reactions Attributed to Microaggregate Debris Microaggregate debris ranges in diameter from 20 to 120 microns and consists of nonviable platelets, white cells, and strands of fibrin that form in blood during storage.91,92 Because of their size, microaggregates are not removed from transfused blood with the standard 170- to 260-micron screen filters. A variety of

adverse events have been attributed to the presence of microaggregate debris after large-volume and massive transfusion. Studies in the 1960s showed that patients undergoing openheart surgery with cardiopulmonary bypass experienced postperfusion syndrome during the postoperative period. This symptom complex consisted of cerebral and renal dysfunction attributed in part to occlusion of end-organ capillaries with microaggregate debris. Cotton wool (Swank) microaggregate blood filters capable of retaining particles or debris with a size of 40 microns or more appeared to eliminate many of these reactions. During the Vietnam War, some soldiers who underwent massive transfusion experienced respiratory distress syndrome (shock lung). At autopsy, the cause was presumed to be the material found in soldiers’ lungs that was positive on a periodic acid-Schiff (PAS) test. Because microaggregate debris stains PAS-positive, this was taken at the time as evidence of the pathologic nature of microaggregate debris. During the 1970s and 1980s, studies were undertaken to determine whether removal of microaggregate debris from blood was clinically significant.91 Several studies showed that microaggregate filtration of up to 6 units of blood during either hip or cardiac surgery provided no benefit. Collins et al93 concluded that the underlying clinical condition rather than the infusion of the microaggregate debris in blood led to the development of the respiratory distress syndrome reported earlier among patients undergoing massive transfusions. Microaggregate blood filters today are used mostly in conjunction with cardiopulmonary bypass pumps and are only rarely used otherwise. With the widespread adoption of leukocyte reduction filtration, routine leukocyte-reduced red cell transfusions are no longer complicated by microaggregate debris because these filters can remove not only leukocytes, but also the larger microaggregate particles. Current studies are investigating the utility of washing stored blood to remove microaggregate debris that accumulates during storage.94

Circulatory Overload Hypervolemia, termed transfusion-associated circulatory overload, is a possible consequence of transfusion in the care of patients with cardiac insufficiency, renal impairment, or already expanded blood volumes, such as patients with chronic anemia. Moreover, patients with restricted blood volumes (such as infants and small children) are at risk of TACO if transfused blood is not reduced to an amount proportional to body mass and intravascular blood volume. The risk of TACO increases with rapid infusion. Circulatory overload increases central venous pressure, causes congestion of the pulmonary vasculature, and decreases lung compliance, manifesting as dyspnea, tachycardia, acute hypertension, and in the extreme, pulmonary edema and left- or right-sided heart failure. Other signs and symptoms of circulatory overload include tachypnea, dry cough, chest or throat tightness, jugular venous distention, and pulmonary rales. Laboratory measurements of circulatory overload include PaO2, atrial natriuretic peptide and B-type natriuretic peptide.95,96 The

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latter has a reported 81% sensitivity for detecting circulatory overload in the appropriate clinical setting. Diagnosing TACO can be difficult and confounded by other concomitant pathology. There is no accepted clinical definition of TACO and the symptoms of TACO can overlap significantly with other transfusion reactions such as TRALI (Table 53-1).97,98 Some rapid methods of differentiating the overlapping symptoms include blood pressure, response to diuretic therapy, white cell count, and heart auscultation for an S3 (third heart sound). If symptoms of overload appear, the transfusion should be stopped, and intravascular volume reduction through diuresis should be instituted as needed (eg, administration of 40 mg furosemide intravenously). The patient should be placed in an upright (reverse Trendelenburg) position, if possible, with supplemental oxygen as necessary. Phlebotomy may be considered for severe volume overload, although this is not usually prudent in the setting of anemia or hypoxemia. Rapid transfusion of any blood component into a patient who is not actively hemorrhaging produces no benefit and can cause harm. As a general guide, infusion should be at a rate not to exceed 2 to 4 mL/kg/hour, and the rate should be lower (⬃1 mL/kg/hour) for patients at high risk of circulatory overload.99 In neonates, a slower blood infusion rate increases the hematocrit and decreases cardiac demand without affecting pulmonary artery pressure. More rapid infusion rates are associated with decreased lung compliance and increased pulmonary airflow resistance.100,101 For patients with volume overload caused by medical reasons existing before transfusion, furosemide can be given prophylactically, and transfusion should proceed slowly. The rate of transfusion can be even further slowed, if necessary, by dividing a unit of RBCs or another component into smaller

aliquots and transfusing a portion at a time over as much as 4 hours, the maximum allowable time a blood component should be kept outside blood-bank-monitored storage. For RBCs and thawed plasma, the unused portion can be stored in the blood bank at 1ºC to 6ºC for up to 24 hours while the initial aliquot is administered. Platelet aliquots can be sampled from a single apheresis platelet unit, and with this practice, donor exposures can be minimized. It is important that transfusion of all or part of a blood component be completed within 4 hours and that any unused portion be stored under regulated blood bank conditions because of concerns about increased risk of bacterial contamination during improper storage. RBC units, apheresis platelets, and whole-blood-derived platelet pools can be further concentrated by means of centrifugation and plasma removal, if other measures to prevent volume overload are inadequate.

Toxic Reactions Resulting from Blood Manufacture or Processing Hypotensive Reactions Hypotension has been reported among patients receiving bedside, leukocyte-reduced platelets who are also medicated with angiotensin-converting enzyme (ACE) inhibitors.102 These reactions appear to be caused by generation of bradykinin in transfused blood just as it is being passed through negatively charged leukocyte reduction filters. The mechanism is believed to involve the formation of activated Factor XIIa when Factor XII, a contact factor, is exposed to the negatively charged filter surface. The filter surface can mimic exposed, negatively charged subendothelium, which is the natural activating stimulus for the contact factors

Table 53-1. Features in TRALI and TACO Feature

TRALI

TACO

Body temperature Blood pressure Respiratory symptoms Neck veins Auscultation Chest radiograph Ejection fraction PA occlusion pressure Pulmonary edema fluid Fluid balance Response to diuretic White count BNP Leukocyte antibodies

Fever can be present Hypotension Acute dyspnea Unchanged Rales Diffuse, bilateral infiltrates Normal, decreased 18 mmHg or less Exudate Positive, even, negative Minimal Transient leukopenia ⬍200 pg/mL Donor leukocyte antibodies present, crossmatch incompatibility between donor and recipient

Unchanged Hypertension Acute dyspnea Can be distended Rales, S3 may be present Diffuse, bilateral infiltrates Decreased Greater than 18 mmHg Transudate Positive Significant Unchanged ⬎1200 pg/mL Donor leukocyte antibodies may or may not be present, positive results can suggest TRALI even with true TACO cases

The typical patterns that would be expected for cases of transfusion-related acute lung injury (TRALI) or transfusion-associated circulatory overload (TACO) are represented. A given case of TRALI or TACO may lack some of the typical features. Also, a case of TRALI may have some features suggesting TACO or vice versa, and TRALI and TACO can be present together. The best strategy is to develop a full clinical profile of the case using the feature list above, and determine which diagnosis is most supported. BNP ⫽ brain natriuretic peptide; PA ⫽ pulmonary artery.

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Chapter 53: Febrile, Allergic, and Nonimmune Transfusion Reactions

of the intrinsic coagulation pathway after blood vessel damage in vivo. Factor XIIa converts prekallikrein to kallikrein, which cleaves high-molecular-weight kininogen to form bradykinin. The biologic activity of the infused bradykinin is prolonged in transfusion recipients who are also receiving ACE inhibitors (eg, captopril, enalapril), which inhibit kininase II, the enzyme that breaks down bradykinin. The combination of bradykinin generation just as the blood is being infused with inhibition of the transfusion recipient’s ability to break down bradykinin produces prolonged bradykinin activity conducive to hypotensive reactions. These reactions are less likely with use of prestorage leukocyte-reduced blood components, because the bradykinin is broken down rapidly in the component bag during storage before transfusion. Although hypotensive reactions have been reported more frequently with negatively charged bedside leukocyte reduction filters, they also have been rarely reported with positively charged filters. This can be explained in part by the possibility that patients taking ACE inhibitors may be more prone to hypotensive reactions in general because of their relative inability to rapidly break down bradykinin generated in vivo by any allergic mechanism. Hypotensive reactions to bedside leukocyte reduction among patients taking ACE inhibitors can be prevented by use of prestorage leukocyte-reduced blood components or by means of temporary discontinuation of ACE inhibitor treatment. Apheresis procedures are also associated with hypotensive reactions and the literature has described hypotensive reactions in both adult and pediatric apheresis patients.103,104 Apheresis may contribute to hypotensive reactions through several mechanisms including the potentiation of bradykinin-mediated effects by albumin and secondary to hypocalcemia.104,105 Data suggest that calcium infusions can mitigate some atypical apheresis reactions, while withholding ACE inhibitor medications 24 to 48 hours before apheresis may also contribute to lessening reactions.

with increasing exposure. The DEHP metabolite, mono(2ethylhexyl)phthalate (MEHP), also accumulates during storage.108 Infusion of blood that contains DEHP results in deposition of DEHP in various tissues; the greatest accumulation is in body fat. Results of some studies with animals have suggested that DEHP is toxic and may even be carcinogenic in large quantities.109 Other studies with animals have shown that MEHP is associated with formation of peroxisomes, indicating tissue alteration and toxicity. Although there have been no reports of transfusion-related plasticizer toxicity among humans, results of some in-vitro experiments suggest that high concentrations of MEHP have a negative inotropic effect and can cause irregular contractions in isolated human myocardial cells. Some clinical data have described the production of antiplasticizer IgE in transfusion recipients and the incorporation of plasticizer into red cells during storage.110 Despite the possible adverse effects of DEHP plasticizers, other data indicate that these substances stabilize red cell membranes and improve the morphologic features of platelets during storage.111,112 No good evidence exists, however, of actual improvement in posttransfusion outcomes as the result of these effects. Formulations for plastic blood bags are being developed with plasticizers other than DEHP that have a decreased capacity to leach into plasma. For example, one polyvinyl-chloride-based material is made with plasticizer butyryl tri-n-hexyl citrate (BTHC). Although BTHC also leaches into blood components, it does so at a significantly slower rate than does DEHP. It also provides an antihemolytic effect similar to that of DEHP.113 Studies have shown this citrate-based plasticizer is suitable for storage of both RBCs and platelets.114 Given the concerns over the risk of DEHP toxicity, other plasticizers and storage materials continue to be sought in an attempt to replace traditional storage materials. The possible human toxicity of DEHP continues to be evaluated.115-117 (See also Chapter 4.)

Ocular Reaction to Leukocyte-Reduced Blood Components: Red Eye Syndrome

Dimethyl Sulfoxide Toxicity During Infusion of Cryopreserved Progenitor Cells

Some patients receiving transfusions of RBCs prestorage leukocyte reduced with a specific filtration system (LeukoNet Prestorage Leukocyte Reduction Filtration System, HemaSure, Marlborough, MA) sustained bilateral conjunctival erythema (red eye syndrome).106 The conjunctival erythema occurred within 24 hours of transfusion. Resolution occurred spontaneously within 2 to 21 days with a median duration of 5 days. The implicated prestorage leukocyte reduction system has been discontinued, and red eye syndrome has not been reported with other leukocyte reduction filters.107 The red eye symptoms are hypothesized to be an allergic or toxic reaction to cellulose acetate derivatives that leached from the filter membrane.

Dimethyl sulfoxide is a versatile solvent that has been used as the principal cryopreservative for mononuclear cells since the 1950s. It is widely used as a cryopreservative for marrow and PBSCs used in human hematopoietic progenitor cell transplantation. Despite this, DMSO is not approved by the Food and Drug Administration as a pharmacologic agent for intravenous administration, and guidelines for intravenous administration are obscure. Toxicologic studies, however, have established the general safety of intravenous DMSO infusion.118,119 The metabolism of DMSO yields a characteristic harmless odor, described as a malodorous garlic or sulfur-like smell. Because of the exceptional solvent properties of the compound, DMSO is distributed throughout all tissues after administration. The two metabolites of DMSO are dimethyl sulfdioxide and dimethyl sulfide (DMS). Dimethyl sulfdioxide is an odorless compound excreted by the kidney, and DMS is excreted through the lungs and through other tissues and contributes to the characteristic odor. The clinical toxicity of DMSO in marrow transplantation has been studied. Anaphylactoid symptoms attributable to the release

Plasticizer Toxicity Plasticizers are chemicals used to make rigid polyvinyl chloride plastics more malleable. The traditional plasticizer for blood storage bags is di(2-ethylhexyl)phthalate (DEHP), which leaches over time from the plastic into the blood and blood components

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of histamine and other mediators are common. Other toxic clinical signs and symptoms include hemolysis with hemoglobinuria, hyperosmolality, increased serum transaminase values, nausea, vomiting, abdominal cramping, fever, chills, tachypnea, cough, diarrhea, flushing, and headache.120,121 Patients who have been conditioned with chemotherapy or who have smaller body mass (⬍70 kg), seem more likely to experience nausea and vomiting after infusion of DMSO-preserved cells. Cardiovascular toxicities include decreased heart rate and bradycardia, occasionally increased heart rate and tachycardia, ectopic heartbeat, heart block, hypotension, hypertension, and other lesser blood pressure changes.120,122 Some studies, however, raise the question whether there is any significant cardiovascular toxicity of DMSO.123 It is possible that some adverse effects attributed to DMSO may be caused by the cellular infusion itself. The mechanism of the clinical toxicities associated with DMSO infusion has not been well established. Histamine receptor binding of DMSO, histamine release, direct vagal tonic effects, cold thermal vagal responses, and renal failure secondary to hemolysis explain many of the symptoms observed during cryopreserved cellular infusion.120,124 Increases in thrombinantithrombin complex, β-thromboglobulin, platelet factor 4, and von Willebrand factor caused by DMSO have been described. Several measures can be taken to prevent or reduce DMSO toxicity. Antihistamine prophylaxis is recommended routinely before any administration of DMSO. Intravenous DMSO should be given as a 10% to 40% solution to avoid local irritation. The recommended maximum daily dose of DMSO is 1 g/kg/day. Slowing an infusion containing DMSO or increasing the time between infusions of multiple aliquots greatly diminishes DMSO-related toxicity, which appears to be a dose-dependent but short-lived response. However, because DMSO is toxic to thawed mononuclear cells, hematopoietic progenitor cells can tolerate exposure to 10% DMSO for only as long as 1 hour.125 This limits how much the infusion rate can be slowed. Antiemetics and sedatives can help to ameliorate symptoms, and cellular products can be carefully washed before infusion to remove DMSO and other substances.

Reactions in Special Transfusion Settings Granulocyte Transfusion Reactions Granulocyte transfusions remain in use as a treatment option for neutropenic patients because of improved granulocyte collection yields after donor treatment with steroids and granulocyte colony-stimulating factor (G-CSF). Febrile nonhemolytic transfusion reactions after granulocyte transfusion are common. Severe reactions can be accompanied by pulmonary complications (eg, TRALI, dyspnea, pulmonary infiltrates, and hypoxia), hypotension, and even cardiovascular collapse.126 In a recent study of dexamethasone- and G-CSF-stimulated granulocyte transfusions, 37% of patients (7% of transfusions) experienced chills, 32% of patients (7% of transfusions) experienced a fever, and 11% of patients (2% of transfusions) experienced hives

840

or itching during a course of therapy.127 Oxygen desaturation of greater than 3% occurred in 7% of transfusions, and severe desaturation of greater than 6% occurred in three of 11 patients experiencing oxygen desaturation. In addition, granulocyte transfusions carry further risk of leukocyte alloimmunization and cytomegalovirus infection.128 Concurrent administration of amphotericin B and granulocytes has been linked to severe pulmonary reactions, although the association has not been confirmed and remains in doubt. Nevertheless, it is prudent to separate amphotericin B administration and granulocyte therapy by at least 6 hours to avoid confusion about the cause of a severe reaction, which can occur with either of these reaction-prone treatments. Because of the relatively high rate and severity of febrile, pulmonary, and allergic reactions, it is prudent to give premedication with acetaminophen and diphenhydramine to recipients of granulocyte transfusions. Hydrocortisone may be added as premedication in the treatment of patients with severe reactions who otherwise cannot tolerate granulocyte transfusion, although the immunosuppressive effects of this agent are unwelcome among patients who need granulocyte transfusions to fight serious and life-threatening infections. Granulocyte concentrates should be transfused slowly.

Autologous Transfusion Reactions A variety of reactions to autologous blood occur despite the complete compatibility. In a study involving 596 hospitals, the rate of reported FNHTRs to autologous blood was 0.12% and the rate of allergic reactions was 0.01% per transfused unit.129 Such rates are approximately fivefold to 10-fold lower than those reported for allogeneic units. The cause of autologous transfusion reactions has not been clearly established. Mechanisms in many cases presumably are the same as for allogeneic transfusions. For example, autologous units can be contaminated with bacteria as can allogeneic units, and contamination leads to febrile reactions or septic complications.130 Because autologous donors are patients, not healthy volunteers, they may have various medical problems that put them at increased risk of bacteremia. Accumulation of inflammatory mediators in blood component bags during storage, released from passenger leukocytes or platelets, may result in the infusion of pyrogenic substances. Autologous leukocytes, rather than allogeneic leukocytes, can also generate endogenous pyrogens. Allergic reactions may be provoked by histamine generation during storage of blood components or by chemicals leached from blood storage containers or filters. Moreover, autologous transfusions may contribute to volume overload and hypervolemic reactions through mechanisms identical to allogeneic transfusions.

Summary A variety of acute, nonhemolytic and noninfectious reactions are reported after transfusion (Table 53-2). Many of these reactions

Table 53-2. Transfusion Reaction Summary Cause

Signs and Symptoms

Treatment

Prevention

Febrile, nonhemolytic

Recipient antibodies against leukocytes or Chills, fever (⬎1ºC increase in body platelets in donor blood components; temperature); rigors in severe cytokines in plasma or supernatant portion reactions of stored components; undetected bacterial contamination of blood component

Stop transfusion, notify physician and blood bank, maintain IV line, monitor vital signs; physician may order acetaminophen

Premedicate with acetaminophen (or glucocorticoid for refractory cases); give leukocyte-reduced RBCs

Allergic

Allergen is a soluble substance in donor plasma

Localized or generalized urticaria, Hold transfusion, notify physician, monitor erythema and pruritus; if severe, may vital signs; physician may order have laryngeal or facial angioedema, antihistamines or restart of transfusion if and hypotension mild urticaria clears and no other symptoms in 30 minutes

Premedicate with H1 blocking antihistamine; add H2 blocker or glucocorticoid for refractory cases; consider washed RBCs and platelets for repeated or severe reactions

Anaphylactic or anaphylactoid

Recipient antibodies to a soluble substance in donor plasma; infusion of plasma with IgA into IgA-deficient recipient with anti-IgA

Uticaria, flushing, angioedema, stridor, wheezing, tachycardia, hypotension, shock, abdominal pain, diarrhea, pelvic pain

Stop transfusion; maintain IV line; notify physician and blood bank, monitor vital signs; physician may order antihistamines, epinephrine, oxygen, IV crystalloid, or glucocorticoids

Premedicate with antihistamines and glucocorticoid; transfuse washed RBCs and platelets for recurrent reactions; use IgAdeficient donors or washed RBCs and platelets for sensitized patients with IgA deficiency

Transfusion-associated circulatory overload (TACO)

Blood volume too large or infusion too fast for compromised cardiovascular system

Dyspnea, orthopnea, systolic hypertension, headache, peripheral edema, coughing, cyanosis

Slow or stop transfusion; keep IV line open; notify physician; monitor vital signs and input and output; physician may order diuretics and oxygen

Transfuse slowly; use split units; consider premedication with diuretics; carefully monitor aged, debilitated, or cardiac patients

Hypothermia

Core body temperature ⬍35ºC caused by rapid infusion of cold blood products, such as RBCs, FFP, cryoprecipitate

Decreased body temperature, chills, cardiac arrhythmia (ventricular fibrillation)

Slow or stop transfusion; use an approved blood warmer, blankets, and other patient warming techniques (warm lavages, lamps)

Transfuse slowly, use an approved blood warmer

Citrate toxicity

Excessive infusion of citrate during apheresis procedure or massive or rapid transfusion; patients with liver failure are at increased risk

Perioral or peripheral paresthesia, Slow or stop transfusion; slow or stop apheresis tingling, buzzing, teeth chattering, procedure; give IV calcium chloride or bed or chair moving, cramps, nausea, gluconate (for PBSC apheresis: 0.5 mg vomiting, arrhythmia, bradycardia, Ca2⫹/1.0 mL ACD-A), or check ionized hypotension, prolongation of QT Ca2⫹ and dose per results; monitor relief of interval, tetany symptoms; consider hypomagnesemia

More likely in lightweight patients (⬍70 kg) and patients with liver dysfunction, renal failure, or less skeletal muscle; observe patients closely for any symptoms, give IV calcium (for PBSC apheresis: 0.5 mg Ca2⫹/1.0 mL ACD-A). (Continued)

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Type

Section IV: Part III

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Table 53-2. (Continued) Type Electrolyte disorder

Cause

Signs and Symptoms

Treatment

Prevention

Hyperkalemia: transfusion of older blood components or massive transfusion of RBCs

Hyperkalemia: cardiac arrhythmia, Hyperkalemia: give calcium to protect ECG changes—peaked T waves, against cardiac effect, alkalinize blood, prolongation of PR interval (if severe, D50 plus insulin, sodium polystrene flat or lost P wave), widened QRS, sulfonate; dialysis ventricular arrhythmia

Hyperkalemia: give fresh products (⬍7 days old), or washed products

Hypokalemia: massive or rapid transfusion of citrate and metabolic alkalosis

Hypokalemia: cardiac arrhythmia, ECG changes—ST depression, U waves

Hypokalemia: give potassium

Hypokalemia: give Adsol-preserved RBCs (not Nutricel).

Hypotensive

Bradykinin generation with use of negatively Hypotension; sometimes also flushing, charged bedside leukocyte reduction filters respiratory distress, nausea, in patients taking angiotension-converting abdominal pain, and loss of enzyme (ACE) inhibitors; apheresis consciousness procedures using albumin, especially in patient’s taking ACE inhibitors; see also citrate toxicity

Stop transfusion, notify physician and blood bank; support blood pressure.

Avoid use of bedside leukocyte reduction filters in patients taking ACE inhibitors; use prestorage leukocyte-reduced components or discontinue ACE inhibitor before transfusion or apheresis procedure; if during apheresis, correct electrolyte disorder such as hypocalcemia

DMSO toxicity

Cryopreservative for bone marrow, PBSCs, Flushing, nausea, vomiting, abdominal donor lymphocyte infusions, or any frozen cramping, throat tightness and cellular component; toxicity with DMSO cough, hypotension, hypertension, ⬎1.0 g/kg/day arrhythmia, fever, chills, headache, hemoglobinuria, hyperosmolality, increased liver enzymes

Antihistamines; antiemetics; slow or stop the infusion; supportive care; wait between infusions for symptoms to clear

Antihistamines; washed or plasma or volume depleted cellular infusions; antiemetics

IV ⫽ intravenous; RBCs ⫽ Red Blood Cells; FFP ⫽ Fresh Frozen Plasma; PBSC ⫽ peripheral blood stem cell; ACD-A ⫽ acid-citrate-dextrose-adenine; D50 ⫽ dextrose 50% in water; ECG ⫽ electrocardiogram; DMSO ⫽ dimethyl sulfoxide.

Chapter 53: Febrile, Allergic, and Nonimmune Transfusion Reactions

have an immune basis and represent inflammatory or allergic responses to infused cells (eg, many FNHTRs) or plasma (eg, some FNHTRs, allergic, and anaphylactic reactions). Although urgent transfusion can be lifesaving, it is important to recognize that a large volume of blood components given too quickly can itself have adverse chemical or physical effects, such as hypothermia, hyperkalemia, hypokalemia, hypocalcemia, and circulatory overload. Some reactions also are caused by unintended consequences of blood storage conditions or processing, such as generation of bradykinin by the contact of blood with some filter surfaces, leaching of toxic chemicals from filters or containers, and use of the chemical DMSO during hematopoietic progenitor cell preservation. It is important that these reactions and toxicities be recognized rapidly by the patient care team and blood bank personnel so that appropriate treatment and preventive measures can be instituted quickly. Care providers who administer transfusions must recognize that some symptoms of transfusion reactions, such as fever, are nonspecific and may be early manifestations of potentially life-threatening reactions, such as hemolysis or sepsis (Table 53-3). For that reason, the guiding rule regarding most transfusion reactions is to err on the side of conservatism and stop the transfusion immediately. Transfusion of a blood component that causes a reaction before complete infusion should not be restarted, with the possible exception of mild urticarial reactions. Several strategies are available to prevent repeated reactions among patients who are reaction-prone. These include leukocyte reduction for the prevention of FNHTR, cell washing for the prevention of allergic and anaphylactic reactions and possibly some FNHTRs, and various premedication regimens. Table 53-3. Overlapping Signs and Symptoms of Transfusion Reactions Sign or Symptom

Possible Reaction

Most Likely > Less Likely Blood Component*

Fever, chills

Febrile nonhemolytic Septic Acute hemolytic TRALI

Platelets (especially septic) ⬎RBCs ⬎Plasma

Urticaria, pruritus

Allergic Anaphylactic

Plasma ⬎Platelets ⬎RBCs

Dyspnea

TACO TRALI Anaphylactic

Any

Hypotension

Septic Hypotensive Acute hemolytic Anaphylactic

Platelets ⬎plasma ⬎RBCs

*Granulocytes would very likely cause febrile reactions and dyspnea. However, granulocytes are not listed in the table because they are infrequently transfused compared to other blood components. RBCs ⫽ Red Blood Cells; TRALI ⫽ transfusion-related acute lung injury; TACO ⫽ transfusion-associated circulatory overload.

Disclaimer The author has disclosed no conflicts of interest.

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62. Shimode N, Yasuoka H, Kinoshita M, et al. Severe anaphylaxis after albumin infusion in a patient with ahaptoglobinemia. Anesthesiology 2006;105:425-6. 63. Gilstad CW. Anaphylactic transfusion reactions. Curr Opin Hematol 2003;10:419-23. 64. Lieberman P, Kemp SF, Oppenheimer J, et al. The diagnosis and management of anaphylaxis: An updated practice parameter. J Allergy Clin Immunol 2005;115(Suppl):S483-523. 65. Holcomb JB. Damage control resuscitation. J Trauma 2007;62 (Suppl):S36-7. 66. Malone DL, Hess JR, Fingerhut A. Massive transfusion practices around the globe and a suggestion for a common massive transfusion protocol. J Trauma 2006;60(Suppl):S91-6. 67. Johansson PI, Stensballe J, Rosenberg I, et al. Proactive administration of platelets and plasma for patients with a ruptured abdominal aortic aneurysm: Evaluating a change in transfusion practice. Transfusion 2007;47:593-8. 68. Perkins RM, Aboudara MC, Abbott KC, Holcomb JB. Resuscitative hyperkalemia in noncrush trauma: A prospective, observational study. Clin J Am Soc Nephrol 2007;2:313-19. 69. Spinella PC, Perkins JG, Grathwohl KW, et al. Risks associated with fresh whole blood and red blood cell transfusions in a combat support hospital. Crit Care Med 2007;35:2576-81. 70. Dzik WH, Kirkley SA. Citrate toxicity during massive blood transfusion. Transfus Med Rev 1988;2:76-94. 71. Bolan CD, Wesley RA, Yau YY, et al. Randomized placebo-controlled study of oral calcium carbonate administration in plateletpheresis: I. Associations with donor symptoms. Transfusion 2003;43:1403-13. 72. Bolan CD, Cecco SA, Wesley RA, et al. Controlled study of citrate effects and response to i.v. calcium administration during allogeneic peripheral blood progenitor cell donation. Transfusion 2002;42:935-46. 73.Bolan CD, Cecco SA, Yau YY, et al. Randomized placebo-controlled study of oral calcium carbonate supplementation in plateletpheresis: II. Metabolic effects. Transfusion 2003;43:1414-22. 74. Diaz J, Acosta F, Parrilla P, et al. Citrate intoxication and blood concentration of ionized calcium in liver transplantation. Transplant Proc 1994;26:3669-70. 75. Jawan B, de Villa V, Luk HN, et al. Ionized calcium changes during living-donor liver transplantation in patients with and without administration of blood-bank products. Transpl Int 2003;16:510-14. 76. Haddad S, Leitman SF, Wesley RA, et al. Placebo-controlled study of intravenous magnesium supplementation during large-volume leukapheresis in healthy allogeneic donors. Transfusion 2005;45:934-44. 77. Hester JP, Ayyar R. Anticoagulation and electrolytes. J Clin Apher 1984;2:41-51. 78. Korbling M, Huh YO, Durett A, et al. Allogeneic blood stem cell transplantation: Peripheralization and yield of donor-derived primitive hematopoietic progenitor cells (CD34⫹ Thy-1dim) and lymphoid subsets, and possible predictors of engraftment and graftversus-host disease. Blood 1995;86:2842-8. 79. Ronquillo J, Yau Y, Stevens W, et al. Acute and sub-acute citrate mediated effects and responses to IV calcium in healthy apheresis donors (abstract). Transfusion 2007;47(Suppl):14A. 80. Latham JT, Jr., Bove JR, Weirich FL. Chemical and hematologic changes in stored CPDA-1 blood. Transfusion 1982;22:158-9. 81. Carvalho B, Quiney NF. ‘Near-miss’ hyperkalaemic cardiac arrest associated with rapid blood transfusion. Anaesthesia 1999;54:1094-6. 82. Woodforth IJ. Resuscitation from transfusion-associated hyperkalaemic ventricular fibrillation. Anaesth Intens Care 2007;35:110-13.

83. Bansal I, Calhoun BW, Joseph C, et al. A comparative study of reducing the extracellular potassium concentration in red blood cells by washing and by reduction of additive solution. Transfusion 2007;47:248-50. 84. Swindell CG, Barker TA, McGuirk SP, et al. Washing of irradiated red blood cells prevents hyperkalaemia during cardiopulmonary bypass in neonates and infants undergoing surgery for complex congenital heart disease. Eur J Cardiothorac Surg 2007;31:659-64. 85. Liu EA, Mannino FL, Lane TA. Prospective, randomized trial of the safety and efficacy of a limited donor exposure transfusion program for premature neonates. J Pediatr 1994;125:92-6. 86. Buchholz DH, Borgia JF, Ward M, et al. Comparison of Adsol and CPDA-1 blood preservatives during simulated massive resuscitation after hemorrhage in swine. Transfusion 1999;39:998-1004. 87. Boyan CP, Howland WS. Blood temperature: A critical factor in massive transfusion. Anesthesiology 1961;22:559-63. 88. Hrovat TM, Passwater M, Palmer RN for the Scientific Section Coordinating Committee. Guidelines for the use of blood warming devices. Bethesda, MD: AABB, 2002. 89. Dubick MA, Brooks DE, Macaitis JM, et al. Evaluation of commercially available fluid-warming devices for use in forward surgical and combat areas. Mil Med 2005;170:76-82. 90. Cohn SM, Stack GE. In vitro comparison of heated saline-blood admixture with a heat exchanger for rapid warming of red blood cells. J Trauma 1993;35:688-90. 91. Snyder EL, Bookbinder M. Role of microaggregate blood filtration in clinical medicine. Transfusion 1983;23:460-70. 92. Rentas FJ, Macdonald VW, Rothwell SW, et al. White particulate matter found in blood collection bags consist of platelets and leukocytes. Transfusion 2004;44:959-66. 93. Collins JA, James PM, Bredenberg CE, et al. The relationship between transfusion and hypoxemia in combat casualties. Ann Surg 1978;188:513-20. 94. Westphal-Varghese B, Erren M, Westphal M, et al. Processing of stored packed red blood cells using autotransfusion devices decreases potassium and microaggregates: A prospective, randomized, single-blinded in vitro study. Transfus Med 2007;17:89-95. 95. Fiebig EW, Wu AH, Krombach J, et al. Transfusion-related acute lung injury and transfusion-associated circulatory overload: Mutually exclusive or coexisting entities? Transfusion 2007;47:171-2. 96. Zhou L, Giacherio D, Cooling L, Davenport RD. Use of B-natriuretic peptide as a diagnostic marker in the differential diagnosis of transfusion-associated circulatory overload. Transfusion 2005;45:1056-63. 97. Skeate RC, Eastlund T. Distinguishing between transfusion related acute lung injury and transfusion associated circulatory overload. Curr Opin Hematol 2007;14:682-7. 98. Rana R, Fernandez-Perez ER, Khan SA, et al. Transfusion-related acute lung injury and pulmonary edema in critically ill patients: A retrospective study. Transfusion 2006;46:1478-83. 99. Marriott HLKA. Volume and rate in blood transfusion for the relief of anaemia. Br Med J 1940;1:1043-6. 100. Sasidharan P, Heimler R. Alterations in pulmonary mechanics after transfusion in anemic preterm infants. Crit Care Med 1990;18:1360-2. 101. Nelle M, Hoecker C, Linderkamp O. Effects of red cell transfusion on pulmonary blood flow and right ventricular systolic time intervals in neonates. Eur J Pediatr 1997;156:553-6.

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102. Cyr M, Eastlund T, Blais C Jr., et al. Bradykinin metabolism and hypotensive transfusion reactions. Transfusion 2001;41:136-50. 103. Michon B, Moghrabi A, Winikoff R, et al. Complications of apheresis in children. Transfusion 2007;47:1837-42. 104. Weinstein R. Prevention of citrate reactions during therapeutic plasma exchange by constant infusion of calcium gluconate with the return fluid. J Clin Apher 1996;11:204-10. 105. Owen HG, Brecher ME. Atypical reactions associated with use of angiotensin-converting enzyme inhibitors and apheresis. Transfusion 1994;34:891-4. 106. Adverse ocular reactions following transfusions—United States, 1997-1998. MMWR Morb Mortal Wkly Rep 1998;47:49-50. 107. Alonso-Echanove J, Sippy BD, Chin AE, et al. Nationwide outbreak of red eye syndrome associated with transfusion of leukocyte-reduced red blood cell units. Infect Control Hosp Epidemiol 2006;27:1146-52. 108. Rock G, Secours VE, Franklin CA, et al. The accumulation of mono-2-ethylhexylphthalate (MEHP) during storage of whole blood and plasma. Transfusion 1978;18:553-8. 109. Kluwe WM, Haseman JK, Huff JE. The carcinogenicity of di(2ethylhexyl) phthalate (DEHP) in perspective. J Toxicol Environ Health 1983;12:159-69. 110. Salkie ML, Hannon JL. Anti-plasticizer specific IgE is present in the serum of transfused patients. Clin Invest Med 1995;18:419-23. 111. Rock G, Tocchi M, Ganz PR, Tackaberry ES. Incorporation of plasticizer into red cells during storage. Transfusion 1984;24:493-8. 112. Labow RS, Tocchi M, Rock G. Platelet storage. Effects of leachable materials on morphology and function. Transfusion 1986;26:351-7. 113. Jaeger RJ, Rubin RJ. Plasticizers from plastic devices extraction, metabolism, and accumulation by biological systems. Science 1970;170:460-2. 114. Snyder EL, Hedberg SL, Napychank PA, et al. Stability of red cell antigens and plasma coagulation factors stored in a non-diethylhexyl phthalate-plasticized container. Transfusion 1993;33:515-19. 115. Latini G, Del Vecchio A, Massaro M, et al. In utero exposure to phthalates and fetal development. Curr Med Chem 2006;13:2527-34. 116. Burkhart HM, Joyner N, Niles S, et al. Presence of plasticizer di-2(ethylhexyl)phthalate in primed extracorporeal circulation circuits. ASAIO J 2007;53:365-7.

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117. Ito Y, Yamanoshita O, Asaeda N, et al. Di(2-ethylhexyl)phthalate induces hepatic tumorigenesis through a peroxisome proliferator-activated receptor alpha-independent pathway. J Occup Health 2007;49:172-82. 118. Brobyn RD. The human toxicology of dimethyl sulfoxide 45. Ann N Y Acad Sci 1975;243:497-506. 119. Jacob SW, Wood DC. Dimethyl sulfoxide (DMSO). Toxicology, pharmacology, and clinical experience. Am J Surg 1967;114:414-26. 120. Davis JM. Clinical toxicity of cryopreserved bone marrow graft infusion. Blood 1990;75:781-6. 121. Kessinger A, Schmit-Pokorny K, Smith D, Armitage J. Cryopreservation and infusion of autologous peripheral blood stem cells. Bone Marrow Transplant 1990;5(Suppl 1):25-7. 122. Zambelli A. Clinical toxicity of cryopreserved circulating progenitor cells infusion 1. Anticancer Res 1998;18:4705-8. 123. Lopez-Jimenez J. Cardiovascular toxicities related to the infusion of cryopreserved grafts: Results of a controlled study. Bone Marrow Transplant 1994;13:789-93. 124. Keung YK. Cardiac arrhythmia after infusion of cryopreserved stem cells. Bone Marrow Transplant 1994;14:363-7. 125. Rowley SD. Effect of DMSO exposure without cryopreservation on hematopoietic progenitor cells. Bone Marrow Transplant 1993;11:389-93. 126. Klein HG, Strauss RG, Schiffer CA. Granulocyte transfusion therapy. Semin Hematol 1996;33:359-68. 127. Price TH, Bowden RA, Boeckh M, et al. Phase I/II trial of neutrophil transfusions from donors stimulated with G-CSF and dexamethasone for treatment of patients with infections in hematopoietic stem cell transplantation. Blood 2000;95:3302-9. 128. Meyer-Koenig U, Hufert FT, Duffner U, et al. G-CSF-mobilised granulocyte transfusion to an ALL patient complicated by cytomegalovirus transmission. Bone Marrow Transplant 2004;34:1095-6. 129. Domen RE. Adverse reactions associated with autologous blood transfusion: Evaluation and incidence at a large academic hospital. Transfusion 1998;38:296-300. 130. Benavides S, Nicol K, Koranyi K, Nahata MC. Yersinia septic shock following an autologous transfusion in a pediatric patient. Transfus Apher Sci 2003;28:19-23.

54

Transfusion-Associated Graft-vs-Host Disease John E. Levine1 & James L. M. Ferrara2 1

Professor of Pediatrics and Internal Medicine, and Clinical Director, Pediatric Blood and Marrow Stem Cell Transplantation Program, University of Michigan, Ann Arbor, Michigan, USA 2 Professor of Pediatrics and Internal Medicine, and Director, Combined Blood and Marrow Transplantation Program, University of Michigan, Ann Arbor, Michigan, USA

Transfusion-associated graft-vs-host disease (TA-GVHD) is a phenomenon that can occur after blood transfusion in which donor T cells, responding to proteins on host cells, proliferate and target host organs, primarily the skin, liver, intestinal tract, and marrow. Although this complication is uncommon, considerable effort has been devoted to GVHD prevention, because of the lack of effective treatment and very high mortality rate. Immunocompromised patients are at greatest risk for TA-GVHD; therefore, prevention strategies initially focused on this population. More recently it has become recognized that TA-GVHD can develop in the apparently immunocompetent transfusion recipient. Patients most likely to develop TA-GVHD include very low birthweight premature infants, critically ill patients receiving extracorporeal membrane oxygenation, patients who require cardiopulmonary bypass, and recipients of blood transfusions from family members. This chapter outlines the pathophysiology and clinical manifestations of TA-GVHD, defines the risk factors, and identifies measures to prevent TA-GVHD.

Pathophysiology of GVHD In 1966, Billingham postulated that three requirements existed for the development of GVHD: the graft must contain immunologically competent cells, there must be antigenic differences between the donor and the recipient, and the recipient must be unable to mount an effective immunologic response to eradicate the transplanted immunologically competent cells.1 T cells are the immunologically competent cells that induce GVHD and can be transferred from one person to another in a variety of clinical settings, including blood component transfusions and organ and marrow transplants. Under most circumstances, the recipient immune system is able to eliminate the transferred T cells. However, when the recipient is immunosuppressed (either from Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

inherited or acquired immunodeficiency or induced immunosuppression, as is the case in transplant recipients) GVHD can develop. Immunocompetent individuals can develop TA-GVHD, although this is a very rare event. Most of our understanding of the pathophysiology of GVHD comes from the hematopoietic stem cell transplant (HSCT) setting. In HSCT, presentation of host antigen to donor cells often occurs in the context of host tissue damage and inflammation, from a combination of factors including the underlying disease, infection, and the HSCT conditioning regimen, which often includes high-dose chemotherapy and radiation therapy. Donor cells are transplanted into an environment of ubiquitous foreign antigens, activated cytokines, enhanced expression of adhesion molecules, and cell surface recognition molecules. Donor cells that participate in the resulting immune process include T cells, natural killer (NK) cells, and monocytes. A modern paradigm for GVHD development considers three consecutive phases: 1) the presentation of host protein to donor T cells by antigen-presenting cells (APCs); 2) donor T-cell activation, proliferation, and migration; and 3) host target tissue damage (Fig 54-1). Host APCs are both necessary and sufficient to stimulate donor T cells to proliferate in response to host antigen.2-5 T-cell receptors (TCRs) recognize fragments of proteins known as minor histocompatibility antigens bound to Class I and II major histocompatibility complex (MHC) molecules on the surface of APCs. The Class I MHC molecules (HLA-A, -B, -C) stimulate CD8⫹ T cells, while the Class II MHC molecules (HLA-DR, -DP, -DQ) present to CD4⫹ T cells.6,7 The additional presence of “danger signals”—such as lipopolysaccharide (LPS) or inflammatory cytokines—help APCs activate T cells8,9 and the presence/absence of “danger signals” may make the difference between an immune response and tolerance.10 Activated T cells can have proinflammatory (Th1) or antiinflammatory (Th2) phenotypes. Th1 cells secrete interleukin (IL)-2, interferon (IFN)-γ, and tumor necrosis factor alpha (TNF-α) while Th2 cells secrete IL-4, IL-5, IL-10, and IL-13.11 Th1 cells are important in the pathogenesis of acute GVHD and targeting these cells reduced acute GVHD in an experimental

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GVHD pathology

(I) Recipient conditioning tissue damage

Host tissues Small intestine

IL-1 IL-6 LPS

TNF-α

Host APC

Donor T-cell

TNF-α IL-1 IL-12

Target cell apoptosis CTL

(II) Donor T-cell activation

IFN-γ IL-2

NK

model.12 In clinical practice, decreasing IL-2 production by donor T cells is a primary target of current GVHD prophylaxis strategies by using the calcineurin inhibitors cyclosporine or tacrolimus. In contrast, the Th2 cytokine IL-10 plays a key role in suppression of immune responses and may have a role in regulating acute GVHD severity.13,14 Regulatory T cells, a third subset of T cells, also influence the development of GVHD. In animal models, adding regulatory T cells to the donor graft suppresses the proliferation of conventional T cells, and prevents GVHD.15 Murine regulatory T cells, which are identifiable by Foxp3 expression, normally constitute 5% of the CD4⫹ T-cell population. These cells secrete IL-10 and transforming growth factor beta (TGF-β), which inhibit the immune response. The regulatory T cells also inhibit APCs by a contact-dependent pathway.15 Migration of T cells from lymphoid tissues to target organs is controlled by chemokines. In experimental GVHD, proinflammatory chemokines, such as macrophage inflammatory protein-1 alpha (MIP-1α), CCL2-5, CXCL2, CXCL9-11, CCL27 are overexpressed. The chemokines may have organ-specific roles. For example, CXCR3⫹ cells cause acute GVHD in the liver and intestine.16 In animal models, migration of CCR5⫹ regulatory T cells to lymphoid tissues and target organs is necessary for GVHD control.17 Experimental and clinical data indicate that target organ injury results from both soluble inflammatory mediators and cell-mediated cytolysis. The principal effector mechanism by which cytotoxic T lymphocytes (CTLs) and NK cells lyse target cells are the Fas/Fas

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(III) Effector

Figure 54-1. The pathophysiology of acute GVHD. In Step I, the conditioning regimen of chemotherapy, radiotherapy, or both leads to host tissue damage, especially in the intestines. This allows translocation of lipopolysaccharide (LPS) from the intestine into the circulation, stimulating the secretion of inflammatory cytokines, including interleukin(IL)-1, IL-6, and tumor necrosis factor alpha (TNF-α). In Step II, cytokinestimulated host antigen-presenting cells (APCs) present host antigen to donor T cells, resulting in activation of donor T cells with a predominance of the Th1 phenotype that secrete interferon-γ (IFN- γ) and IL-2. In Step III, effector cells, cytotoxic T cells, and natural killer cells, induce target cell apoptosis both by direct cell-mediated lysis and the secretion of inflammatory cytokines that independently induce target cell apoptosis.

ligand (FasL) and perforin/granzyme pathways.18,19 Fas is an apoptosis-signaling receptor molecule on the surface of a number of different cells. When FasL binds to Fas, a death-inducing signaling complex is formed, which results in the subsequent activation of caspases.20 CTLs and NK cells store perforin with granzyme in cytotoxic granules. Perforin inserts itself into the target cell membrane, forming pores that allow granzymes to enter the cells and induce apoptosis through various downstream effector pathways.21 Inflammatory cytokines synergize with CTLs, resulting in amplification of local tissue injury and development of target organ dysfunction. A key inflammatory cytokine, especially for intestinal GVHD, is TNF-α, which plays a major role in the amplification and propagation of the “cytokine storm” characteristic of acute GVHD developing early after HSCT.22 TNF-α, which is produced by both donor and host cells, promotes GVHD and target organ damage by three different ways: first, it activates dendritic cells and enhances alloantigen presentation; second, it recruits effector cells to target organs via the induction of inflammatory chemokines; and third, it directly causes tissue necrosis, as its name suggests.23-25

Features of TA-GVHD Incidence of TA-GVHD The incidence of TA-GVHD is unknown because the diagnosis can be missed; other conditions, such as infection or drug

Chapter 54: Transfusion-Associated Graft-vs-Host Disease

reactions, may have similar clinical features. However, changes in practice appear to have lowered the already low incidence even further. The use of cellular components that have been gamma irradiated to 25 Gy appears to offer essentially complete protection to patients known to be at high risk for TA-GVHD.26 In the 10 year period from 1996 to 2005, 13 cases of TA-GVHD were reported via a national identification program. The implementation of universal leukocyte reduction in the United Kingdom (UK) in 1999 appears to have resulted in a statistically significant further reduction of TA-GVHD; of the 13 cases, 11 occurred in patients who received non-leukocyte-reduced (LR) components and only two occurred following transfusion of LR components. No cases of TA-GVHD have been reported in the UK since 2001.27

Risk Factors for Developing TA-GVHD As noted above, the transfusion of viable T cells is necessary to develop TA-GVHD. GVHD can develop after transplantation of as few as 4 ⫻ 103 T cells/kg recipient weight,28 although other studies indicate that acute GVHD is not seen when fewer than 1 to 5 ⫻ 105 T cells/kg recipient weight are transplanted.29,30 The average unit of Red Blood Cells (RBCs) contains approximately 2 ⫻ 109 leukocytes, each whole-blood-derived platelet unit contains approximately 4 ⫻ 107 leukocytes, and apheresis platelets contain approximately 1 ⫻ 108 leukocytes.31 Leukocyte reduction reduces the number of passenger T cells by 2 to 3 logs, but has not been able to completely prevent TA-GVHD.32-34 A 2-log reduction of viable leukocytes can be achieved by washing red cells34 and a 2 to 3 log reduction is attained by freezing-deglycerolization.34,35 For obvious reasons, leukocyte reduction is not performed with therapeutic white cell transfusions, and these can lead to TA-GVHD.36,37 Irradiating these cells can prevent GVHD (as discussed below), but donor leukocyte transfusions given to induce a graft-vs-leukemia effect following post-HSCT relapse are not irradiated and GVHD often results.38 TA-GVHD has not been reported with the use of cell-free plasma products, including Fresh Frozen Plasma, because the freezing-thawing process destroys T cells.39 Unfrozen plasma has caused TA-GVHD,40 but is now rarely used. Because TA-GVHD is caused by the engraftment of viable T lymphocytes, the primary risk factor for development of TAGVHD is an inability to reject donor T lymphocytes. The mechanisms are the same as those discussed for HSCT GVHD. It is of interest that TA-GVHD has been reported in only one patient with human immunodeficiency virus (HIV)/AIDS.41 Despite their immunodeficiency, HIV/AIDS patients appear to be resistant to engraftment of donor T lymphocytes, for unclear reasons.42

Fetuses and Neonates Maternal lymphocytes regularly cross over into fetal circulation, but under most circumstances GVHD does not develop, indicating that the fetus has immunologic defense mechanisms against maternal lymphocyte engraftment.43 Nonstimulated bidirectional mixed lymphocyte cultures have shown that neonatal lymphocytes specifically dominate against maternal lymphocytes, which may partly account for the neonatal protection against

maternal lymphocytes.44 However, the fetal immune system does not completely protect against adult lymphocytes as demonstrated by the development of TA-GVHD following intrauterine transfusion (IUT). Liley performed the first successful intraperitoneal IUT to treat hemolytic disease in 1963.45 Not long afterwards, it became apparent that IUT could lead to TA-GVHD.46,47 As a result, transfusion with LR and irradiated blood was initiated to prevent TA-GVHD. However, up to one-third of recipients of nonirradiated, non-LR transfusions engrafted donor leukocytes, without any GVHD or other apparent disorder of the immune system, and these donor leukocytes were detectable up to 25 years following IUT.48 The mechanisms behind this selective tolerance for the donor cells are unclear, but may involve downregulation of molecules essential for immune recognition. It is also possible that tolerance between the fetus and the donor may develop because of the following:49 1. Lack of host immune surveillance because of immaturity. 2. Inadequate expression of target antigens in fetal tissue. 3. Presence of maternal lymphocytes that protect against GVHD. TA-GVHD in neonates transfused postnatally is very rare. Most cases of TA-GVHD in immunocompetent newborns occur following transfusions from haploidentical or homozygous donors. Therefore, it is not surprising that most reports come from Japan, where there is a high degree of HLA homozygosity. Another risk factor may be prematurity. In one Japanese study, 20 of 27 newborns who developed TA-GVHD were premature.50 Most of the newborns received a blood component from a relative. TA-GVHD developed a median of 28 days from transfusion with fever typically the presenting sign, followed by GVHD rash. The mortality rate was 100%. The implementation of routine irradiation of blood and blood components before transfusion in newborns; congenital immunodeficient recipients; elderly recipients; and cardiac, cancer, and severe trauma surgery patients, has reduced the number of TA-GVHD cases in Japan to just a few cases in recent years.51

TA-GVHD in Immunodeficient Patients TA-GVHD occurs in T-cell deficient patients, who lack the ability to reject the donor T cells, usually caused by a congenital immunodeficiency syndrome, treatment for malignancy, or solid-organ transplantation (Table 54-1).

Congenital Immunodeficiency Syndromes TA-GVHD has been reported in a wide array of congenital immunodeficiency syndromes, including severe combined immunodeficiency (SCID) syndrome, Wiskott-Aldrich syndrome, purine nucleoside phosphorylase deficiency, and DiGeorge syndrome.52-56 As expected, a common feature to these disease entities is abnormal or absent T-cell function. Malignancies TA-GVHD has been reported to occur following nonirradiated transfusion in patients undergoing treatment for cancer.37,57-69 It is not clear if the presence of malignancy alone increases the

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Table 54-1. Patient Groups at Risk for TA-GVHD Neonates ● Intrauterine transfusion (IUT) ● Postnatal transfusion in recipients of IUT ● Very low-birthweight premature neonates ● Neonatal alloimmune thrombocytopenia Congenital Immunodeficiency ● Severe combined immunodeficiency ● Wiskott-Aldrich syndrome ● DiGeorge syndrome ● Other T-cell defects Malignancies and Immunosuppression Caused by Treatment ● Leukemia and lymphoma ● Neuroblastoma ● Rhabdomyosarcoma ● Other solid tumors Purine Analog Therapy ● Fludarabine Hematopoietic Cell Transplantation ● Allogeneic ● Autologous ● Syngeneic Solid-Organ Transplant ● Lung ● Liver ● Heart ● Kidney ● Pancreas Surgery ● Cardiac surgery ● Extracorporeal membrane oxygenation

risk of TA-GVHD, or if the problem is caused by the immunosuppressive effects of treatment. Most cases of TA-GVHD have been reported for patients with hematologic malignancies such as leukemia or lymphoma but there have been rare cases of TA-GVHD in patients with solid tumors such as neuroblastoma and rhabdomyosarcoma. This observation suggests that it is the immunosuppressive effect of the cancer treatment that increases the risk of TA-GVHD because antileukemia and antilymphoma treatment tends to be more immunosuppressive than treatment for solid tumors. However, there has been no systematic attempt to correlate the extent or duration of therapy to the development of TA-GVHD. One specific chemotherapeutic agent, the purine analog fludarabine, has been associated with TA-GVHD.69-71 Fludarabine very efficiently depletes T cells and is used to treat a variety of indolent lymphomas, especially chronic lymphocytic leukemia.72 Immunosuppressive therapy with fludarabine, even in the absence of malignancy, has also been associated with the development of TA-GVHD.73 Because fludarabine is often used as a single agent, the connection between its use and TA-GVHD may be easier to discern. It appears prudent to consider the use of any immunosuppressive chemotherapy agent to be a risk factor for TA-GVHD. In any event, the routine use of preventive

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measures against TA-GVHD in cancer patients regardless of diagnosis or treatment appears to have reduced the incidence of TA-GVHD. A review of the literature finds very few reports of TA-GVHD in cancer patients since 1997,70,74,75 although this may, in part, reflect underreporting. Autologous stem cell collections are also at risk for contamination with donor lymphocytes if nonirradiated blood components are given during the marrow or peripheral stem cell collection. This can lead to the subsequent development of TA-GVHD following reinfusion of the autologous stem cells and passenger donor lymphocytes.76 A syndrome consisting of features reminiscent of GVHD, occurring generally around the time of engraftment following autologous stem cell transplant, has been reported.77 This syndrome, termed engraftment syndrome, presents with noninfectious fever, skin rash, capillary leak, pulmonary infiltrates, and hypoxia. Biopsies of the skin rash are often equivocal with respect to establishing a diagnosis. TA-GVHD has not been ruled out in all of these cases, but it is generally felt that this syndrome represents changes associated with proinflammatory cytokine release around the time of engraftment. Likewise, the phenomena of autologous GVHD, which occurs after autologous stem cell transplant, can be induced by administration of cyclosporin A for a short period following transplantation, leading to the generation of autoreactive T cells.78 One way to distinguish between autologous GVHD and TA-GVHD is to demonstrate engraftment of transfused donor lymphocytes through techniques such as HLA Class I or II antigen differences or other genetic variations such as differences in short tandem repeat.79

Solid-Organ Transplantation GVHD in solid-organ transplantation is usually caused by donor lymphocytes transported with the donor organ but can also occur from transfusions given when the recipient is intensely immunosuppressed to prevent organ rejection. GVHD has been reported following cadaveric liver, heart, kidney, and pancreas-spleen transplants.80-86 In one study,82 12 cases of GVHD occurred in 1082 liver transplants performed at a single institution. GVHD symptoms (fever, skin rash, diarrhea, or pancytopenia) typically developed within 2 to 6 weeks after the transplant. Mortality was over 90%. Another risk factor for GVHD following organ transplant is HLA homology between the donor and the recipient.87,88 Matching at the HLA-B locus appears to particularly dispose patients to the development of GVHD following liver transplantation.89

TA-GVHD in Immunocompetent Patients HLA Homozygosity One risk factor for TA-GVHD in immunocompetent patients is transfusion from a donor who is HLA homologous to the recipient. This situation is most likely to occur when the donor is a family member,90-92 but there are also many reports from Japan, where a genetically homogenous population increases the risk of transfusion from a partially HLA-matched random

Chapter 54: Transfusion-Associated Graft-vs-Host Disease

donor.91,93 The transfusion recipient is less likely to reject donor lymphocytes when the donor is homozygous for an HLA haplotype for which the recipient is heterozygous. In this situation, the donor does not carry HLA antigens that trigger allogeneic recognition on the part of the recipient and thus are able to engraft. However, the recipient possesses HLA antigens that are foreign to the donor, and thus alloreaction culminating in TA-GVHD can occur.94,95 Most recent reports of TA-GVHD come from settings or locales where blood irradiation is not routine.95

Surgery TA-GVHD has been reported in immunocompetent individuals undergoing cardiac surgery.96-98 Cardiopulmonary bypass may increase the risk of TA-GVHD through the induction of transient immunodeficiency as evidenced by reduced mitogenic lymphocyte transformation and reduced production of the proimmune response cytokines, TNF-α, and IL-2.99-101 Likewise, TA-GVHD has been seen following extracorporeal membrane oxygenation.102 An additional risk factor contributing to the development of TA-GVHD is transfusion of cellular blood components from donors who share HLA antigens with the recipient, such as related donors and donor-recipient dyads from areas with low HLA diversity, such as Japan.97,99,103,104 Fresh Blood Another risk factor appears to be the use of freshly donated blood. In a series of 2686 consecutive adult patients who underwent cardiac surgery between 1980 and 1996, four cases of TA-GVHD developed, all following transfusion of cellular blood components donated within 48 hours.104 Transfusion of fresh blood components has been associated with TA-GVHD in other studies, as well.94,95,105 Fresh blood presumably predisposes patients to TA-GVHD because it contains maximal numbers of viable lymphocytes. During storage the number of viable lymphocytes declines106 as does the expression of cell-surface lymphocyte activation antigens.107 The decrease in cell surface lymphocyte antigen expression directly correlates with a reduced ability to activate T cells as measured by a mixed lymphocyte culture by Day 3 of storage. This finding helps explain the rarity of TA-GVHD following transfusion of blood that is more than 4 days old.103,107 In summary, in immunocompetent recipients, the factors most likely to lead to TA-GVHD are as follows: 1. HLA-homozygous donor to haploidentical recipient, particularly likely if the donor is a relative or transfusion takes place in a region with low HLA genetic diversity. 2. Cardiac bypass. 3. Use of blood less than 4 days old.

Clinical Presentation and Diagnosis of TA-GVHD Unlike GVHD occurring after HSCT, which can take weeks to months to develop (median onset: 23 days),108-111 GVHD following transfusion typically occurs sooner, usually within a

Table 54-2. Comparison of GVHD Following Hematopoietic Stem Cell Transplantation with Transfusion-Associated GVHD* Manifestation

HSCT GVHD

TA-GVHD

Median onset (range) Fever Skin rash Liver involvement Gastrointestinal involvement Pancytopenia Occurrence Response to therapy Mortality

23 days (12-100 days) Often ⫹ ⫹/⫺ ⫹/⫺ Rare 25%-50% 35%-50% 10%-25%

10 days (2-30 days) Usually ⫹ ⫹ ⫹/⫺ Almost always Rare Rare 90%-100%

*Modified from Brubaker.113

(A)

(B)

Figure 54-2. A patient with Stage III skin GVHD following hematopoietic cell transplantation. (A) Generalized skin erythema with the characteristic maculopapular rash. (B) Two days later, the rash is more intense with areas of confluence.

few days, but up to 4 weeks (median onset: 10 days) from transfusion95,112 (Table 54-2113). Fever is often the first symptom, but is soon followed by target organ involvement of skin, gastrointestinal tract, and/or the liver.114 The host hematopoietic system is also a target in GVHD resulting in pancytopenia, a major contributor to mortality, in the transfusion-associated version.115 Pancytopenia is less commonly seen in GVHD following allogeneic HSCT because the transplanted donor hematopoeitic stem cells are spared from donor attack. Consistent with the observation that the hematopoietic system is a specific target in TA-GVHD, newborns with TA-GVHD reveal striking thymic damage.50 There is no grading system for pancytopenia, but other target organs are staged using a scale developed for HSCTassociated GVHD (see Chapter 35). The skin is usually the first organ involved. The characteristic maculopapular rash is pruritic, often involves the palms of the hands and soles of the feet, and spreads throughout the body (Fig 54-2). In severe cases the skin may blister and ulcerate.111,114 Apoptosis at the base of epidermal rete pegs is a characteristic pathologic finding. Other features include dyskeratosis, exocytosis of lymphocytes, satellite lymphocytes adjacent to dyskeratotic epidermal keratinocytes, and a perivascular lymphocytic infiltration in the dermis.116,117

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Gastrointestinal tract GVHD usually presents as diarrhea but may also include vomiting, anorexia, and/or abdominal pain when severe.116 The diarrhea of GVHD is secretory and often voluminous (greater than 2 L/day). Bleeding occurs as a result of mucosal ulceration and can lead to hemorrhagic death.118 Endoscopic examination with biopsies can confirm gastrointestinal tract involvement. Histologic features include lymphocytic infiltration, patchy ulcerations, apoptosis of crypt epithelial cells, crypt abscesses, and loss as well as flattening of the surface epithelium.119,120 Liver involvement in GVHD is characterized by laboratory and clinical features of cholestasis, with elevated bilirubin levels in the blood and clinical jaundice.111 The histologic features of hepatic GVHD are bile duct damage, portal and lobular inflammation, endothelialitis, and canalicular cholestasis.121 However, the liver is rarely biopsied because thrombocytopenia greatly increases the risks of the procedure. Diagnosis of TA-GVHD is usually made on clinical grounds, but detection of donor lymphocytes in the recipient’s circulating blood confirms the diagnosis. Techniques to establish donor lymphocyte engraftment are based on genetic differences between the donor and the host and include chromosome differences by standard cytogenetics,122 fluorescent in situ hybridization of a biopsy,123 detection of small genetic differences in highly polymorphic regions,79 and differences in tissue typing.61,67,97 A source of recipient chromosomes or DNA is necessary and can be obtained by buccal scraping or skin biopsy.

Treatment of GVHD Transfusion-associated GVHD carries a very poor prognosis with mortality rates over 90% despite aggressive treatment that has included agents such as high-dose steroids, azathioprine, methotrexate, cyclosporine, intravenous immunoglobulin, and growth factors,39,74,105 The principles of treatment for TA-GVHD come primarily from the HSCT literature. Steroids, with their potent antilymphocyte and antiinflammatory activity, are the gold standard for treatment of GVHD. However, steroid therapy results in durable complete or partial remission in only around 50% of patients, primarily those with less severe GVHD.108,124 Antithymocyte globulin (ATG) has been used together with steroids to improve outcome. In a prospective randomized study of 100 patients, the combination of steroids and ATG was no more effective than steroids alone as front-line therapy for GVHD; however, there was a better-than-expected response rate to steroids alone.124 In a retrospective study, 1-year survival was better when ATG was added within 4 to 7 days of steroid therapy for those patients who showed early signs of steroid resistance.126 Other studies have not shown benefit when ATG is added to steroid therapy to treat GVHD.127,128 Because ATG increases the risk of infection in patients with GVHD, it is not used routinely.125,127,128 This increased risk of infection is of particular importance in the treatment of TA-GVHD, because death from overwhelming infection is common.94-96

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One treatment that may be less immunosuppressive is extracorporeal photopheresis (ECP). During ECP, the patient’s white cells are collected by apheresis, incubated with the DNA-intercalating agent, 8-methoxypsoralen, exposed to ultraviolet A (UVA) light, and returned to the patient. ECP is known to induce cellular apoptosis, which has strong antiinflammatory effects in a number of systems, including prevention of rejection of solidorgan grafts.129 A Phase II study of ECP in steroid-dependent or steroid-refractory GVHD showed complete and durable resolution of GVHD in a large majority of patients. High response rates in the skin (82%), liver (61%), and intestine (61%) translated into 50% long-term survival in this very high-risk group.130 Another possible strategy to treat GVHD is inhibition of the inflammatory cytokine TNF-α, a molecule that plays a critical role in the development of GVHD as discussed earlier. A Phase II trial of etanercept, a solubilized dimeric TNF-receptor-2, showed significant efficacy when added to systemic steroids as primary therapy for acute GVHD. Seventy percent of patients had complete resolution of all GVHD symptoms within 1 month, with 80% complete responses in the gastrointestinal tract and the skin. The authors also showed that plasma levels of TNF-receptor-1 were a significant biomarker for clinical GVHD.131 Given the very high mortality from TA-GVHD, only a few cases of successful treatment have been reported. An infant with X-linked SCID developed TA-GVHD following nonirradiated blood transfusion. The TA-GVHD was initially controlled with high-dose steroid therapy. A haploidentical transplant from his mother was initially rejected by the engrafted blood donor T lymphocytes, but on second transplant engraftment was achieved with eventual eradication of blood transfusion donor T cells. The blood donor, patient, and marrow donor all shared an HLA haplotype.54 In two cases, GVHD was successfully treated by infusion of patient lymphocytes.71,132 Anti-CD3 treatment, together with cyclosporine, showed effectiveness in one case.133 A spontaneous remission has also been reported.134

Prevention of TA-GVHD γ-Irradiation Although TA-GVHD is a preventable disease, there remains considerable controversy regarding the optimal prevention strategies, primarily because of the rarity of the diagnosis. Treating cellular blood components with 25 Gy γ-irradiation reliably inactivates donor lymphocytes through cross-linkage of DNA, and prevents TA-GVHD.39,135 Irradiation does not damage platelets or granulocytes, and therefore can be performed safely at any time following the collection.112 However, irradiation does shorten the shelf life of red cell components and it is recommended that irradiated red cells be used within 28 days from the time of irradiation.39 The effects of irradiation on red cells include increased plasma membrane permeability, with potassium leakage, hemolysis, and problems with adenosine triphosphate maintenance.136 Prestorage leukocyte reduction may lessen some of the effects of

Chapter 54: Transfusion-Associated Graft-vs-Host Disease

irradiation on red cell storage parameters.137 Irradiation incurs additional cost and is not available at all hospitals. Therefore, although universal irradiation of all cellular components would eliminate TA-GVHD, this strategy has not been adopted in all countries.138,139 In Japan, TA-GVHD has been a particularly significant issue because of the genetically homogenous population. In 2000, Japanese experts recommended blood component irradiation for the following identified risk situations: transfusions from blood relatives, granulocyte transfusions, and all transfusions to immunodeficient recipients including any cancer patients, newborns, elderly patients, and those undergoing cardiac surgery.51 Subsequently, universal blood component irradiation was adopted in Japan.139 In the United States and Europe, there is no well-established consensus on the indications for blood irradiation. Most transfusion services perform their own assessment of the risk and develop a list of indications for which blood irradiation is provided routinely. The indications most frequently included in such a list are: transfusions from blood relatives; intrauterine transfusions; granulocyte transfusions (except when given to patients with congenital neutrophil disorders); patients with congenital cellular immunodeficiency syndromes, Hodgkin disease, and other leukemias and lymphomas; patients taking fludarabine; and patients undergoing HSCT. Many authorities irradiate fresh blood (less than 24 hours old) when used for transfusion. Other indications are assessed differently at different institutions. Blood irradiation can be performed with commercial irradiators designed specifically for this purpose. The most appropriate dose for blood component irradiation is 25 Gy at midplane.135,140 Commercial irradiators incorporate one to four gamma ray sources, generally cesium-137, and deliver 300 to 1000 cGy per minute.141 Linear accelerators and cobalt-60 can also be used as a source of γ-irradiation.135,141 Alternatives to cesium-137 as a radiation source are preferred because of concerns about security risks, despite the increased costs of such alternatives. Radioactive cesium chloride used in blood irradiators and other medical equipment is widely used in significant quantities, is soluble and easily dispersible, and often lacks secure disposal options.142

Nonirradiation Prevention Strategies Leukocyte reduction has been shown to reduce the risk of TA-GVHD, especially in a genetically diverse population,27 but is not a substitute for irradiation in at-risk populations. Photochemical treatment using psoralens and long-wavelength ultraviolet irradiation inactivates T cells and has also been shown to inactivate blood-borne pathogens, such as cytomegalovirus.143,144

Summary Transfusion-associated GVHD is a rare and preventable disease that occurs when sufficient numbers of viable T cells are transfused into a susceptible host. Immunodeficient states, either inherited or

acquired, are well recognized as a risk factor, but TA-GVHD can develop in immunocompetent patients, especially if fresh blood components, with their high numbers of viable leukocytes, are transfused. Immunocompetent recipients are generally only at risk if they receive HLA haploidentical T cells, which is most likely to happen if the donor is a relative, or if the patient lives in a region with low HLA genetic diversity. TA-GVHD usually develops within days of the transfusion and follows a fulminant course with death from severe pancytopenia and infection. The disease is generally recognized by classical features of rash, diarrhea, and liver dysfunction. Irradiation of blood components is highly effective at preventing this disease, but is not universally employed. Careful attention must be paid to identifying at-risk patients, because, once established, the disease is almost always fatal.

Disclaimer The authors have disclosed no conflicts of interest.

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88. Kamei H, Oike F, Fujimoto Y, et al. Fatal graft-versus-host disease after living donor liver transplantation: Differential impact of donor-dominant one-way HLA matching. Liver Transpl 2006;12:140-5. 89. Key T, Taylor CJ, Bradley JA, Taylor AL. Recipients who receive a human leukocyte antigen-B compatible cadaveric liver allograft are at high risk of developing acute graft-versus-host disease. Transplantation 2004;78:1809-11. 90. Shivdasani RA, Anderson KC. HLA homozygosity and shared HLA haplotypes in the development of transfusion-associated graft-versus-host disease. Leuk Lymphoma 1994;15:227-34. 91. Petz LD, Calhoun L, Yam P, et al. Transfusion-associated graftversus-host disease in immunocompetent patients: Report of a fatal case associated with transfusion of blood from a seconddegree relative, and a survey of predisposing factors. Transfusion 1993;33:742-50. 92. McMilin KD, Johnson RL. HLA homozygosity and the risk of related-donor transfusion-associated graft-versus-host disease. Transfus Med Rev 1993;7:37-41. 93. Otsuka S, Kunieda K, Kitamura F, et al. The critical role of blood from HLA-homozygous donors in fatal transfusion-associated graft-versus-host disease in immunocompetent patients. Transfusion 1991;31:260-4. 94. Triulzi D, Duquesnoy R, Nichols L, et al. Fatal transfusion-associated graft-versus-host disease in an immunocompetent recipient of a volunteer unit of red cells. Transfusion 2006;46:885-8. 95. Agbaht K, Altintas ND, Topeli A, et al. Transfusion-associated graft-versus-host disease in immunocompetent patients: Case series and review of the literature. Transfusion 2007;47:1405-11. 96. Rososhansky S, Badonnel MC, Hiestand LL, et al. Transfusionassociated graft-versus-host disease in an immunocompetent patient following cardiac surgery. Vox Sang 1999;76:59-63. 97. Ahya R, Douglas JG, Watson HG. Transfusion associated graft versus host disease in an immunocompetent individual following coronary artery bypass grafting. Heart 1998;80:299-300. 98. Serefhanoglu K, Turan H, Saba T, et al. Transfusion-associated graft-versus-host disease in an immunocompetent individual following cardiac surgery. J Natl Med Assoc 2005;97:418-20. 99. Hisatomi K, Isomura T, Kawara T, et al. Changes in lymphocyte subsets, mitogen responsiveness, and interleukin-2 production after cardiac operations. J Thorac Cardiovasc Surg 1989;98:580-91. 100. Borgermann J, Friedrich I, Flohe S, et al. Tumor necrosis factoralpha production in whole blood after cardiopulmonary bypass: Downregulation caused by circulating cytokine-inhibitory activities. J Thorac Cardiovasc Surg 2002;124:608-17. 101. Hauser GJ, Chan MM, Casey WF, et al. Immune dysfunction in children after corrective surgery for congenital heart disease. Crit Care Med 1991;19:874-81. 102. Hatley RM, Reynolds M, Paller AS, Chou P. Graft-versus-host disease following ECMO. J Pediatr Surg 1991;26:317-19. 103. Ohto H, Anderson KC. Survey of transfusion-associated graft-versus-host disease in immunocompetent recipients. Transfus Med Rev 1996;10:31-43. 104. Yasuura K, Okamoto H, Matsuura A. Transfusion-associated graftversus-host disease with transfusion practice in cardiac surgery. J Cardiovasc Surg (Torino) 2000;41:377-80. 105. Aoun E, Shamseddine A, Chehal A, et al. Transfusion-associated GVHD: 10 years’ experience at the American University of BeirutMedical Center Transfusion 2003;43:1672-6.

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106. Qu L, Triulzi DJ, Rowe DT, et al. Stability of lymphocytes and Epstein-Barr virus during red blood cell storage. Vox Sang 2007;92:125-9. 107. Chang H, Voralia M, Bali M, et al. Irreversible loss of donor blood leucocyte activation may explain a paucity of transfusion-associated graft-versus-host disease from stored blood. Br J Haematol 2000;111:146-56. 108. Martin PJ, Schoch G, Fisher L, et al. A retrospective analysis of therapy for acute graft-versus-host disease: Initial treatment. Blood 1990;76:1464-72. 109. Sullivan KM, Agura E, Anasetti C, et al. Chronic graft-versus-host disease and other late complications of bone marrow transplantation. Semin Hematol 1991;28:250-9. 110. Sullivan KM, Mori M, Sanders J, et al. Late complications of allogeneic and autologous marrow transplantation. Bone Marrow Transplant 1992;10 (Suppl 1):127-34. 111. Deeg HJ, Antin JH. The clinical spectrum of acute graft-versushost disease. Semin Hematol 2006;43:24-31. 112. Perrotta PL, Snyder EL. Non-infectious complications of transfusion therapy. Blood Rev 2001;15:69-83. 113. Brubaker DB. Transfusion-associated graft-versus-host disease. Hum Pathol 1986;17:1085-8. 114. Vogelsang GB, Lee L, Bensen-Kennedy DM. Pathogenesis and treatment of graft-versus-host disease after bone marrow transplant. Annu Rev Med 2003;54:29-52. 115. Oto OA, Paydas S, Baslamisli F, et al. Transfusion-associated graftversus-host disease. Eur J Intern Med 2006;17:151-6. 116. Ferrara JL, Deeg HJ. Graft-versus-host disease. N Engl J Med 1991;324:667-74. 117. Goker H, Haznedaroglu IC, Chao NJ. Acute graft-vs-host disease: Pathobiology and management. Exp Hematol 2001;29:259-77. 118. Nevo S, Enger C, Swan V, et al. Acute bleeding after allogeneic bone marrow transplantation: Association with graft versus host disease and effect on survival. Transplantation 1999;67:681-9. 119. Snover DC, Weisdorf SA, Vercellotti GM, et al. A histopathologic study of gastric and small intestinal graft-versus-host disease following allogeneic bone marrow transplantation. Hum Pathol 1985;16:387-92. 120. Bombi JA, Nadal A, Carreras E, et al. Assessment of histopathologic changes in the colonic biopsy in acute graft-versus-host disease. Am J Clin Pathol 1995;103:690-5. 121. Quaglia A, Duarte R, Patch D, et al. Histopathology of graft versus host disease of the liver. Histopathology 2007;50:727-38. 122. Matsushita H, Shibata Y, Fuse K, et al. Sex chromatin analysis of lymphocytes invading host organs in transfusion-associated graftversus-host disease. Virchows Arch B Cell Pathol Incl Mol Pathol 1988;55:237-9. 123. Tanei R, Ohta Y, Ishihara S, et al. Transfusion-associated graftversus-host disease: An in situ hybridization analysis of the infiltrating donor-derived cells in the cutaneous lesion. Dermatology 1999;199:20-4. 124. MacMillan ML, Weisdorf DJ, Wagner JE, et al. Response of 443 patients to steroids as primary therapy for acute graft-versushost disease: Comparison of grading systems. Biol Blood Marrow Transplant 2002;8:387-94. 125. Cragg L, Blazar BR, Defor T, et al. A randomized trial comparing prednisone with antithymocyte globulin/prednisone as an initial systemic therapy for moderately severe acute graft-versus-host disease. Biol Blood Marrow Transplant 2000;6:441-7.

Chapter 54: Transfusion-Associated Graft-vs-Host Disease

126. MacMillan ML, Weisdorf DJ, Davies SM, et al. Early antithymocyte globulin therapy improves survival in patients with steroid-resistant acute graft-versus-host disease. Biol Blood Marrow Transplant 2002;8:40-6. 127. McCaul KG, Nevill TJ, Barnett MJ, et al. Treatment of steroidresistant acute graft-versus-host disease with rabbit antithymocyte globulin. J Hematother Stem Cell Res 2000;9:367-74. 128. Arai S, Margolis J, Zahurak M, et al. Poor outcome in steroidrefractory graft-versus-host disease with antithymocyte globulin treatment. Biol Blood Marrow Transplant 2002;8:155-60. 129. Barr ML, Meiser BM, Eisen HJ, et al. Photopheresis for the prevention of rejection in cardiac transplantation. Photopheresis Transplantation Study Group. N Engl J Med 1998;339:1744-51. 130. Greinix HT, Knobler RM, Worel N, et al. The effect of intensified extracorporeal photochemotherapy on long-term survival in patients with severe acute graft-versus-host disease. Haematologica 2006;91:405-8. 131. Levine JE, Paczesny S, Mineishi S, et al. Etanercept plus methylprednisolone as initial therapy for acute graft-versus-host disease. Blood 2008;111:2470-5. 132. Kuball J, Theobald M, Ferreira EA, et al. Control of organ transplant-associated graft-versus-host disease by activated host lymphocyte infusions. Transplantation 2004;78:1774-9. 133. Yasukawa M, Shinozaki F, Hato T, et al. Successful treatment of transfusion-associated graft-versus-host disease. Br J Haematol 1994;86:831-6. 134. Mori S, Matsushita H, Ozaki K, et al. Spontaneous resolution of transfusion-associated graft-versus-host disease. Transfusion 1995;35:431-5. 135. Pelszynski MM, Moroff G, Luban NL, et al. Effect of gamma irradiation of red blood cell units on T-cell inactivation as assessed by

136. 137.

138.

139.

140.

141.

142.

143.

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limiting dilution analysis: Implications for preventing transfusionassociated graft-versus-host disease. Blood. 1994;83:1683-9. Holme S. Current issues related to the quality of stored RBCs. Transfus Apher Sci 2005;33:55-61. Wagner SJ, Myrup AC. Prestorage leucoreduction improves several in vitro red cell storage parameters following gamma irradiation. Transfus Med 2006;16:261-5. Anderson K. Broadening the spectrum of patient groups at risk for transfusion-associated GVHD: Implications for universal irradiation of cellular blood components. Transfusion 2003;43:1652-4. Klein HG. Transfusion-associated graft-versus-host disease: Less fresh blood and more gray (Gy) for an aging population. Transfusion 2006;46:878-80. Moroff G, Luban NL. The irradiation of blood and blood components to prevent graft-versus-host disease: Technical issues and guidelines. Transfus Med Rev 1997;11:15-26. Goes EG, Borges JC, Covas DT, et al. Quality control of blood irradiation: Determination T cells radiosensitivity to cobalt-60 gamma rays. Transfusion 2006;46:34-40. Report finds alternatives for blood irradiators, other radiation sources to improve national security. AABB Weekly Report 2008;14(8):1. Luban NL. Prevention of transfusion-associated graft-versus-host disease by inactivation of T cells in platelet components. Semin Hematol 2001;38:34-45. Roback JD, Conlan M, Drew WL, et al. The role of photochemical treatment with amotosalen and UV-A light in the prevention of transfusion-transmitted cytomegalovirus infections. Transfus Med Rev 2006;20:45-56.

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55

Transfusional Iron Overload Sujit Sheth Associate Clinical Professor, Department of Pediatrics, Columbia University Medical Center, New York, New York, USA

Tissue iron overload inevitably results in patients who receive regular red cell transfusions for congenital or acquired anemias. Iron is deposited initially in the liver and monocyte-macrophage (reticuloendothelial) system, but as these tissues become saturated, iron begins to deposit in other organs such as the endocrine glands and the heart. The body lacks an effective means of eliminating this excess iron, and without therapy, cirrhosis, heart disease, diabetes, and other disorders develop; death is usually the result of cardiac failure. Transfusional iron overload, with its resulting morbidity and mortality, is an important health issue in patients of all ages. Children with thalassemia major, or other inherited disorders such as Diamond-Blackfan anemia (DBA), congenital dyserythropoietic anemia, and congenital aplastic anemia are dependent on regular red cell transfusions to maintain their well being. Children with sickle cell disease who are found to be at increased risk of stroke may be placed on a chronic transfusion program to reduce this risk substantially.1 Some adults with aplastic anemia or myelodysplasic syndrome (MDS) may also become dependent on regular red cell transfusions. In addition, with chemotherapeutic regimens becoming more intense and myelosuppressive, patients undergoing treatment for various forms of cancer, particularly leukemias and lymphomas, may also receive a large number of red cell transfusions. Some of these individuals may also undergo stem cell transplantation, during the course of which they may receive numerous such transfusions. With advances in the medical management of some of these conditions, including improved chelation regimens, patients also have a better survival probability, thus increasing the number of individuals in whom transfusional iron overload requires close medical attention. Until recently, the most long-term experience in the pathogenesis and management of transfusional iron overload came from studies in patients with thalassemia major. Although these individuals differ from other

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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transfusion-dependent patients in some important ways, experience with patients with thalassemia helps guide many of the recommendations made here.2

Pathophysiology Iron is an essential nutrient required by every cell. It is present in the human body in hemoglobin, myoglobin, and several mitochondrial respiratory enzymes. It serves as a carrier for oxygen and electrons and acts as a catalyst for a variety of oxygenation reactions. It is able to perform these functions in part because of its ability to reversibly and readily cycle between its ferrous and ferric forms. This very property also makes it potentially toxic and capable of producing free radicals, which can cause cellular damage (Fig 55-1). The normal body iron concentration is approximately 40 to 50 mg/kg body weight; women have lower amounts and men somewhat higher.4 Three-quarters of this iron, about 30 mg/kg, is contained in the circulating red cell compartment as hemoglobin. Approximately 5 to 6 mg/kg is present in functional form in a variety of heme compounds (myoglobin and cytochromes) and iron-dependent enzymes. The remainder (5 mg/kg in women, 10-12 mg/kg in men) is held in reserve in two primary

Exogenous or from electron transport and other oxidation reactions NAD NADH H2O2
Fe2


Fe3

(OH
)
OH

Protein bound or from labile iron pool

DNA damage Cell death, mutations

Figure 55-1. Iron-induced oxidative tissue damage via the Fenton Reaction.3

Chapter 55: Transfusional Iron Overload

storage forms (ferritin and hemosiderin) in the liver, marrow, spleen, and muscle—readily available when required for erythropoiesis. Iron balance is maintained by controlling iron absorption; iron stores and iron absorption are reciprocally related so that as stores increase, absorption declines. These processes are closely managed by the iron regulatory “hormone” hepcidin,5 which plays a critical role in iron metabolism. It regulates the absorption of iron from the gastrointestinal tract through its effect on levels of ferroportin, the protein responsible for transporting iron out of the enterocyte. Red cells are normally broken down by cells of the monocyte-macrophage system, and the iron contained in hemoglobin is usually returned via transferrin to the erythroid precursors in the marrow, for recycling into new red cells. Hepcidin controls the release of iron from macrophages, an effect also mediated by ferroportin. In the healthy host, when the body has adequate or increased amounts of iron, hepcidin is upregulated, iron absorption from the intestine is inhibited, and iron is sequestered in its storage sites, the macrophages and hepatocytes. The body lacks any effective mechanism for the excretion of excess iron. Iron exchange is limited so that the adult male absorbs and loses only about 0.01 mg/kg/day. Iron overload results from repeated blood transfusion, excessive iron absorption from the gastrointestinal tract, or of a combination of these processes.4 In patients with severe congenital anemias (such as the thalassemias, DBA, or congenital dyserythropoietic anemia), death from severe anemia in infancy is averted by a regular transfusion program which, if adequate, allows for normal growth and development during the first decade of life. Individuals with MDS or aplastic anemia may begin transfusions later in life. As in healthy individuals, macrophages process senescent red cells (transfused in these patients) and extract iron from heme. With no effective means for the excretion of this iron, there is gradual tissue accumulation of this iron, which eventually exceeds the body’s capacity for safe storage. As the amount of iron increases, there is virtually no change in the amount that is contained within the functional or transport (transferrinbound) pools, and almost all of the excess iron is stored. Initially, the iron is sequestered in the cells of the monocyte-macrophage system, but with continued accumulation, iron spills over and is deposited in other tissues, usually the liver (hepatocytes), heart, pancreas, and other endocrine organs. Transfusion-dependent individuals may be broadly categorized as those having ineffective erythropoiesis (such as individuals with thalassemia or some forms of sideroblastic anemia), those with effective erythropoiesis (such as individuals with sickle cell anemia or hereditary spherocytosis), and those with decreased or absent erythropoiesis (such as patients with aplastic anemia or DBA). The “effectiveness” of erythropoiesis, with the ability to incorporate iron into hemoglobin in mature red cells, is a critical determinant of the pathophysiology of iron loading in transfusion-dependent patients. Once transfused red cells are phagocytosed by macrophages, hepcidin levels determine the fate of iron released from heme. If hepcidin levels are high, more iron remains sequestered in these storage cells, and less is released for

potential deposit in other tissues. Iron stored in the hepatocyte is similarly retained. In contrast, if hepcidin levels are low, more iron is released from these cells and may be available for deposition in other parenchymal tissues. Thus, hepcidin helps determine the partition and internal redistribution of the excess iron between the monocyte-macrophage and parenchymal sites,6 another crucial factor in tissue toxicity. Ineffective erythropoiesis—and, to a lesser degree, anemia— result in a downregulation of hepcidin expression and enhanced absorption of iron from the intestine.7 Patients with thalassemia major or intermedia, paradoxically have relatively low levels of hepcidin even in the presence of iron overload, as a result of their marked ineffective erythropoiesis. In patients with sickle cell disease, who have intact erythropoiesis, hepcidin expression varies inversely with erythropoietic drive,8 with untransfused patients excreting less hepcidin in the urine. Similarly, patients with DBA or aplastic anemia, who have almost no erythropoiesis, likely have appropriately elevated hepcidin levels when iron loading occurs. Thus, patients with thalassemia major or intermedia not only have higher iron burdens because of increased intestinal absorption, but also have a greater risk of parenchymal tissue deposition than the latter groups. In fact, patients with thalassemia intermedia who have received very few or no red cell transfusions may have profound iron overload because of marked hepcidin inhibition7 as a result of their ineffective erythropoiesis. In patients with thalassemia major or intermedia, regular transfusion suppresses ineffective erythropoiesis to some extent, and hepcidin levels may rise somewhat. However, the levels remain inappropriately low in relation to the higher body iron burden, resulting in iron loading through gastrointestinal absorption. Recently, a growth factor (GDF15) has been identified that is secreted during erythroblast maturation, and suppresses hepcidin production. There is increased expression of this factor in thalassemia, likely because of the ineffective erythropoiesis.9 In contrast, levels of this factor are not elevated in patients with homozygous sickle cell anemia. Therefore, although the volume of red cells transfused is similar, variability in the hepcidin response to the underlying erythropoietic status produces differences in the partition and distribution of the excess iron. While hepcidin levels are critical in modifying the pathophysiology of iron loading in transfusion-dependent patients, several other variables are important in determining the morbidity related to the increased body iron burden. These include 1) the age at which transfusion therapy began, 2) the duration of transfusion therapy, and 3) the initiation and maintenance of effective chelation. Additional modifying factors include other genetic determinants, alcohol use, coexisting viral hepatitis, and other drugs and medications that the patient may be taking. The complex interplay between the underlying disorder and these factors in each individual plays a key role in the pathophysiology of iron toxicity. Even though the body has the ability to safely store increased amounts of iron, deposition in nonstorage tissues eventually occurs as the body iron burden rises. Without treatment,

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patients with thalassemia, who begin transfusions in infancy, develop growth retardation, diabetes, and other endocrine disturbances in the first decade of life, and death from iron loading of the myocardium occurs during the second decade. Patients with sickle cell disease, who receive regular transfusions to prevent the occurrence of strokes, may begin these transfusions later in childhood, and may achieve normal growth and sexual maturation before the threshold for tissue damage is reached. A comparative study of regularly transfused patients with thalassemia major and sickle cell disease with similar iron burdens showed that the latter group had fewer cardiac and endocrine complications.10-12 The patients with sickle cell disease began regular transfusions at a later age, and had been transfused for approximately half as long as the thalassemia patients, possibly contributing to the observed differences in clinical manifestations. These observations emphasize the relevance of the age at which transfusions are begun, particularly with regard to growth, development, and sexual maturation. As the duration of transfusion increases, severe iron loading may occur in all transfusiondependent patients who are not effectively chelated. Diabetes and cardiac disease may develop, the latter remaining the major cause for morbidity in all of these patients. As the body accumulates iron from transfused red cells, chelation therapy is usually begun to prevent deposition and damage to parenchymal tissues. Just as the rate of uptake of iron by these tissues is influenced by transfusion parameters and hepcidin levels, the variability of the chelation regimen also plays an important role. The timing of initiation, choice of chelator and dosing, and compliance with the regimen are some of these variables. When transfusion and chelation are occurring simultaneously, there is likely a complex and dynamic balance between transfusional iron loading and chelator-induced iron purging.13 Different organs may take up iron at different rates and likely have different thresholds for when tissue damage occurs. It has been demonstrated that in transfused and chelated patients with thalassemia and sickle cell disease, the relationship the liver iron concentration (LIC) and myocardial iron levels is nonlinear and variable.13,14 In summary, the pathophysiology of iron metabolism in the condition underlying the transfusion-dependent anemia, as well as the factors directly related to the initiation, intensity, duration, and effectiveness of both the transfusion regimen and the chelation regimen, contribute to the pathology of transfusional iron overload. Storage iron is present in the body predominantly in two high-molecular-weight forms, ferritin and hemosiderin. Both forms are stained on pathologic specimens by Prussian blue, via the Pearl’s reaction. Hemosiderin is insoluble and is deposited in aggregates of varying sizes, while ferritin is soluble and more homogeneously distributed within cells. Under physiologic conditions, iron is distributed approximately equally between these storage forms, but as the body’s iron load increases, the proportion stored as hemosiderin increases dramatically. A small amount of iron is present in a low-molecular-weight intracellular

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pool—likely the most toxic form of iron, because of its propensity for producing free radicals, which cause oxidative damage. It is likely that the ferritin fraction is in closer equilibrium with the low-molecular-weight iron pool.15 As the magnitude of iron excess grows, some stored iron may be mobilized into the circulation from macrophages or hepatocytes. As transferrin is increasingly saturated, some plasma non-transferrin-bound iron (NTBI) appears. This iron may be more easily taken up by parenchymal tissues that do not normally store iron, leading to toxicity. Thus, it is critical to ensure that effective chelation therapy is ongoing, to bind this iron and prevent its entry into cells, thereby avoiding organ damage. Histopathologic examination of the liver in mild transfusional iron overload reveals predominant Kuppfer cell iron deposition, with preserved hepatocyte architecture and no fibrosis. As iron loading continues, the hepatocytes also demonstrate increased iron uptake; there is some disruption of the architecture and some scarring/fibrosis. Until this point, these changes are reversible with appropriate chelation. With massive overload, there is bridging fibrosis, and progression to cirrhosis, which may or may not be reversible.16 Myocardial iron distribution is heterogeneous, with more in the left heart than the right, more in the ventricles than the atria, and more in the epicardium than the endocardium.17 It has been suggested, based on studies in the iron-loaded gerbil model, that iron in myocardial fibers may cause impaired repolarization of the myocytes.18 This may interfere with generation and orderly propagation of the electrical impulse and lead to a variety of arrhythmias. With progressive loading, fibers may not contract normally, and a restrictive cardiomyopathy with impaired filling and diastolic dysfunction may result. This can eventually lead to congestive heart failure. Heart failure is reversible in part, and with improved chelation, cardiac function may be recovered substantially.19,20 Therefore, long-term monitoring of transfused and chelated patients should include serial measurement of myocardial iron deposition as well as the LIC.

Transfusional Iron Burden The total amount of iron that enters the body via red cell transfusion can be estimated. Because the magnitude of the iron burden is critical in the development of complications, it is of great importance to maintain accurate records, for each patient, of the amount of red cells transfused. The total amount of transfused red cells is calculated as the total amount of blood in mL multiplied by the hematocrit of each unit (as a percentage) divided by 100. Each mL of red cells contains 1.08 mg of iron, and the total amount of iron introduced (Kin) is calculated as follows: Kin  (total amount of red cells transfused, in mL)  1.08 Alternatively, if the exact volume is not available, the iron burden can be estimated from the number of units transfused.

Chapter 55: Transfusional Iron Overload

Each Red Blood Cell (RBC) unit transfused contains 200 to 250 mg of iron. The total iron burden can be calculated with use of one of these methods. Transfusion-dependent patients usually require 200 to 300 mL/kg/year of blood, an amount equivalent to 0.25 to 0.40 mg/kg/day of iron. With no physiologic means of excreting this excess iron, and the inapplicability of phlebotomy in patients who are regularly transfused, these individuals must receive iron chelation therapy in order to facilitate excretion. A minimum chelator-induced excretion of 0.25 to 0.40 mg/kg/day would maintain iron balance in such individuals. Higher levels of excretion would be necessary produce a negative iron balance. Iron balance should be monitored during chelation therapy to assess the efficacy of such treatment. The total body iron (Us) at any point in time (t) may be extrapolated from the LIC (in mg/g dry weight) using the formula published by Angelucci et al21 as follows: Us(t)  10.6  LIC  (body weight in kg) For patients who are being chelated as well, there is an ongoing change in the body iron balance as the iron entering the body through transfused red cells is removed by the chelator. In these situations, it is important to monitor the total body iron excretion between LIC measurements. This can be calculated based on the amount of red cells transfused (Kin  iron introduced in mg), and on the changes in total body iron between measurements of liver iron concentrations at baseline (t0) and a later time (t), expressed as milligrams of iron excreted per day.22 Total body iron excretion  (Kin
[Us(t0)  Us(t)]) / (t  t0)

Clinical Features Although iron deposition in tissues begins soon after the initiation of regular transfusions, signs and symptoms of iron toxicity usually occur after a few years of iron loading. By then the usual storage sites for iron have been almost saturated, and there is deposition in other tissues that do not normally store iron. Organs suffer damage, often irreversible, resulting in significant morbidity. Early death is sometimes seen, generally as a result of cardiac failure.23,24 In a recent cross-sectional study of patients with thalassemia born after 1970 in Italy, it was found that 7% had heart failure, 6% had diabetes, 11% had hypothyroidism, and 55% had hypogonadism.25 A third of these patients had died, the cause of death in 68% being heart failure. Liver disease of transfusional iron overload may be manifested as hepatomegaly, abnormalities in function, fibrosis (which may lead to scarring, bridging fibrosis, and micronodular regeneration and cirrhosis), and hepatocellular carcinoma.25,26 Patients are asymptomatic, or have mild to moderate icterus, and with progression, symptoms and signs of cirrhosis and hepatic failure.

Heart failure remains the leading cause of mortality in transfusional iron overload. Unfortunately, symptoms do not appear until the myocardium has large amounts of deposited iron, and routine testing by conventional echocardiography and Holter monitoring early in the course almost always yields normal results. With progressive deposition, patients manifest symptoms. Early signs may include a variety of arrhythmias including supraventricular or ventricular tachycardias, premature or extra systoles, heart block, and atrial fibrillation. Contractile dysfunction affects diastole early,28 likely the reason that conventional echocardiography, which assesses mainly systolic function, indicates “normal” function. The onset of cardiac failure is late, and often sudden, and is an ominous sign. Both complications are often refractory to treatment. Therefore, prevention of myocardial iron loading is critical. In patients who begin receiving transfusions in early childhood, the developing endocrine organs are particularly susceptible to iron deposition if effective chelation is not initiated at the appropriate time. The anterior pituitary is most often involved, resulting in slow growth, delayed sexual maturation, and often infertility, the latter being the result of direct iron deposition in the gonads as well. Supplemental growth hormone or sex hormone therapy is often required to treat these complications. The pancreatic islet cells are also susceptible to the toxic effects of iron. Impaired insulin secretion and abnormal glucose tolerance usually precedes the development of insulin-dependent (type 1) diabetes and may occur in highly loaded individuals at any age.29,30 Older males may experience dysfunction in sexual performance, and females may develop secondary amenorrhea related to gonadal iron deposition and disturbances in sex hormone secretion. Thyroid dysfunction, although uncommon, may also manifest at any age.26 Other manifestations include arthropathy and skin pigmentation, which are directly related to iron deposition in these tissues. Patients with marked hemosiderosis appear bronzed initially, and with progression, may appear darker, or grayish. Complicating the manifestations of iron toxicity are the clinical signs and symptoms of chelator side effects, which are described below.

Measurement of Iron Burden The goal of therapy for individuals with transfusional iron overload is to maintain iron balance at low levels of tissue iron, thereby preventing the development of overload and complications. When there is little or no ineffective erythropoiesis, simply keeping track of the volume of red cells transfused provides a measure of the body iron burden in transfused patients who are not being chelated. Different tissues load iron at different rates and the balance between loading via transfusion and removal by chelation is also a determinant in the differential deposition of iron. Thus, monitoring iron deposition in any one organ is not representative of the others. The ideal means of monitoring iron overload is to periodically assess tissue iron levels in each of the

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different organs affected, but this is currently feasible only in the liver and heart. The LIC has been used to assess the body iron burden. In patients with thalassemia major, who had been cured by stem cell transplantation, phlebotomy was used as a means of removing excess iron that had accumulated from years of red cell transfusions. A serial decline in the LIC correlated well (R  0.98; p  0.001) with the amount of iron removed by phlebotomy, supporting the idea that the LIC is a reflection of total body iron load.21 Patients with thalassemia who had LICs of 6 to 13 mg/g dry weight had an almost twofold risk of progression of fibrosis; in those with liver iron concentrations of 13 to 41 mg/g dry weight, the hazard rate was almost ninefold.31 Coexisting infection with the hepatitis C virus (HCV) independently increased the risk for progression of fibrosis threefold. These observations confirmed the results of previous studies: LIC 7 mg/g dry weight is associated with an increased risk of hepatic fibrosis, diabetes, and other complications of iron overload.32 Maintaining the LIC in the suggested “ideal range” of 3 to 7 mg/g dry weight in regularly transfused and chelated patients should minimize iron deposition in nonstorage parenchymal sites, and prevent significant toxicity. Because correlation between hepatic and cardiac iron concentrations is not satisfactory,13,14 independent assessment of myocardial iron should be performed, as discussed in the next section. Whereas the LIC can be measured directly by biopsy or noninvasive means, direct quantification of myocardial iron is not feasible. A surrogate for cardiac iron burden, myocardial T2*, has been shown to correlate with function,33 and is currently used in several centers. In the absence of methods for assessing iron deposition in other tissues such as the pancreas and anterior pituitary, current ideal monitoring includes periodic assessment of hepatic and myocardial iron deposition. Serial measurement of the LIC over time is an accurate and reliable means of monitoring the progression of iron loading and the efficacy of chelation therapy. The ideal method for measuring the LIC is one that is safe, accurate, noninvasive, and valid over a wide range of concentrations. Magnetic resonance (MR)based methods meet almost all of these criteria, although access to centers offering them may be limited. The reference method has been the quantitative chemical estimation of nonheme iron in liver tissue obtained by biopsy. To obtain accurate results by this method, several requirements must be met: the patient should not have cirrhosis or focal lesions, the biopsy must weigh at least 1 mg dry weight (or 4 mg wet weight), and should be processed using strictly iron-free methods. Serial biopsies would ideally allow monitoring not only of the rate of iron loading and the efficacy of chelation therapy in the liver, but also the development and rate of progression of fibrosis or scarring. Liver biopsy is an invasive procedure with considerable risks, and is not ideal for the frequent measurement of LIC that is necessary for the monitoring of regularly transfused and chelated individuals. A recent workshop by the National Institute of Diabetes and Digestive and Kidney Diseases concluded that a clinical need

862

was evident for quantitative means of measuring body storage iron that are noninvasive, safe, accurate, and readily available to improve the diagnosis and management of patients with iron overload, including hereditary hemochromatosis (HH), thalassemia major, sickle cell disease, aplastic anemia, myelodysplasia, and other disorders.34 Several noninvasive methods have been used to estimate the LIC. These include direct methods such as magnetic biosusceptometry, and indirect methods such as MR. Recently, there has been tremendous interest in using MR techniques to study tissue iron.35-37 A brief description of the application of these techniques is presented here; a more in-depth analysis has recently been published.38 Hemosiderin and ferritin iron affect the relaxation of hydrogen atoms present in the nuclei of tissue water molecules. Initial limitations in the technology have been overcome and more sensitive instruments capable of measuring more rapid signal decay (very short relaxation times) are now used to quantify hepatic and cardiac iron concentration. Several such methods have been used to assess the hepatic iron concentration.39-41 Two widely used techniques are a gradient-echo T2*-based sequence that measures the signal intensity ratio of liver to skeletal muscle,42 and a stimulated spin-echo R2 sequence-based scan (http://www.resonancehealth. com).43 Measurements of hepatic iron concentration obtained by this technique correlate well with hepatic iron chemically estimated directly from liver tissue. T2* sequences have been used to quantify hepatic iron, but the correlation is less robust.33 The relationship between estimates of LIC by MR methods and the serum ferritin has been variable. Iron concentrations in the myocardium are much lower than those in the liver, and the iron is distributed nonhomogeneously in different areas of the heart. A multi-echo spin-echo T2* technique is the most widely used method of assessing myocardial iron.44 Although no direct correlation of T2* with tissue iron has been demonstrated in humans, data are available from the iron-loaded gerbil model.45 All individuals with clinical cardiac disease had T2* measures of 20 msec, and all subjects with T2* values above 20 msec had normal cardiac function.33 The threshold for “severe” cardiac disease has also been reduced to a T2* value of 8 msec, below which there is very high risk for development of cardiac disease.46 Several studies have compared the efficacy of different chelators in removing myocardial iron using this method.20,47,48 Magnetic resonance techniques are being developed to study iron deposition and its effects in other organs as well, including the anterior pituitary and pancreas, enabling better surveillance for development of complications and closer monitoring of the efficacy of chelation in preventing or ameliorating iron deposition. Magnetic biosusceptometry has been validated to be a safe, accurate, and noninvasive method of quantitatively measuring the LIC over a wide range of body iron burdens. The principle is that storage iron (both hemosiderin and ferritin) is paramagnetic, ie, when a steady magnetic field is applied, these molecules will induce a field change that is proportional to the number of

Chapter 55: Transfusional Iron Overload

iron atoms present. An extremely sensitive sensor, or superconducting quantum interference device (SQUID), measures this response. A strong linear correlation (R  0.99) between LIC determined by biopsy and by SQUID susceptometry has validated this technique.49 SQUID susceptometers have been used extensively in studies of iron-loaded individuals to assess iron burden and guide chelation therapy.22,32,50-52 Use of this method has been limited because of the complexity and high cost of building, installing, and maintaining the instrument. Currently, only four centers worldwide are using this technique. Other limitations include use in individuals who are obese, are very small, or have metal implants or devices that may not be removed before the study. In addition, susceptometric methods cannot be used to quantify iron in other organs that may be loaded with iron, such as the heart, pancreas, and other endocrine organs. A new high-transition temperature device, cooled using liquid nitrogen and with better resolution of the signal from surrounding tissues, is under development.53 Traditionally, the serum ferritin level has been used as an indirect estimate of the body iron burden. Several studies have shown a significant correlation between the serum ferritin level and the LIC estimated by biopsy in patients with thalassemia.32,54-55 However, the coefficient of correlation in most of these was poor, with marked scatter around the correlation line. A recent study in patients with sickle cell disease also showed poor correlation between serum ferritin levels and the LIC measured in biopsy tissue in patients with sickle cell disease.57 There are several reasons for this variability in the serum ferritin level, which does not necessarily reflect a change in the body iron burden. Ferritin is well-known to be an acute-phase reactant. Serum levels may be increased by infection, inflammation, hepatic dysfunction, tissue injury, hemolysis, and decreased when there is chronic hypoxia or a deficiency of ascorbic acid. Thus, it is at best an indirect marker of iron overload. Although practical and easy to measure, ferritin is not reliable as an indicator of the total body iron burden or as a parameter for the monitoring of chelator efficacy, and does not in any way identify the differential iron loading that may exist between the liver and the heart. Without the wide availability of noninvasive measures of hepatic and cardiac iron, there is still reliance on the serum ferritin level as a means of monitoring the iron burden and the efficacy of chelation. However, it may actually be misleading in individual patients, remaining stable in the face of rising body iron burden and ongoing deposition in the heart and endocrine organs. Similarly, measurements of serum iron, transferrin or transferrin saturation, and urinary iron excretion do not provide reliable indication of the level of body iron stores. Plasma A NTBI has been suggested to be a marker of the “toxic iron pool,”58,59 but its measurement is very complex and is currently still used for research purposes only. These limitations in serum measurement of iron-related moeities underscore the need for assessment of iron in specific tissues. As discussed previously, there is variability in iron deposition between the heart and the liver, as a result of the changing

balance between transfusional iron loading and chelation.13,14 Thus, optimal monitoring of iron burden should include assessment of both—the hepatic iron concentration by biopsy, MR, or susceptometry and the myocardial iron by the surrogate T2* estimate. These noninvasive monitoring methods are not yet widely available, but special effort should be devoted to securing such testing for patients in order to chelate them effectively and prevent the development of complications.

Management The primary goal of therapy is to prevent tissue deposition of excess iron, thereby preventing organ damage and the resulting morbidity and mortality. Maintaining safe levels of tissue iron requires achieving a balance between the amount of iron entering the body and that being removed. Unlike management of HH patients, phlebotomy to remove excess iron is not an option for those with transfusional iron overload unless the patient has successfully undergone stem cell transplantation, or has completed treatment with intensive chemotherapy regimens. If transfusion therapy is ongoing, iron-overloaded patients must be treated with a chelating agent capable of sequestering iron and permitting its excretion from the body. As discussed previously, a minimum chelator-induced excretion of 0.25 to 0.40 mg/ kg/day is necessary to maintain iron balance. The ideal chelator should form a high-affinity 1:1 uncharged complex with iron; be able to chelate intracellular iron; be orally effective, with a long half-life, high chelator efficiency, and low toxicity profile; and be effective in removing iron from (or preventing deposition in) all organs that may be affected with long-term usage. Such a chelator is not yet available. Chelators in current use are described in brief below; more detailed reviews of chelation are available elsewhere.60,61

Chelating Agents Deferoxamine mesylate (Desferal, Novartis, East Hanover, NJ) (Fig 55-2), a naturally occurring trihydroxamic acid produced by Streptomyces pilosus, was first introduced almost 40 years ago and has been found to be a generally safe and effective means of managing iron overload, ameliorating or preventing ironinduced organ damage, and reducing morbidity and mortality. In fact, patients with thalassemia major who were effectively chelated survived significantly longer than those whose chelation was ineffective.62 Deferoxamine is a hexadentate chelator that forms a charged 1:1 complex with iron. This complex does not readily enter or leave cells and is excreted almost equally by the liver in bile and by the kidneys. Unfortunately, deferoxamine is not absorbed intact when taken orally, has a very short half-life (approximately 15 minutes), and must be administered by subcutaneous infusion using a portable syringe pump over 8 to 12 hours daily for maximal chelation efficiency.63 It has been shown that levels of NTBI (the form of iron that more easily enters parenchymal cells) rise in the plasma within 16 hours

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Section IV: Part III

of the end of the infusion,63 and maintaining chelator levels in the body is critical to prevent toxic effects of this iron. Patients who receive the drug in this manner often have pain, experience swelling and redness at the site of infusion, and may develop injection abscesses. Rare toxicities occur when deferoxamine is present in excess of chelatable iron, including effects on hearing (usually high-frequency hearing loss) and vision (leading to blindness in severe instances).63,64 For all of these reasons, compliance with this regimen is not very good, and morbidity from organ deposition and dysfunction is often the result. The dosage range for deferoxamine is between 30 and 50 mg/kg administered over 8 to 12 hours by subcutaneous infusion five to seven times a week.64 In severely loaded individuals, such as those with iron induced cardiac disease,19 it may be administered by continuous intravenous infusion, although dosing above 50 mg/kg/day does not increase iron excretion further,63 and may result in toxicity. Although cardiac function typically improves relatively quickly (2-3 months), such continuous therapy often has to be maintained for many months or even years before there is a substantial decline in the LIC or improvement in the myocardial T2*.20 Deferiprone (1,2-dimethyl-3-hydroxypyridin-4-one) (Fig 55-2) is a bidentate chelator that forms an uncharged 3:1 complex with iron, enabling it to cross membranes easily and remove toxic iron from cells. It is effective orally, but has a short halflife (2 hours) and must be taken three or four times a day. Excretion occurs in the urine. It is less effective than deferoxamine in reducing the LIC, but seems to have greater effectiveness in removing myocardial iron.47 A retrospective study of patients who had undergone long-term treatment with deferiprone showed that there were significantly fewer cardiac events in those patients compared to patients who had been treated with deferoxamine alone.24 Serious idiosyncratic complications have been reported with its use, including neutropenia (up to 5%), agranulocytosis (up to 0.5%), the development of an erosive

O (CH2)5 NH3

(CH2)2 NH (CH2)5 N O HO

(CH2)2 NH (CH2)5 N O HO Deferoxamine B mesylate (Desferal)

N HO

O

O

OH

OH

N

CH3

O

Deferiprone (L1)

N

N

OH

HO

Deferasirox (Exjade)

Figure 55-2. Chemical structure of the different chelator molecules.

864

O

Initiation of Chelation

N

CH3

CH3

arthropathy (5% to 20%), and the development of a neurologic syndrome of cerebellar and psychomotor retardation.65 In spite of adherence to the suggested weekly monitoring of blood counts, one death from agranulocytosis has been reported.66 A Canadian study described progression of hepatic fibrosis in several patients who were undergoing long-term therapy.67 A subsequent study did not confirm this finding68 but has been criticized.69 Deferiprone is prescribed at 75 to 100 mg/kg/day divided into three doses, with careful monitoring of blood counts every week. The ability of this drug to move across the cell membrane led to the idea of using it in combination with deferoxamine, with deferiprone acting as a siphon to bind iron and transport it extracellularly. Although one study showed that patients treated with combination deferoxamine and deferiprone showed significantly greater improvements in T2* than those on deferoxamine alone,48 the significance of this finding is limited by the small number of subjects and the short duration of evaluation. This drug is not approved for use in the United States, and the conclusion of a recent independent review by the Cochrane Collaboration70 was that “deferiprone is indicated for the treatment of iron overload in patients with thalassemia major when deferoxamine is contraindicated or inadequate.” Deferasirox (Exjade, Novartis) (Fig 55-2) is a tridentate N-substituted bis-hydroxyphenyltriazole chelator that forms a 2:1 complex with iron that is primarily excreted in bile. With a plasma half-life of 12 to16 hours, once-daily administration means the drug is present in the circulation throughout the day, enabling constant effective scavenging of iron. Phase III studies of this agent, comparing it to deferoxamine, have only recently been reported.32 At the suggested and subsequently approved starting dose of 20 mg/kg, most patients were not able to achieve negative iron balance in this study, although most but not all did appear to maintain their iron balance. Although it was able to cause negative iron balance (reduction in LIC) in the majority of patients at doses of 30 mg/kg/day, there was a high incidence (up to 39%) of hepatic and/or renal toxicity at this dose level. Other side effects include gastrointestinal upset (15%), rash (11%), and, rarely, neutropenia. Patients who are taking this agent should be monitored carefully for hepatic and renal function, and appropriate dose adjustments should be made. As yet, there are no published data on the efficacy of deferasirox in removing myocardial iron, but studies are in progress. Trials using 30 to 40 mg/kg/day of deferasirox are under way for highly iron-loaded patients who are intolerant to deferoxamine. Combination trials with deferoxamine and deferasirox are currently being designed.

The optimal range recommended for maintaining the LIC is 3 to 7 mg/g dry weight, as discussed previously. When this range is reached after regular transfusion, chelation therapy should be initiated. There are no immediate clinically apparent consequences of not instituting chelation when the patient reaches this range of iron overload, because such complications usually result at higher levels and after more prolonged loading.

Chapter 55: Transfusional Iron Overload

The goal of therapy is to prevent these complications and chelation should be instituted well before they occur. The range for initiation of chelation is usually reached when the total transfused red cell volume reaches 150 to 180 mL/kg. If records of transfused volumes are not available, the LIC (as measured by biopsy) or noninvasive methods (such as SQUID or MR) should be used to guide the decision. In children in whom iron is required for growth, aggressive chelation could potentially reduce the amount of available iron. A conservative LIC of 4 to 5 mg/g dry weight is usually used as the threshold for initiating chelation (Table 55-1). Although an oral chelating agent would be much preferred by patients compared to a parenterally administered one, there is still not enough experience or data with regard to the long-term use of deferasirox as a first-line agent, and deferiprone is recommended only if deferoxamine is contraindicated.71 Deferoxamine is still recommended as the choice for initiation of chelation.71 Patient education, compliance, and preference should also be taken into consideration.

Compliance Compliance with chelation therapy is the primary determinant of morbidity and survival in such patients, and the responsibility of the medical team caring for such patients in reinforcing this cannot be emphasized enough. If compliance with chelation is poor, iron loading exceeds removal and iron accumulates first in the liver and then in the other parenchymal tissues including the heart. A recent meta-analysis of 18 studies involving patients with thalassemia confirmed that noncompliance results in significant cardiac and endocrine morbidity, and increased risk of death from heart disease.72 When transfusion and chelation are begun in early childhood, it is usually the responsibility of the parent to ensure that the chelation regimen is followed. In adolescence and young adulthood, when this responsibility shifts to the patient, various distractions and life-changing events may result in poor compliance. Often a lack of physical symptoms in spite of significant iron overload lulls such individuals into a false sense of wellness and invulnerability. This is a particularly critical time for all caregivers to monitor these patients very carefully. Similarly, older individuals who have begun regular transfusion

later in life may feel that it is unnecessary to chelate because the onset of symptoms may be years later. Education and constant reinforcement is necessary to ensure compliance and reduce morbidity and mortality. Patients undergoing chelation therapy should be vigilant for symptoms and signs of infection, particularly if they have undergone splenectomy. Chelation may be temporarily suspended if there is any suspicion of a bacterial or fungal illness, until this is appropriately managed.

Intensification of Chelation Noncompliance resulting in a positive iron balance and an increase in the LIC or shortening of the T2*, would necessitate changes in the chelation regimen to prevent progression of organ damage. The compliance record should be closely examined and consultation with an expert in the management of iron overload should be undertaken before intensification of chelation is begun. Dosage of deferoxamine can be increased to a maximum of 50 mg/kg/day, beyond which there seems to be no additional increment in urinary iron excretion.63 Higher doses have also been associated with the development of a pulmonary syndrome.73 Increasing the frequency of subcutaneous administration to 7 days per week is a more effective strategy (Table 55-1). For patients with symptomatic heart disease, continuous intravenous chelation with deferoxamine at 50 mg/kg/day has been shown to be effective in reversing heart failure, improving cardiac function, and reducing the LIC more effectively19 (Table 55-1). Generally, an indwelling catheter is required, because deferoxamine may cause sclerosis of peripheral veins. Close monitoring for toxicity and efficacy is necessary. Because noncompliance is the primary cause of severe loading, this regimen may not be acceptable to the patient. Although a combination of deferoxamine and deferiprone has been reported to be effective in reducing the iron burden in the liver and the heart,48 the number of patients in the study was small, and the duration of follow-up was short. Longer prospective studies with larger numbers of patients are required before combination therapy can be recommended. Results of long-term, prospective, randomized trials of higher doses of deferasirox are not available. Intravenous deferoxamine

Table 55-1. Guidelines for Chelation Therapy Indication

Choice of Product/Regimen

Initiation of chelation

LIC 3-7 mg Fe/g dry weight, or total red cell volume transfused 150-180 mL/kg

Deferoxamine 35-40 mg/kg by subcutaneous infusion over 8-12 hours five to seven times/ week; only if deferoxamine is unacceptable, deferasirox 20 mg/kg/day on empty stomach, daily

Intensification (in conjunction with expert consultation and advice)

LIC 8-15 mg Fe/g dry weight, or T2* 20 msec, without clinical cardiac disease

Deferoxamine 50 mg/kg/day by subcutaneous infusion daily

LIC 15 mg Fe/g dry weight, or T2* 10 msec, or onset of clinical cardiac disease

Deferoxamine 50 mg/kg/day by continuous infusion via central venous catheter

LIC  liver iron concentration.

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as described above should also be considered for any patient with a severe iron burden, corresponding to LICs above 15 mg/g dry weight even in the absence of clinical cardiac deposition, and for those with myocardial T2* values below 10 msec.

Monitoring Chelation Therapy Once chelation is begun, regular noninvasive assessment of iron deposition in the heart (MRI T2*) and liver (MRI R2 or SQUID) every 12 to 18 months is recommended (Table 55-2) to ensure the efficacy of chelation and give warning of rising levels, which may be associated with significant iron toxicity. Higher LICs predispose to progression of fibrosis,31 and the development of cirrhosis; keeping the iron level in the ideal range of 3 to 7 mg/g dry weight is an important preventive measure. Simultaneously, monitoring the cardiac T2* for myocardial deposition is also critical. A liver biopsy is indicated if there is coexisting hepatitis or liver dysfunction suggestive of fibrosis or progression to cirrhosis. Adjustments in the chelation regimen should be based on changes in the LIC or the cardiac T2*. More frequent measurements may be indicated after such changes have been made to ensure optimal efficacy of the regimen. In addition to monitoring iron burden and adherence to chelation therapy, organ function must also be monitored for the development of abnormalities related to the transfusion and

chelation regimen (Table 55-2). The ideal method for assessing cardiac function is MR assessment of chamber dimensions, ventricular filling, and ejection fractions. This would provide indicators of systolic as well as diastolic function. Conventional echocardiography does not provide adequate assessment of diastolic function, and is suboptimal. Cardiac dysfunction in iron-induced cardiac disease is mainly diastolic and systolic function is preserved until late in the course of the ironinduced restrictive cardiomyopathy. Normal results often lead to a false sense of well being and noncompliance with chelation. Serial measurements may provide useful information on trends and may signal the need for closer evaluation by MRI.74 Despite preserved global function, tissue Doppler echocardiography-detected regional wall motion abnormalities represent an early sign of cardiac disease.75 These special echocardiographic techniques may be of limited use, but annual MR-based cardiac function assessment, performed at the same time as T2* measurement, is the ideal method of monitoring. Holter monitoring for development of arrhythmias, is also recommended annually. Regular assessment of hepatic and renal function should also be part of comprehensive care. In addition to screening for transfusion-associated viral infections, frequent assessment is also indicated to monitor for chelator toxicity. Comprehensive annual endocrine evaluation including thyroid and parathyroid

Table 55-2. Recommended Comprehensive Evaluation of Regularly Transfused and Chelated Individuals System

Test

Monitoring of iron load

●

LIC by biopsy, SQUID, or MRI

Every 12-18 months; more frequently if iron burden is high and patient is undergoing intensive chelation

●

Cardiac T2*

Annually

●

MRI for assessment of function Annually (echocardiography with tissue Doppler if MRI not possible)

●

Holter monitoring

Annually

●

Bilirubin levels and AST/ALT monitoring

Every transfusion visit if patient is taking deferasirox or if HBV or HCV infection is active; less frequent if patient is taking deferoxamine

●

HBV, HCV serology

Annually

Renal

●

BUN and serum creatinine

Every transfusion visit if patient is taking deferoxamine or deferasirox

Endocrine

●

Thyroid and parathyroid function

Annually

●

Bone density

Annually

●

Glucose tolerance

Annually

●

hGH, testicular and ovarian function

Based on age and clinical indications

●

HIV serology

Annually

●

Vision and hearing

Annually if patient is taking deferoxamine

●

Pulmonary function

Every 2 years

Cardiovascular

Hepatobiliary

Other

Frequency

LIC  liver iron concentration; SQUID  superconducting quantum interference device; MRI  magnetic resonance imaging; AST  aspartate transaminase; ALT  alamine transaminase; HBV  hepatitis B virus; HCV  hepatitis C virus; BUN  blood urea nitrogen; hGH  human growth hormone; HIV  human immunodeficiency virus.

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Chapter 55: Transfusional Iron Overload

function, bone density assessment, glucose tolerance, and gonadal function testing are recommended, especially for growing children and adolescents. Vision and hearing should be evaluated annually in patients on deferoxamine. Testing of pulmonary function is also recommended, although not as frequently. Monitoring for chelator toxicity should also be followed as shown in Table 55-2.

Summary Iron overload is a significant cause of transfusion-related morbidity and mortality. With improved survival of patients with inherited or childhood transfusion-dependent anemias, older individuals with marrow failure, and cancer patients who have undergone intensive chemotherapeutic treatments or stem cell transplantation, the prevalence of this entity is likely to continue to grow. New technologies are now available for the noninvasive assessment of the body iron burden, making it easier to quantify the amount of excess storage iron in different tissues and to monitor the efficacy of chelation regimens. New oral drugs have made it easier for patients to be compliant with the chelation therapy that must be instituted in order to prevent tissue iron deposition and ameliorate the toxicity that would result from such deposition. As more information becomes available on the long-term use of deferasirox, combination therapy with deferoxamine may change the way chelation is prescribed. Research continues on the development of newer oral chelators. These advances hold promise for a new era of effective diagnosis, monitoring, and treatment of transfusional iron overload.

Disclaimer

7. 8. 9.

10.

11.

12.

13. 14.

15.

16.

17. 18.

The author has disclosed no conflicts of interest.

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27. Cunningham MJ, Macklin EA, Neufeld EJ, Cohen AR for the Thalassemia Clinical Research Network. Complications of betathalassemia major in North America. Blood 2004;104:34-9. 28. Wood JC, Enriquez C, Ghugre N, et al. Physiology and pathophysiology of iron cardiomyopathy in thalassemia. Ann N Y Acad Sci 2005;1054:386-95. 29. De Sanctis V, Eleftheriou A, Malaventura C for the Thalassaemia International Federation Study Group on Growth and Endocrine Complications in Thalassaemia. Prevalence of endocrine complications and short stature in patients with thalassaemia major: A multicenter study by the Thalassaemia International Federation (TIF). Pediatr Endocrinol Rev 2004;2(Suppl 2):249-55. 30. Gamberini MR, Fortini M, De Sanctis V, et al. Diabetes mellitus and impaired glucose tolerance in thalassaemia major: Incidence, prevalence, risk factors and survival in patients followed in the Ferrara Center. Pediatr Endocrinol Rev 2004;2(Suppl 2):285-91. 31. Angelucci E, Muretto P, Nicolucci A, et al. Effects of iron overload and hepatitis C virus positivity in determining progression of liver fibrosis in thalassemia following bone marrow transplantation. Blood 2002;100:17-21. 32. Olivieri NF, Brittenham GM. Iron-chelating therapy and the treatment of thalassemia. Blood 1997;89:739-61. 33. Anderson LJ, Holden S, Davis B, et al. Cardiovascular T2-star (T2*) magnetic resonance for the early diagnosis of myocardial iron overload. Eur Heart J 2001;22:2171-9. 34. Brittenham GM, Badman DG. Noninvasive measurement of iron: Report of an NIDDK workshop. Blood 2003;101:15-19. 35. Cohen AR, Galanello R, Pennell DJ, et al. Thalassemia. Hematology Am Soc Hematol Educ Program 2004;14-34. 36. Hershko C. Purging iron from the heart. Br J Haematol 2004;125: 545-51. 37. Jensen PD. Evaluation of iron overload. Br J Haematol 2004;124: 697-711. 38. Wood JC. Magnetic resonance imaging measurement of iron overload. Curr Opin Hematol 2007;14:183-90. 39. Bonkovsky HL, Rubin RB, Cable EE, et al. Hepatic iron concentration: Noninvasive estimation by means of MR imaging techniques. Radiology 1999;212:227-34. 40. Jensen PD, Jensen FT, Christensen T, et al. Evaluation of myocardial iron by magnetic resonance imaging during iron chelation therapy with deferrioxamine: indication of close relation between myocardial iron content and chelatable iron pool. Blood 2003;101:4632-9. 41. Wood JC, Enriquez C, Ghugre N, et al. MRI R2 and R2* mapping accurately estimates hepatic iron concentration in transfusiondependent thalassemia and sickle cell disease patients. Blood 2005;106:1460-5. 42. Gandon Y, Olivie D, Guyader D, et al. Non-invasive assessment of hepatic iron stores by MRI. Lancet 2004;363:357-62. 43. St. Pierre TG, Clark PR, Chua-anusorn W, et al. Noninvasive measurement and imaging of liver iron concentration using proton magnetic resonance. Blood 2005;105:855-61. 44. Westwood M, Anderson LJ, Firmin DN, et al. A single breath-hold multiecho T2* cardiovascular magnetic resonance technique for diagnosis of myocardial iron overload. J Magn Reson Imaging 2003;18:33-9. 45. Wood JC, Otto-Duessel M, Aguilar M, et al. Cardiac iron determines cardiac T2*, T2, and T1 in the gerbil model of iron cardiomyopathy. Circulation 2005;112:535-4.

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46. Pennell D. MRI and iron-overload cardiomyopathy in thalassaemia. Circulation 2006; 113:f43-4. 47. Pennell DJ, Berdoukas V, Karagiorga M, et al. Randomized controlled trial of deferiprone or deferoxamine in beta-thalassemia major patients with asymptomatic myocardial siderosis. Blood 2006;107:3738-44. 48. Tanner MA, Galanello R, Dessi C, et al. A randomized, placebocontrolled, double-blind trial of the effect of combined therapy with deferoxamine and deferiprone on myocardial iron in thalassemia major using cardiovascular magnetic resonance. Circulation 2007;115: 1876-8. 49. Brittenham GM, Farrell DE, Harris JW, et al. Magnetic-susceptibility measurement of human iron stores. N Engl J Med 1982;307:1671-5. 50. Fischer R, Tiemann CD, Engelhardt R, et al. Assessment of iron stores in children with transfusion siderosis by biomagnetic liver susceptometry. Am J Hematol 1999;60:289-99. 51. Nielsen P, Engelhardt R, Duerken M, et al. Using SQUID biomagnetic liver susceptometry in the treatment of thalassemia and other iron loading diseases. Transfus Sci 2000;23:257-8. 52. Vichinsky E, Onyekwere O, Porter J, et al. Deferasirox in Sickle Cell Investigators. A randomised comparison of deferasirox versus deferoxamine for the treatment of transfusional iron overload in sickle cell disease. Br. J. Haematol 2007;136:501-8. 53. Farrell DE, Allen CJ, Whilden MW, et al. Magnetic measurement of liver iron stores: Engineering aspects of a new scanning susceptometer based on high-temperature superconductivity. IEEE Transactions on Magnetics 2007;43:4030-6. 54. Letsky EA, Miller F, Worwood M, Flynn DM. Serum ferritin in children with thalassaemia regularly transfused. J Clin Pathol 1974;27:652-5. 55. Angelucci E, Baronciani D, Lucarelli G, et al. Needle liver biopsy in thalassaemia: Analyses of diagnostic accuracy and safety in 1184 consecutive biopsies. Br J Haematol 1995;89:757-61. 56. Telfer PT, Prestcott E, Holden S, et al. Hepatic iron concentration combined with long-term monitoring of serum ferritin to predict complications of iron overload in thalassaemia major. Br J Haematol 2000;110:971-7. 57. Karam LB, Disco D, Jackson SM, et al. Liver biopsy results in patients with sickle cell disease on chronic transfusions: Poor correlation with ferritin levels. Pediatr Blood Cancer 2007;50:62-5. 58. Singh S, Hider RC, Porter JB. A direct method for quantification of non-transferrin-bound iron. Anal Biochem 1990;186:320-3. 59. Evans RW, Rafique R, Zarea A, et al. Nature of non-transferrinbound iron: Studies on iron citrate complexes and thalassemic sera. J Biol Inorg Chem 2008;13:57-74. 60. Neufeld EJ. Oral chelators deferasirox and deferiprone for transfusional iron overload in thalassemia major: New data, new questions. Blood 2006;107:3436-4. 61. Maggio A. Light and shadows in the iron chelation treatment of haematological diseases. Br J Hematol 2007;138:407-21. 62. Brittenham GM, Griffith PM, Nienhuis AW, et al. Efficacy of deferoxamine in preventing complications of iron overload in patients with thalassemia major. N Engl J Med 1994;331:567-73. 63. Porter, JB, Abeysinghe RD, Marshall L, et al. Kinetics of removal and reappearance of non-transferrin-bound plasma iron with deferoxamine therapy. Blood 1996;88:705-13. 64. Porter JB. Deferoxamine pharmacokinetics. Semin Hematol 2001;38(Suppl):63-8. 65. Important safety information: Risks of fatal agranulocytosis and neurological disorders with the use of ferriprox (letter). Toronto,

Chapter 55: Transfusional Iron Overload

66. 67.

68.

69. 70.

71.

Canada: Apotex, 2007. [Available at http://www.thalassemia.org/ bodies/body309.php (accessed June 6, 2008).] Henter JI, Karlen J. Fatal agranulocytosis after deferiprone therapy in a child with Diamond-Blackfan anemia. Blood 2007;109:5157-9. Olivieri NF, Brittenham GM, McLaren CE, et al. Long-term safety and effectiveness of iron-chelation therapy with deferiprone for thalassemia major. N Engl J Med 1998;339:417-23. Cohen AR, Galanello R, Piga A, et al. Safety and effectiveness of long-term therapy with the oral iron chelator deferiprone. Blood 2003;102:1583-7. Brittenham GM, Nathan DG, Olivieri NF, et al. Deferiprone and hepatic fibrosis. Blood 2003;101:5089-90. Roberts DJ, Brunskill SJ, Doree C, et al. Oral deferiprone for iron chelation in people with thalassaemia. Cochrane Database Syst Rev 2007;(3):CD004839. Roberts DJ, Rees D, Howard J, et al. Desferrioxamine mesylate for managing transfusional iron overload in people with

72.

73.

74.

75.

transfusion-dependent thalassaemia. Cochrane Database Syst Rev 2007;(4):CD004450. Delea TE, Edelsberg J, Sofrygin O, et al. Consequences and costs of noncompliance with iron chelation therapy in patients with transfusion-dependent thalassemia: A literature review. Transfusion 2007;47:1919-29. Freedman MH, Grisaru D, Olivieri N, et al. Pulmonary syndrome in patients with thalassemia major receiving intravenous deferoxamine infusions. Am J Dis Child 1990;144:565-9. Davis BA, O’Sullivan C, Jarritt PH, et al. Value of sequential monitoring of left ventricular ejection fraction in the management of thalassemia major. Blood 2004;104:263-9. Vogel M, Anderson LJ, Holden S, et al. Tissue Doppler echocardiography in patients with thalassaemia detects early myocardial dysfunction related to myocardial iron overload. Eur Heart J 2003;24:113-9.

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56

Transfusion-Related Acute Lung Injury Jonathan P. Wallis1 & Ulrich J.H. Sachs2 1 2

Consultant Haematologist, Department of Haematology, Freeman Hospital, Newcastle upon Tyne, United Kingdom Head, The Platelet and Granulocyte Laboratory, Institute for Clinical Immunology and Transfusion Medicine, Justus Liebig University, Giessen, Germany

Acute noncardiogenic pulmonary edema occurring immediately after transfusion of donor plasma known to contain leukoagglutinins was first clearly described by Brittingham in 1957.1 Scattered case reports of “allergic pulmonary edema” or “anaphylactic pulmonary edema” followed. In 1985 Popovsky and Moore2 published the first prospective study of this complication that—recognizing the similarity in pathophysiology with the syndrome of acute lung injury (ALI) seen in critically ill patients—they called transfusion-related acute lung injury (TRALI). They reported a much higher incidence of the complication than had previously been considered likely, and following their report, increasing numbers of individual cases and small case series were published. However, with the development of hemovigilance schemes in the late 1990s, it became apparent that TRALI was not only an important cause of transfusion-related morbidity but, with the reduction in infective complications of transfusion, the leading cause of transfusion-related mortality. In the first decade of this century, recognition and understanding of the condition has further increased and the first serious efforts at prevention have been made.

Clinical Features Clinical reports of TRALI describe a sudden deterioration in lung function closely related to blood transfusion. The changes occur rapidly and generally begin within 2 hours and nearly always within 6 hours of the subsequently implicated transfusion. A small number of reports suggest that the onset may rarely be delayed until 12 hours or more after transfusion.3 The conscious patient describes a tightness in the chest, feels short of breath, develops a dry cough, and may also experience nausea, dizziness, and rigors. On examination the patient is hypoxic, is often Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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hypotensive, and has tachypnea and tachycardia. Widespread crepitations are heard on auscultation of the chest. Rigors and fever are commonly reported but are not always present and fever may develop only some hours after the transfusion. On intubation, or on suction of the already ventilated patient, a typical finding is of a copious frothy tracheal exudate, much like lightly whipped egg white. The nature and quantity of this exudate are often remarked on by attending anesthetists and it may be considered one of the hallmarks of severe TRALI. Arterial blood gasses show hypoxia and hypercapnia that is often severe. Chest x-rays show nodular shadowing typically in the “bat’s wing” pattern of acute respiratory distress syndrome (ARDS) (Fig 56-1). Physiologic measurements such as pulmonary artery wedge pressure or esophageal Doppler study of the left atrium show systemic hypovolemia that may be marked. Further diagnostic tests of value are described below. These cases represent the severe end of the spectrum of the disorder. Most early published reports describe severe cases such as these, but with more knowledge of the condition many milder cases are being recognized. Milder cases are proportionately less dramatic. Recovery of respiratory function starts as early as 6 hours after the onset in milder cases but in severe cases deterioration may continue until 24 hours or beyond. Some authors have reported a slightly different clinical picture in which the chief signs are rigors and fever with transient respiratory dysfunction, hypertension rather than hypotension, and usually occurring within 30 minutes of transfusion. Pulmonary edema is not always demonstrated radiologically and recovery is within 1 or 2 hours. These reactions have typically been associated with cellular blood components. It has been suggested that these cases may represent a different and non-antibody-related etiology (see below) from the “classical” severe cases.4 However, a similar spectrum of mild to moderate reactions of this nature has also been documented with transfusion of plasma containing antibody to a neutrophil antigen, anti-HNA-2a.5 Acute lung injury with noncardiogenic pulmonary edema is common among critically ill patients and is generally

Chapter 56: Transfusion-Related Acute Lung Injury

(A)

(B) Figure 56-2. Sections of lung from a fatal case of TRALI. Note the presence of granulocytes in the capillaries (arrow indicates neutrophils).

Pathophysiology Acute Lung Injury

Figure 56-1. Chest x-rays from a 33-year-old man with severe TRALI, taken 2 hours (A) and 24 hours (B) after onset of symptoms, following transfusion of FFP containing HLA Class II antibodies. Note the “bat’s wing” pattern of edema with sparing of the lung bases, and the air bronchograms clearly visible on the first radiograph, and the more confluent airspace shadowing but still with basal sparing in the second x-ray.

ALI is the result of a capillary endothelial leak that allows fluid to pass from the pulmonary vessels, initially into the interstitial space and subsequently into the alveolar space. Because this edema is distinct from hydrostatic edema caused by cardiac failure or volume overload, it is sometimes known as nonhydrostatic edema. Numerous stimuli have been suggested to contribute to the likelihood of developing nonhydrostatic pulmonary edema including sepsis, trauma, aspiration of gastric contents, disseminated intravascular coagulation, and high tidal volume ventilation. In some cases of TRALI the transfusion appears to be the only probable cause of the lung injury, whereas in other cases it may be only one of several possible factors present. Histopathology, clinical findings, and experimental work have helped elucidate the nature of the stimulus from the transfused blood and the mechanism of the lung damage.

Histopathology

considered to have a multifactorial pathogenesis. Many sick patients receive transfusion, especially after multiple trauma. There is evidence that TRALI is a significant contributor to ALI and that plasma-rich components from female donors are particularly implicated. The relationship between ALI and TRALI is further discussed below.

Histopathologic findings from fatal TRALI cases are consistent with those for early ARDS, showing interstitial and intraalveolar edema2,3,6-10 and extravasation of neutrophils into the interstitial and air spaces (Fig 56-2).3,6,7,10 Hyaline membranes and destruction of the pulmonary architecture have been reported.6,7 A consistent finding in TRALI is the presence of increased numbers of neutrophils within the pulmonary capillary vasculature and small pulmonary vessels.9,10 On electron microscopic pictures, neutrophils were degranulated and focally in direct contact with

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denuded stretches of the capillary wall. A positive correlation has been reported between capillary leukostasis and desquamated epithelial cells and between the degree of capillary leukostasis and the amount of proteinaceous fluid within the alveolar air spaces.10 From these observations it appears that the neutrophil is central to the occurrence of lung damage. After sequestration in the early stages of TRALI, neutrophils and endothelial cells of the pulmonary microvasculature establish close contact. Activation of the neutrophils leads to endothelial damage and capillary leakage. The transit of proteinaceous fluid from the vessels into the air spaces results in acute pulmonary edema. In the later stage, especially of severe TRALI, neutrophils extravasate from the capillary into the alveoli and induce further pulmonary injury.

Table 56-1. Leukocyte Antibodies Implicated in Transfusion-Related Acute Lung Injury* Antigens

Antibody Specificity

HLA Class I

A2 A9 A23 A24 A11 A28 A68 B7 B8 B12 B44 B45 B35 B57 B62

HLA Class II

DR1 DR4 DR6 DR7 DR8 DR9 DR14 DR17 DR51 DR52 DR53

HNA

HNA-1a HNA-1b HNA-2a HNA-3a

Evidence for an Antibody-Related Etiology The relationship between TRALI and leukocyte antibodies in donor plasma was first noted by Brittingham, who reported that leukoagglutinins present in the plasma of a multitransfused patient induced an acute pulmonary reaction when transfused to a volunteer.1 Severe pulmonary edema was similarly induced in a healthy volunteer who received an experimental gamma globulin concentrate, deliberately prepared from plasma that contained leukocyte, and, in particular, monocyte-reactive antibodies.11 It is likely that this preparation contained high levels of HLA Class II antibodies. In addition to these cases of TRALI resulting from experimental transfusion of plasma containing leukocyte antibodies, there are numerous case reports of TRALI in which a transfused unit has been found to contain antibodies reactive with recipient leukocytes. In two large series of TRALI, where pulmonary infiltrates were apparent in chest x-rays, leukocyte antibodies in the donor of a transfused blood component were detected in 61% to 89% of cases.2,12 Animal models have provided confirmation of this antibodymediated mechanism of TRALI. Severe vascular leakage was reproduced in isolated rabbit lungs by application of HNA-3a antibodies in an ex-vivo rabbit lung model.13 From this experiment it was concluded that leukoagglutinating antibodies and concomitant complement activation are capable of causing TRALI. Other animal experiments have confirmed that antibodies are capable of causing TRALI but suggest that complement is not a prerequisite for TRALI induced by antibodies to CD177 (HNA-2a), because the induction of TRALI was found to be dependent on the density of the cognate antigen and occurred in a complementfree environment.14 Alternative mechanisms leading to TRALI have been proposed and are discussed later, but the available evidence strongly suggests that donor leukocyte antibodies reacting with recipient antigens are the predominant mechanism.

Specificities of Antibodies Identified as Causing TRALI Antibodies to HLA Class I and II antigens and to neutrophil antigens have all been clearly implicated as causing TRALI. Evidence from hemovigilance schemes and laboratories specializing in TRALI investigation has shown that the majority of cases (75-90%) are associated with HLA antibodies, and that with

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*Only antibody specificities that matched the antigens of the transfusion recipient are listed.

improved detection techniques about 50% of these are directed against Class II antigens.15 Antibodies to neutrophil antigens are found in about 10% to 25% of cases. The spectrum of antibody specificities is shown in Table 56-1.

Mechanisms of Lung Damage in TRALI Priming and Activation of Neutrophils In TRALI the endothelial and alveolar cell damage appears to be mediated chiefly by activated neutrophils. Neutrophils activated by injurious agents will respond by the release of preformed granular enzymes and proteins and by the de-novo synthesis of a range of highly toxic reactive oxygen species (ROS). Neutrophils may be activated directly by one sufficiently strong stimulus, but the process often requires two or more stimuli. The first or “priming” stimulus will potentiate the response to a second or “activating” stimulus. A large percentage of patients who develop TRALI are sick, and there is in-vivo evidence that surgical

Chapter 56: Transfusion-Related Acute Lung Injury

(A) 6-8 µm 2-15 µm

(B)

(C)

Figure 56-3. The neutrophil’s passage through the pulmonary microvasculature. Approximately 50% of all pulmonary capillaries surrounding the alveoli have a smaller diameter than the spheric neutrophil. (A) In order to pass through the capillary, neutrophils are forced to pause, deform, and assume a “sausage shape” that allows transit of the capillary. (B) Neutrophil priming is associated with a decrease of deformability (called “stiffening”), which results in local trapping of the cell and a prolonged overall transit time. (C) Activation of the endothelial cells of the capillaries results in the upregulation of surface ligands, which also results in local trapping of the cell and a prolonged overall transit time.

procedures and active infections induce neutrophil priming.16-18 In response to priming agents, neutrophils undergo polarization, a process that leads to “stiffening” of the cell.19 This “stiffening” augments mechanical retention of neutrophils within the pulmonary capillary bed and prolongs their passage through the lungs (see below).20 Prolonged, close contact between neutrophils and the endothelium provides a micro-environment in which transmembrane receptors and released mediators of each cell type can interact closely. Sequestered neutrophils, having been primed in the circulation, and endothelial cells can be activated by exogenous stimuli present in the blood bag. These transfused stimuli include antibodies, cytokines, and bioactive lipids (see below).21,22 Antibodies to neutrophil antigens involved in TRALI cases are able to prime and to activate neutrophils in some cases without additional stimuli,14,23-25 explaining why even completely healthy individuals can develop TRALI if the antibody stimulus is sufficient.11

Aspects of Neutrophil Passage through the Pulmonary Microvasculature The alveolar capillary bed is a complex interconnecting network of short capillary segments. The path of a neutrophil from arteriole to venule crosses up to eight or more alveolar walls and encounters more than 50 capillary segments. Approximately half of these pulmonary capillaries are narrower than the diameter of a spherically shaped neutrophil (Fig 56-3). This forces neutrophils to slow and to deform before passing through the narrow capillary segment.26 The transit time of neutrophils through the

pulmonary microvasculature is mainly affected by their deformation time, and slow transit accounts for significant accumulation of neutrophils in the lungs.27 The pulmonary circulation normally contains about 28%, the “marginated pool,” of the total blood neutrophil pool.28 The stimulus-induced decrease in deformability appears to be more important than selectin-mediated rolling, a key mechanism of neutrophil adhesion within other capillary beds, but changes in surface receptors in primed neutrophils will also lead to molecular adhesion to endothelial cells.27,29,30 Under physiologic conditions, primed and locally trapped neutrophils migrate from the capillaries into the alveoli as part of a local inflammatory reaction. In TRALI the primed and trapped neutrophil encounters a further activation signal in the form of transfused antibody or other transfusion stimulus, activates its microbicidal arsenal, and induces endothelial damage.

Activation of Pulmonary Endothelial Cells TRALI can also be triggered by activated pulmonary endothelium. In addition to constitutively expressed surface receptors, activated endothelial cells upregulate surface membrane receptors that facilitate neutrophil adhesion and primimg.31-34 Primary activation of endothelial cells has been suggested as the mechanism responsible for TRALI induction after infusion of bioactive lipids.35,36 Neutrophil/Endothelial Cell Interplay The interplay between neutrophils and endothelial cells, regardless of whether it has been started by neutrophil or endothelial cell activation, contributes largely to lung damage. Neutrophils respond to endothelial cell-derived mediators by activating and expressing integrins and by releasing proinflammatory mediators and granule contents. Released mediators activate endothelial cells, which, in turn, mobilize selectins, upregulate adhesion proteins, and produce inflammatory mediators; thereby, they enhance neutrophil adhesion and activation. It is within this interplay that the lung barrier breaks down and allows transit of proteinaceous fluid and, later, of neutrophils into the alveolar space. ROS released from neutrophils may play a relevant role in this process.7,14,37,38

Mechanisms of Lung Injury by Different Mediators in Transfused Blood Components The exact pathway leading to lung damage associated with transfusion depends on the nature of the antibody or other stimulus and the interplay between it and the cellular components.

Antibodies to Human Neutrophil Antigens Serologic work-up of TRALI patients identified antibodies to human neutrophil antigens in a number of cases.39-41 In hemovigilance schemes, they are detected in approximately 10% of cases. As discussed above, the ability of these antibodies to induce TRALI has been shown in ex-vivo models of lung injury.13,14 HNA antibodies, particularly those of HNA-2a, HNA-3a and HNA-4a specificity, are capable of directly activating neutrophils, which appears to be the mechanism by which they induce TRALI [Fig 56-4(A)].14,23,42,43

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(A)

(B)

1

1

1a 1b

2

(D)

(C)

2

2

(E)

(F)

2 1a 1b

1b 1

1a

2

2

Figure 56-4. Proposed mechanisms of TRALI. The activation of neutrophils and the subsequent release of toxic agents that harm the endothelium is a key mechanism in TRALI. Epidemiological, clinical, and experimental data show that most TRALI reactions are induced by antibodies present in a transfused blood component. These antibodies may recognize epitopes on the surface of (A, B) the neutrophil, (C) the monocytes, or (D) endothelial cells. Binding of HNA or HLA Class I antibodies to neutrophils causes direct activation of the cell. Binding of antibodies, especially of HLA Class II antibodies, to monocytes has been proposed to induce the release of mediators that activate the neutrophil. Experimental data

demonstrate that neutrophils may also be activated indirectly when HLA Class I antibodies are bound to the endothelium of the lung, where they can recruit neutrophils via their Fc receptors binding to the Fc parts of these antibodies. Fc receptor cross-linking leads to neutrophil activation. (E-F) Biologically active substances other than antibodies (eg, bioactive lipids and CD40L derived from cellular blood components) are thought to cause some cases of TRALI. These substances are usually too weak to activate neutrophils directly, but may induce TRALI in concert with other factors that activate endothelial cells or neutrophils See text for details.

Antibodies to HLA Class I Antigens Only a few studies have investigated the mechanisms by which major histocompatibility complex (MHC) Class I antibodies induce a TRALI reaction. An elegant study performed on mice by Looney and coworkers44 presented in-vivo data on the mechanism of endothelial cell-dependent TRALI [Fig 56-4(D)]. Transfusion of an MHC Class I monoclonal antibody to mice expressing the cognate antigen induced TRALI and acute peripheral blood neutropenia. Mice lacking neutrophils and mice lacking the Fcγ-receptor were resistant to MHC Class I antibody-induced TRALI. Transfer of wild-type neutrophils into FcRγ⫺/⫺ mice restored TRALI following antibody infusion. This model is consistent with binding of the antibody directly to endothelial cells, in the first vascular bed encountered after injection, and recruitment of neutrophils through binding of the immunoglobulin Fc portion to the neutrophil Fcγreceptor. The protection observed in FcRγ⫺/⫺ mice argues against direct neutrophil activation by the antibody. A clinical example of TRALI in keeping with such a mechanism was reported by Dykes et al45 in a patient who had undergone lung transplantation several weeks earlier and who developed lung injury in the transplanted lung only following transfusion. An antibody to HLA-B44 (HLA Class I antigen) was present in the donor of one of the transfused units, and the cognate antigen was expressed on the transplanted lung but not on the patient’s native tissues. In this case direct Fab-dependent interaction between MHC Class I antibodies and the neutrophils could not have

contributed to the neutrophil recruitment and subsequent lung injury. However, proof that HLA Class I antibody may also cause TRALI by direct binding to neutrophil antigens comes from case reports of “inverse TRALI.” In one well-documented case infusion of human granulocytes caused severe lung injury in a patient who had Class I antibodies.50 The antibodies cannot have reacted with the native endothelial cells but did react in vivo with the donor granulocytes, in keeping with a mechanism of TRALI by direct activation of neutrophils [Figs 56-4(A) and 4(B)]. In summary, cases of TRALI caused by HLA Class I antibody may be by specific antibody binding directly to endothelial cells with Fc-induced neutrophil aggregation, by specific antibody binding to leukocytes, or by a combination of both.

874

Antibodies to HLA Class II Antigens The biological mechanism by which HLA Class II antibodies induce TRALI remains to be fully elucidated. Corresponding Class II antigens are not expressed on resting human neutrophils, although they may be expressed upon neutrophil stimulation.46 Expression of HLA Class II antigens on activated endothelium has been described, but HLA Class II antigen expression was not found on vascular endothelium of pulmonary capillaries or intravascular neutrophils in a patient who experienced fatal TRALI caused by an HLA Class II antibody.38 Blood monocytes and intraalveolar macrophages normally express HLA Class II, and one possibility is that binding of antibodies to HLA Class

Chapter 56: Transfusion-Related Acute Lung Injury

II causes release of cytokines from monocytes and activation of neutrophils within the pulmonary circulation [Fig 56-4(C)].47,48 It is uncertain at present whether local monocytes are able to produce sufficient amounts of cytokines to significantly alter the activity of neutrophils and/or the endothelium. It is unlikely that transfused antibodies have direct access to the alveolar space through an intact endothelium in sufficient concentration to induce release of cytokines and subsequent activation of neutrophils and/or endothelial cells, but where there is already some damage to the pulmonary endothelium, such a reaction may exacerbate ALI. It remains possible that detection of antibodies to HNA and HLA could be surrogates for antibodies to yet unknown antigens on other cell types, eg, on monocytes. Alloantibodies to these or other cells might explain some apparently antibodynegative cases.

Inverse TRALI: Transfusion of Neutrophils In most cases of TRALI, antibodies or neutrophil-priming agents present in the blood component are causative for the pulmonary reaction. However TRALI, as described above, has also been reported in alloimmunized patients receiving blood components that contain neutrophils. Viable neutrophils may still be present in other blood components and Popovsky and Moore estimated that 6% of observed TRALI cases were caused by antibodies present in the recipient.2 As universal leukocyte reduction is introduced in more countries, inverse TRALI caused by leukocytes in platelet concentrates (PCs) and Red Blood Cells (RBCs) will become less important, but will remain of particular relevance to patients receiving granulocyte transfusions.49,50 Bioactive Lipids Blood components may accumulate intermediate metabolic products, such as bioactive lipids, during storage. These substances are breakdown products of membrane lipids, including lyso-phosphatidylcholines [C16, C18 lyso-platelet activating factor (PAF)], and act on neutrophils through the cells’ PAF receptors in order to prime the respiratory burst reaction.51 Because these neutrophil-priming agents do not develop in stored acellular plasma, their generation is dependent on the presence of blood cells. It has been demonstrated that posttransfusion sera from these patients contained significantly more neutrophil-priming activity than the controls,7 and were also reported to induce TRALI after the transfusion of stored autologous blood.52 In an ex-vivo rat lung model of TRALI, addition of both lipopolysaccharide (LPS) and plasma containing neutrophil-priming lipids obtained from stored RBCs was necessary to induce TRALI,7 which is in accordance with the pathophysiologic model that priming precedes efficient activation of neutrophils [Fig 56-4(E) and (F)]. Plasma from stored PCs and containing neutrophil-priming lipids can cause TRALI in an identical animal model.36 In this model, rats were treated with LPS to approximate active infection. Lungs from LPS-treated animals perfused ex vivo with plasma from stored RBCs or stored PCs, but not plasma from identical fresh RBCs or PCs, evidenced

TRALI.51 Lungs pretreated with vehicle instead of LPS did not evidence TRALI with any of the perfusates.

CD40-Ligand CD40 ligand (CD40L) is another neutrophil-priming breakdown product. It is a platelet-derived proinflammatory mediator found in cell-associated and soluble (sCD40L) forms. It may be found in PCs and accumulates during storage.53 sCD40L binds to CD40, which is present on the surface of monocytes, macrophages, and neutrophils.54 This CD40L/CD40 interaction induces neutrophil priming. CD40L has been identified as a possible cofactor in TRALI because its concentration in transfused PCs that were involved in TRALI cases was found to be significantly higher than in control units. In vitro, human microvascular endothelial cells preincubated with LPS experienced severe damage when sCD40L-primed neutrophils were added, whereas unprimed neutrophils did not induce such damage.54 Immune Complexes Incubation of neutrophils with immune complexes (ICs) results in production of tumor necrosis factor alpha (TNF-α) and induced apoptosis of endothelial cells in vitro.55 It remains speculative whether antibodies present in blood components might form ICs with their corresponding soluble HNA or HLA antigens in the recipient’s circulation and so prime or activate neutrophils in the pulmonary circulation. Immunoglobulins Normal IgG has been postulated to activate neutrophils in a patient with osteopetrosis being treated with gamma interferon and granulocyte and monocyte colony-stimulating factor.56 The patient had very low levels of endogenous IgG1 and IgG2 and developed severe lung injury shortly after transfusion of platelets from an untransfused male donor. No leukocyte antibodies could be found either in donor or recipient. It is suggested that transfused IgG binding to the neutrophils, which were already primed by interferon and stimulating factor, was sufficient to cause neutrophil activation and lung injury. This case may be considered to be a good example of “multiple hit” TRALI. Reports of lung injury following intravenous IgG infusion are rare and seem to be associated with high doses or concentrates prepared intentionally with high level of leukocyte antibodies.11,57 It is possible that antibodies are both diluted and neutralized during the preparation of the pooled product as suggested for pooled viricidally treated plasma.58,59

Multiple Hit/Threshold Theory of TRALI Causation Where the stimulus to endothelial and neutrophil activation is sufficient, lung damage can occur in an otherwise healthy individual with no other likely cause of lung injury. Evidence for this comes from reports of TRALI in transfused volunteers as described above and also from reports where plasma has been used for clinical reasons in otherwise healthy individuals. These cases are a minority and most patients receiving transfusion and

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Severe TRALI

Healthy individuals

Patients at risk

(A)

(B)

Mild TRALI

Individual predisposition Strength of transfusionrelated mediators Activated

Primed

Resting

Neutrophils/endothelial cells

Figure 56-5. A TRALI threshold model. The TRALI threshold model proposes that a certain threshold must be overcome to commence a TRALI reaction. The threshold of mild TRALI, in which oxygen supply is sufficient, is lower than that of severe TRALI, in which patients require mechanical ventilation (horizontal lines). In order to overcome this threshold, numerous factors must act in concert. These factors can be summarized as strength of exogenous transfusion-related mediators (light box) and individual predisposition of the patient (grey box). The individual predisposition covers both constitutive (genetic) factors and dynamic or acute influences, ie, acute infection or trauma. A strong exogenous transfusionrelated mediator, such as a strong activatory neutrophil-specific antibody, will precipitate a TRALI reaction even if the influence of the individual predisposition is low (example A, in an otherwise healthy recipient). In contrast (example B, an individual “at risk,” such as a septic patient with an activated pulmonary endothelium), a relatively mild exogenous transfusion-related mediator with low neutrophil-priming activity will be sufficient to overcome the threshold. Used with permission from Bux and Sachs.60

especially transfusion of plasma have significant comorbidities, some or many of which may also result in priming or activation of neutrophils or damage to pulmonary endothelial cells. It has been suggested that TRALI will be more common in such patients. Early reports noted that most patients with TRALI had recently undergone surgery and suggested that this in itself was a sufficient second stimulus in many cases. Some experimental evidence for the theory of “two hit” or “multiple hit” theory has been provided by studies on bioactive lipids described above.36 The theory of “multiple hits” has been further developed by Bux and Sachs,60 who suggest that the neutrophil is central to the pathogenesis of TRALI and that activation of the neutrophil requires sufficient stimuli from one or more sources to reach a certain threshold at which point full activation and lung damage will ensue (Fig 56-5). Depending on the magnitude of the neutrophil response, the lung damage can be mild or severe with corresponding clinical effects.

ALI and Transfusion in Critically Ill Patients Clinical evidence for the theory that “multiple hits” may result in TRALI has been provided by studies involving critically ill patients who are known to be susceptible to ALI. Numerous retrospective studies have suggested a relationship between the amount of blood transfused and morbidity and mortality. All these studies are beset by the difficulty in allowing for the confounding factor of how much blood the patient required also being a marker for the severity of the illness. The Transfusion Requirements in Critical Care (TRICC) trial of transfusion triggers was both prospective and randomized and found a

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significantly higher incidence of pulmonary edema and a higher incidence of ALI in patients receiving more transfusion where the standard component was relatively plasma rich.61 Gajic et al62 studied the clinical associations with ALI developing in patients during mechanical ventilation that was required for management of critical illness. They found a strong association with transfusion of plasma, but not with red cell transfusion, age of transfused red cells, the leukocyte content of transfused red cells, or with platelet transfusion. Further studies identified transfusion of female plasma as being particularly associated with development of ALI in keeping with an antibody type of mechanism.63 A prospective study in intensive care patients comparing male donor and parous female plasma found similar results.64 The incidence of ALI associated with transfusion in these studies was of the order of 1 in 50 to 200 units of female plasma transfused, a far higher incidence than the reported incidence of TRALI in other circumstances. These findings are in keeping with the “multiple hits” or “threshold” model of TRALI in which highly susceptible patients subject to multiple toxic insults to neutrophils and endothelial cells develop lung damage after a relatively mild additional stimulus from transfusion.

Diagnosis and Differential Diagnosis Respiratory Dysfunction The development of new respiratory dysfunction caused by pulmonary edema in association with recent transfusion should be considered as evidence of possible TRALI. Significant respiratory dysfunction consistent with ALI can be defined as a decrease in transcutaneous oxygen saturation to less than 90% or an arterial pO2 of less than 60 mm Hg while breathing room air, or a PaO2/ FiO2 ratio of less than 300 mm Hg. Pulmonary edema can be demonstrated by clinical examination and chest x-ray. Alternative causes of sudden respiratory dysfunction, without edema, include transfusion-related problems such as allergic reactions with bronchospasm, shock associated with a bacterially infected unit or with ABO incompatibility, and causes unrelated to the transfusion such as cardiac arrythmias, infection, and pulmonary embolus (Fig 56-6).

Distinguishing between Hydrostatic and Nonhydrostatic Pulmonary Edema Once pulmonary edema is demonstrated, it is necessary to determine whether it is cardiogenic (hydrostatic) or caused by increased capillary permeability as in TRALI and other forms of ALI (nonhydrostatic). Cardiogenic pulmonary edema may be caused by transfusion-associated cardiac overload (TACO) or may be caused by factors unrelated to transfusion, such as simple overhydration, especially in renal failure. Radiology Radiographic appearances of edema from increased pulmonary capillary permeability are often characteristic with patchy

Chapter 56: Transfusion-Related Acute Lung Injury

Dyspnea/Hypoxia within 6 hours of transfusion Transcutaneous O2 sat ⬍90% or PaO2/FiO2 ⬍300 mm Hg

No

Yes ?evidence of pulmonary edema clinical or radiological

No

Observe for further deterioration

Transfusion-related cause eg, bronchspasm, anaphylaxis, Shock secondary to infected unit or ABO incompatibility Non-transfusion related eg, Infection Pulmonary embolus Myocardial infarct

Yes Yes Is the edema cardigenic/hydrostatic?

Transfusion-related cardiac overload Prior cardiac failure Overhydration in renal failure O2 therapy and diuretics are indicated Cardiac failure plus TRALI not excluded

No

Yes TRALI or ALI Other likely cause of ALI present?

No

Probable TRALI

ALI due to shock, sepsis, aspiration, etc ⫹possible TRALI Treat underlying condition and Support respiration Investigate donors if TRALI considered likely Factors increasing likelihood of TRALI: New neutropenia or monocytopenia Transfusion of plasma-rich components Copious frothy yellow or pink tracheal exudate

Support respiration Investigate donors Figure 56-6. Flow chart for diagnosis of TRALI.

or nodular shadowing, mainly peripheral but sparing both the apices and the costophrenic angles and with the appearance of air bronchograms. This pattern is sometimes likened to a bat’s wing. In contrast, cardiogenic edema typically shows upper lobe venous distension and edema in the perihilar and basal areas. Edema in overhydration or renal failure is typically perihilar, and shows no air bronchograms. In severe TRALI or in later stages, the radiologic appearances may progress to a complete white-out of the lung fields.

Physiologic Measurements Physiologic measurements are aimed primarily at assessing the cardiac status. Measurement of the left atrial pressure or volume may be by pulmonary artery wedge pressure through a Swan-Ganz catheter, via esophageal Doppler ultrasound, or by transthoracic echocardiography. High left atrial filling pressure or volume suggest cardiogenic pulmonary edema or fluid overload.

Normal or low pressure or volume is in keeping with noncardiogenic pulmonary edema. Low levels indicate hypovolemia, a common finding in TRALI. Electrocardiography may also be helpful in detecting cardiac strain patterns or evidence of infarction. Prior or new cardiac failure does not exclude the possibility of TRALI, because both may be present in the same patient. However, cardiac failure does indicate that the use of diuretics, which are otherwise contraindicated in TRALI, may be beneficial.

Laboratory Tests A serum level of B-type naturetic peptide (BNP) of less than 250 pg/mL is consistent with ALI rather than cardiac failure, while a level of greater than 250 pg/mL or a twofold increase from a previous level is consistent with cardiac failure.65 A low level is not completely specific for ALI nor very sensitive, as many patients with ALI also have cardiac dysfunction. A high level may also be seen in renal failure.

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Section IV: Part III Table 56-2. 2004 Consensus Conference Definition of TRALI66 I. TRALI criteria A. ALI 1. Acute onset 2. Hypoxemia a. Research setting: 1) PaO2/FiO2 ⭐ 300, or 2) SpO2 ⬍90% on room air b. Nonresearch setting: 1) PaO2/FiO2 ⭐ 300, or 2) SpO2 ⬍90% on room air, or 3) other clinical evidence of hypoxemia 3. Bilateral infiltrates on frontal chest radiograph 4. No evidence of left atrial hypertension (ie, circulatory overload) B. No preexisting ALI before transfusion C. During or within 6 hours of transfusion D. No temporal relationship to an alternative risk factor for ALI II. Possible TRALI A. ALI B. No preexisting ALI before transfusion C. During or within 6 hours of transfusion D. A clear temporal relationship to an alternative risk factor for ALI

anti-HNA-2a, and with neutrophil agglutinating antibodies such as anti-HNA-3a and anti-HLA-A2, whereas monocytopenia alone is most likely to be seen with HLA Class II antibodies.68 Neutropenia is seen without evidence of lung damage with some neutrophil-specific antibodies.5,69 Donor Tests To implicate a particular donation as a cause of antibody-related TRALI requires leukocyte typing of the recipient and antibody testing of the donors (see below). Samples for neutrophil and HLA typing should be taken early from the patient in case death ensues. The finding of a donor/recipient antibody/antigen match is of value in donor management and will make the diagnosis of TRALI more likely than a negative investigation, especially where only a small number of units have been transfused. Such matches also occur in patients without TRALI, and are of increasing statistical likelihood where a large number of units have been transfused. A positive match does not prove that TRALI has occurred.

Management and Outcome Management of the Patient

A protein concentration in the pulmonary edema of greater than 70% of the serum protein is strong evidence in favor of a capillary to alveolar leak as seen in ALI from any cause.

Implicating Transfusion as a Cause of Nonhydrostatic Edema Consensus Definition of TRALI A consensus definition of TRALI was agreed upon in 2004.66 A clinical diagnosis of TRALI is considered where a new episode of ALI has occurred within 6 hours of a blood component or derivative transfusion. Where no other cause of ALI is found, the diagnosis of TRALI is considered probable whatever further tests show. Where another possible cause of ALI is present, the diagnosis of TRALI can only be considered possible (Table 56-2). These criteria are useful but do not include laboratory tests. Many cases of TRALI will be in patients with competing etiologies for ALI. Further recipient and donor investigation can help determine, often in retrospect, the probability that the lung injury was wholly or partly due to transfusion. Laboratory Tests Changes in Circulating Leukocytes Leukopenia and, in particular, neutropenia and monocytopenia occurring within the first hour after the transfusion and possibly present before the development of clinical lung injury, are supportive of transfusion as opposed to other causes of ALI. The neutropenia is often followed by neutrophilia 5 to 6 hours later.1,67 Monocytopenia is common, may be absolute, and may be more persistent than the neutropenia.67,68 Neither change is completely specific or sensitive. The degree of neutropenia is likely to be greater with neutrophil-specific antibodies such as

878

The mainstay of treatment is respiratory support, either with oxygen alone, or with oxygen and continuous positive airway pressure by mask or mechanical ventilation. In severe cases with systemic hypovolemia, there must be adequate fluid replacement in addition to positive pressure ventilation. This will often require central venous pressure or left atrial volume monitoring. Additional strategies such as high-frequency ventilation and prone ventilation can be of value. Diuretics are contraindicated, because they have been clearly reported to worsen the hypovolemia and hypotension in several cases.49,70 Only where there is concomitant fluid overload from cardiac failure or other causes are diuretics indicated. The use of corticosteroids after the insult is of uncertain value. Where the diagnosis is made within 6 hours and the lung damage is severe, use of high-dose steroids might in theory limit further damage by inhibition of neutrophil activation. Prednisolone, methylprednisolone, and dexamethasone have all been used, but there is no useful clinical evidence regarding efficacy. Where the diagnosis is made later, they are unlikely to be of value. In intractable cases for which maximal ventilation is insufficient, extracorporeal membrane oxygenation has been used to support the patient until recovery.71 The use of plasmapheresis with the intent of removing the causative antibody has been reported.11 Further transfusions should be given if clinically indicated, but plasmarich products from female donors should be avoided.

Clinical Course and Outcome Most patients begin to improve by 24 hours after the initial injury. Milder cases improve as early as 6 hours. Chest x-rays usually show clearance of edema by 48 to 96 hours. Nearly all patients who recover do so without any long-term lung damage. Other organ damage such as acute renal failure is seen in more

Chapter 56: Transfusion-Related Acute Lung Injury

Donor investigations

Probable or possible TRALI

Units transfused within 6 hours

Female and transfused male donors

Recently transfused units

Determine donor gender and transfusion history

Units transfused before 6 hours Withhold investigation unless suspicion high AND other units negative Allow to donate as normal

Male un-transfused donors Withhold investigations unless other tests negative

Ab negative donors

Allow to donate as normal

Allow to donate as normal

HLA and HNA antibody screening

Donor management

Ab positive donors Donor Ab specificity Patients Antigen typing Match Ab & Ag specificities

Ab positive unmatched donors Do not use for plasma rich components and send plasma for fractionation *Defer anti-HNA 3a positive donors from future donation

Matching donors

Ab positive matched donors Permanently defer from all donation or * Do not use for plasma-rich components and send plasma for fractionation

* See text for explanation

Figure 56-7. Flow chart for investigation and management of donors.

severe cases, although it is not clear whether this is all due to hyopoxia and hypovolemia or whether there is damage to capillary beds in organs other than the lungs.67 Mortality in different published series varies between 5% and 30%. This depends in part on how cases are ascertained. Where patients receiving transfusion were assessed prospectively for the complication, the incidence was higher, presumably because of recognition of milder cases, but the mortality was low (6%). Where reports depend on a physician recognizing and reporting the condition, the incidence is lower but mortality higher. As an example, the hemovigilance scheme in the United Kingdom (UK) reported seven deaths out of 28 possible, probable, or likely cases reported in 2002, a mortality of 25%.72 Mortality is more likely in patients receiving larger volumes of single donor plasma [including Fresh Frozen Plasma (FFP)] rather than RBCs and more likely in patients with more comorbidities.

TRALI in Neonates and Children TRALI has been reported in children with the same features as noted in adults, including leukopenia, nonhydrostatic pulmonary edema, and hypovolemia. Of 12 published reports, four ascribed death of the child partly or wholly due to the transfusion reaction.56,73,74 Age range of cases is from 0 months to 16 years but only two neonatal cases have been reported,49,75 and no neonatal cases have been reported to the UK hemovigilance scheme over 10 years. Use of blood components is more common in neonates than older children, and neonates also receive larger volumes of single donor plasma relative to their weight. It is possible that neonates are in some way less susceptible to TRALI.

Directed Donations and TRALI A particular feature of pediatric TRALI is its occurrence after the use of directed donations from the mother. This practice

has been documented to cause TRALI and can be expected to be high risk by virtue of the high probability of the mother having leukocyte antibodies that will match her child’s antigen specificity.76 By the same rationale, directed donations from a wife to a husband, or of a leukocyte-containing component from a child to a mother or a husband to a wife, will be particularly likely to cause TRALI.

Donor Investigation Strategies of donor investigation vary among blood centers. The dual aim of investigation is to help establish the diagnosis and to allow appropriate management of implicated donors. A typical scheme adapted from Su and Kamel 77 is shown in Fig 56-7. Investigation of donors is undertaken only when a diagnosis of TRALI is considered probable or possible. Donations transfused within the 6 hours before the development of lung injury are considered to be under suspicion. Where the number of possibly implicated donors is small (four or fewer) all donors should be investigated. Where the number of possibly implicated donors is greater, the high-risk donors with a history of pregnancy or transfusion are initially investigated. If a donor or donors are found with an antibody that matches a cognate antigen, investigations of the lower risk donors are not undertaken. If no donor is implicated in the initial investigation, further lower risk donors can be tested in those cases that are considered to be probable TRALI. A further selection policy is to initially investigate only those donors of plasma-rich components. Investigations are undertaken at a qualified laboratory with adequate sensitivity for HLA Class I and II antibodies, and HNA antibodies. Recipient HLA and HNA typing may be performed prospectively or only when a donor antibody is found. Tests for recipient antibodies may be undertaken when the lung injury followed transfusion of granulocyte concentrate, or following non-leukocyte-reduced

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Section IV: Part III

component transfusion in which no donor/recipient antibody/ antigen match can be identified. When a nonantibody mechanism is suspected, or if an antibody-related etiology has been largely excluded, tests on the residual donation for abnormal lipids or other bioactive substances may be carried out at a research or reference center.

Management of Implicated Donors Management of implicated donors varies among countries and centers. It is possible to permanently defer all donors who are implicated in a case of TRALI by virtue of having leukocyte antibodies of any specificity. This would undoubtedly exclude many donors unnecessarily and the logical extension of such a policy is that all donors with leukocyte antibodies should be deferred whether or not they have been possibly implicated in a case of TRALI. Alternatively, only those donors with antibody matching a recipient antigen are excluded from future donation. This policy assumes that some feature of the antibody in that donor makes them more likely to have caused TRALI in the index case, and thus to cause TRALI with further donations. Finally, it is possible to defer donors who are either possibly or definitely implicated only from donation of plasma-rich components. In addition, some centers automatically defer permanently any donor found to have anti-HNA-3a because of the high frequency of the antigen in the recipient pool (95%) and the common association with severe TRALI. As more countries restrict the manufacture, of plasma-rich components to male or nulliparous untransfused females, the policy of donor management will become less contentious.

Incidence and Epidemiology

the transfusion will affect the likelihood of developing clinically apparent TRALI. Inherited variables have not been defined but will almost certainly include polymorphisms in the immune response pathways. The presence of high levels of soluble HLA antigens in the recipient’s plasma may be protective.83 Acquired recipient variables include the presence of other lung insults, and other forms of comorbidity as discussed earlier. Transfusion variables include the titer and specificity of antibody, the volume of plasma infused, and the speed of infusion. Plasma-reduced RBCs transfused over 90 minutes rarely cause TRALI, whereas FFP given over 15 minutes—a more than 50-fold higher rate of plasma transfusion—is not uncommonly implicated. Finally, the likelihood of diagnosing and reporting TRALI depends heavily on the physician’s knowledge of the condition.

Reported Frequency of TRALI One single hospital prospective study estimated frequency for all blood components transfused without any donor selection as 1 in 5000 units.2 A retrospective single hospital study estimated the incidence as 1 in 7900 units of FFP only.67 Hemovigilance data from voluntary reports in the UK found a reported incidence of approximately 1 in 30,000 units of FFP transfused.72 Retrospective studies of ALI/TRALI in critically ill patients and a single prospective trial of TRALI after FFP infusion found that as many as 1 in 50 to 200 units of plasma from parous female donors could cause some respiratory dysfunction when compared to plasma from male donors.64,65 It is not possible to give a single figure for incidence, but the chance of female donor plasma containing an antibody matching a recipient antigen is greater than 1 in 50. Severe and clinically distinct TRALI probably occurs about 1 in 2500 to 4000 units of female donor plasma transfused. ALI in which transfusion is a contributory factor in a critically ill patient may be much more common.

Variables Affecting the Incidence of TRALI The frequency of reported TRALI depends upon variables affecting the donors, the recipients, and the physicians. Antibody-associated TRALI, which is the common and severe form, is nearly always associated with blood donations from female donors with a history of pregnancy and is both more common and more severe with transfusion of plasma-rich products, such as FFP or apheresis platelets. HLA antibodies have been found in approximately 15% of all female donors but are more common in those who have had more pregnancies and more recent pregnancies,78,79 and are even more common with more sensitive tests.80 Human neutrophil antibodies are less common. Therefore, incidence of TRALI will depend in part on the donors used by the local blood center. Policies to reduce the number of components containing antibodies are discussed below. Look-back studies for regular donors of blood whose antibody-containing plasma has been implicated in a case of TRALI show that many cases of TRALI are either overlooked or unreported. In addition, infusion of an antibody to a recipient with a matching antigen does not necessarily result in TRALI.81,82 Both inherited and acquired recipient variables and details of

880

Prevention The majority of severe TRALI cases have an antibody-related etiology. Therefore, prevention is largely aimed at reducing the likelihood of transfusion of the causative antibodies. There are several strategies to be considered.

Avoidance of Unnecessary Transfusion of Plasma This includes unjustified transfusion of plasma components such as FFP and of unnecessary transfusion of plasma associated with RBCs. Use of FFP varies considerably among countries and it has been demonstrated that use to correct minor coagulopathies is both ineffective and unnecessary.84,85 Avoidance of unnecessary transfusion of FFP will reduce the potential for transfusion of units containing antibody. RBCs in additive solution containing less than 20 mL residual plasma are uncommon causes of TRALI, and in those recorded cases there have usually been multiple antibodies in the donor plasma matching cognate antigens in the recipient. Whole blood donations, particularly

Chapter 56: Transfusion-Related Acute Lung Injury

40 35 All TRALI cases Highly likely/probable cases TRALI deaths

Figure 56-8. Cases of TRALI reported to the UK hemovigilance scheme from 1996 to 2006. After 1998 all cases were considered by an expert panel and classified as highly likely, probable, possible, or unlikely. From 1996 to 2003 case ascertainment rose steadily with increasing knowledge of the condition among physicians. From October 2003 a policy to procure FFP from male donors only was introduced. Following the change there was a decrease in reports, and a marked decline in highly likely or probable TRALI and in deaths from TRALI (note that cases were recorded by the year that the report was made, and that many cases documented for 2004 occurred in 2003). Data kindly provided by Dr. C Chapman on behalf of SHOT UK.

Number of cases

30 25 20 15 10 5 0 1997

from high-risk donors (parous female or previously transfused donors), can be processed into RBCs in additive solution with minimal remaining plasma. Similarly, use of platelet additive solutions will reduce the volume of plasma with platelets.

High-Risk Donor Exclusion The most straightforward form of this policy is to exclude donations from all female donors from production of plasma-rich components. Some individual blood centers have followed such policies locally for some years.86 The effectiveness of this intervention was demonstrated in the UK, where such a policy was instituted nationally in 2003. An active hemovigilance scheme clearly showed a 66% decrease both in the incidence of probable TRALI and in associated deaths over the next 3 years (Fig 56-8).87 Further developments of this policy include excluding only females with a history of pregnancy and excluding any donor with a history of transfusion or organ grafting.

Antibody Testing By testing donors for HLA and HNA antibodies it is possible to exclude all donors with detectable antibodies, or those with antibodies of certain specificities or titer, either from all blood donation or just from donation of apheresis plasma-rich components. This strategy is attractive in that unnecessary exclusion of valuable donors is avoided but it also depends on the sensitivity and specificity of the antibody tests. It has been used with apparent success in some centers.88

Use of Pooled Plasma Pooled solvent/detergent-treated plasma does not appear to cause TRALI. It is hypothesized that this is because of dilution of antibodies, neutralization of HLA antibodies by soluble HLA antigens in the plasma of other donors, and subsequent removal of the immune

1998

1999

2000

2001

2002

2003

2004

2005

2006

Year of report

complexes in the processing.58,59 Extensive use of solvent/detergenttreated plasma in Europe has not been associated with TRALI.89,90

Nonantibody TRALI and Inverse TRALI Leukocyte reduction reduces the production of cellular activation or breakdown products that have been implicated in TRALI. It also will prevent the rare but distinct cases of inverse TRALI related to bystander granulocyte transfusion.

Conclusion It has taken over 50 years for the full extent of TRALI to be appreciated. It is now recognized as one of the major adverse outcomes of transfusion. Preventive strategies are effective but risk loss of donors. These strategies will be refined over the next decade as more about the condition is learned.

Disclaimer The authors have disclosed no conflicts of interest.

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22. Crockett-Torabi E, Sulenbarger B, Smith CW, Fantone JC. Activation of human neutrophils through L-selectin and Mac-1 molecules. J Immunol 1995;154:2291-302. 23. Sachs UJ, Chavakis T, Fung L, et al. Human alloantibody antiMart interferes with Mac-1-dependent leukocyte adhesion. Blood 2004;104:727-34. 24. Kopko PM. Leukocyte antibodies and biologically active mediators in the pathogenesis of transfusion-related acute lung injury. Curr Hematol Rep 2004;3:456-61. 25. Silliman CC. The two-event model of transfusion-related acute lung injury. Crit Care Med 2006;34:S124-31. 26. Gebb SA, Graham JA, Hanger CC, et al. Sites of leukocyte sequestration in the pulmonary microcirculation. J Appl Physiol 1995;79:493-7. 27. Doerschuk CM. Neutrophil rheology and transit through capillaries and sinusoids. Am.J Respir Crit Care Med 1999;159:1693-5. 28. Peters AM. Just how big is the pulmonary granulocyte pool? Clin Sci (Lond) 1998;94:7-19. 29. Burns AR, Smith CW, Walker DC. Unique structural features that influence neutrophil emigration into the lung. Physiol Rev 2003;83:309-36. 30. Reutershan J, Ley K. Bench-to-bedside review: Acute respiratory distress syndrome—how neutrophils migrate into the lung. Crit Care 2004;8:453-61. 31. Gerritsen ME, Bloor CM. Endothelial cell gene expression in response to injury. FASEB J 1993;7:523-32. 32. Klein CL, Bittinger F, Skarke CC, et al. Effects of cytokines on the expression of cell adhesion molecules by cultured human omental mesothelial cells. Pathobiology 1995;63:204-12. 33. Scholz D, Devaux B, Hirche A, et al. Expression of adhesion molecules is specific and time-dependent in cytokine-stimulated endothelial cells in culture. Cell Tissue Res 1996;284:415-23. 34. Williams MA, Solomkin JS. Integrin-mediated signaling in human neutrophil functioning. J Leukoc Biol 1999;65:725-36. 35. Silliman CC, Voelkel NF, Allard JD, et al. Plasma and lipids from stored packed red blood cells cause acute lung injury in an animal model. J Clin Invest 1998;101:1458-67. 36. Silliman CC, Bjornsen AJ, Wyman TH, et al. Plasma and lipids from stored platelets cause acute lung injury in an animal model. Transfusion 2003;43:633-40. 37. Rattan V, Sultana C, Shen Y, Kalra VK. Oxidant stress-induced transendothelial migration of monocytes is linked to phosphorylation of PECAM-1. Am J Physiol 1997;273:E453-61. 38. Kao GS, Wood IG, Dorfman DM, et al. Investigations into the role of anti-HLA class II antibodies in TRALI. Transfusion 2003;43: 185-91. 39. Yomtovian R, Kline W, Press C, et al. Severe pulmonary hypersensitivity associated with passive transfusion of a neutrophil-specific antibody. Lancet 1984;i:244-6. 40. Nordhagen R, Conradi M, Dromtorp SM. Pulmonary reaction associated with transfusion of plasma containing anti-5b. Vox Sang 1986;51:102-7. 41. Leach M, Vora AJ, Jones DA, Lucas G. Transfusion-related acute lung injury (TRALI) following autologous stem cell transplant for relapsed acute myeloid leukaemia: A case report and review of the literature. Transfus Med 1998;8:333-7. 42. Kopko PM, Curtis BR, Kelher M, et al. Merging the pathogenesis of transfusion-related acute lung injury: The priming activity of the 5b (HNA-3) antibody (abstract). Transfusion 2004;44(Suppl):22A.

Chapter 56: Transfusion-Related Acute Lung Injury

43. Silliman CC, Curtis BR, Kopko PM, et al. Donor antibodies to HNA-3a implicated in TRALI reactions prime neutrophils and cause PMN-mediated damage to human pulmonary microvascular endothelial cells in a two-event, in vitro model. Blood 2006;109:1752-5. 44. Looney MR, Su X, Van Ziffle JA, et al. Neutrophils and their Fc gamma receptors are essential in a mouse model of transfusionrelated acute lung injury. J Clin Invest 2006;116:1615-23. 45. Dykes A, Smallwood D, Kotsimbos T, Street A. Transfusion-related acute lung injury (TRALI) in a patient with a single lung transplant. Br J Haematol 2000;109:674-6. 46. Gosselin EJ, Wardwell K, Rigby WF, Guyre PM. Induction of MHC class II on human polymorphonuclear neutrophils by granulocyte/ macrophage colony-stimulating factor, IFN-gamma, and IL-3. J Immunol 1993;151:1482-90. 47. Kopko PM, Paglieroni TG, Popovsky MA, et al. TRALI: Correlation of antigen-antibody and monocyte activation in donor-recipient pairs. Transfusion 2003;43:177-84. 48. Nishimura M, Hashimoto S, Takanashi M, et al. Role of anti-human leucocyte antigen class II alloantibody and monocytes in development of transfusion-related acute lung injury. Transfus Med 2007;17:129-34. 49. O’Connor JC, Strauss RG, Goeken NE, Knox LB. A near-fatal reaction during granulocyte transfusion of a neonate. Transfusion 1988;28:173-6. 50. Sachs UJ, Bux J. TRALI after the transfusion of crossmatch-positive granulocytes. Transfusion 2003;43:1683-6. 51. Silliman CC, Clay KL, Thurman GW, et al. Partial characterization of lipids that develop during the routine storage of blood and prime the neutrophil NADPH oxidase. J Lab Clin Med 1994;124:684-94. 52. Covin RB, Ambruso DR, England KM, et al. Hypotension and acute pulmonary insufficiency following transfusion of autologous red blood cells during surgery: A case report and review of the literature. Transfus Med 2004;14:375-83. 53. Phipps RP, Kaufman J, Blumberg N. Platelet derived CD154 (CD40 ligand) and febrile responses to transfusion. Lancet 2001;357:2023-4. 54. Khan SY, Kelher MR, Heal JM, et al. Soluble CD40 ligand accumulates in stored blood components, primes neutrophils through CD40, and is a potential cofactor in the development of transfusion-related acute lung injury. Blood 2006;108:2455-62. 55. Nishimura M, Ishikawa Y, Satake M. Activation of polymorphonuclear neutrophils by immune complex: Possible involvement in development of transfusion-related acute lung injury. Transfus Med 2004;14:359-67. 56. Madyastha PR, Jeter EK, Key LL Jr. Cytophilic immunoglobulin G binding on neutrophils from a child with malignant osteopetrosis who developed fatal acute respiratory distress mimicking transfusion-related acute lung injury. Am J Hematol 1996;53:196-200. 57. Rizk A, Gorson KC, Kenney L, Weinstein R. Transfusionrelated acute lung injury after the infusion of IVIG. Transfusion 2001;41:264-8. 58. Sinnott P, Bodger S, Gupta A, Brophy M. Presence of HLA antibodies in single-donor-derived fresh frozen plasma compared with pooled, solvent detergent-treated plasma (Octaplas). Eur J Immunogenet 2004;31:271-4. 59. Sachs UJ, Kauschat D, Bein G. White blood cell-reactive antibodies are undetectable in solvent/detergent plasma. Transfusion 2005;45:1628-31.

60. Bux J, Sachs UJ. The pathogenesis of transfusion-related acute lung injury. Br J Haematol 2007;136:788-99. 61. Hebert PC, Wells G, Blajchman MA, et al. A multicenter, randomized, controlled clinical trial of transfusion requirements in critical care. Transfusion Requirements in Critical Care Investigators, Canadian Critical Care Trials Group. N Engl J Med 1999; 340: 409-17. 62. Gajic O, Rana R, Mendez JL, et al. Acute lung injury after blood transfusion in mechanically ventilated patients. Transfusion 2004;44:1468-74. 63. Dara SI, Rana R, Afessa B, et al. Fresh frozen plasma transfusion in critically ill medical patients with coagulopathy. Crit Care Med 2005;33:2667-71. 64. Palfi M, Berg S, Ernerudh J, Berlin G. A randomized controlled trial of transfusion-related acute lung injury: Is plasma from multiparous blood donors dangerous? Transfusion 2001;41:317-22. 65. Rana R, Fernandez-Perez ER, Khan SA, et al. Transfusion-related acute lung injury and pulmonary edema in critically ill patients: A retrospective study. Transfusion 2006;46:1478-83. 66. Kleinman S, Caulfield T, Chan P, et al. Toward an understanding of transfusion-related acute lung injury: Statement of a consensus panel. Transfusion 2004;44:1774-89. 67. Wallis JP, Lubenko A, Wells AW, Chapman CE. Single hospital experience of TRALI. Transfusion 2003;43:1053-9. 68. Flesch BK, Neppert J. Transfusion-related acute lung injury caused by human leucocyte antigen class II antibody. Br J Haematol 2002;116:673-6. 69. Wallis JP, Haynes S, Stark G, et al. Transfusion-related alloimmune neutropenia: An undescribed complication of blood transfusion. Lancet 2002;360:1073-4. 70. Levy GJ, Shabot MM, Hart ME, et al. Transfusion-associated noncardiogenic pulmonary edema. Report of a case and a warning regarding treatment. Transfusion 1986;26:278-81. 71. Nouraei SM, Wallis JP, Bolton D, Hasan A. Management of transfusion-related acute lung injury with extracorporeal cardiopulmonary support in a four-year-old child. Br J Anaesth 2003;91:292-4. 72. Stainsby D, Jones H, Milkins C, et al. Serious Hazards of Transfusion (SHOT), Annual Report 2003. Manchester, UK: SHOT Office, 2003. [Available at: http://www.shotuk.org.] 73. Church GD, Price C, Sanchez R, Looney MR. Transfusion-related acute lung injury in the paediatric patient: Two case reports and a review of the literature. Transfus Med 2006;16:343-8. 74. Ririe DG, Lantz PE, Glazier SS, Argenta LC. Transfusion-related acute lung injury in an infant during craniofacial surgery. Anesth Analg 2005;101:1003-6. 75. Wu TJ, Teng RJ, Tsou Yau KI. Transfusion-related acute lung injury treated with surfactant in a neonate. Eur J Pediatr 1996;155: 589-91. 76. Yang X, Ahmed S, Chandrasekaran V. Transfusion-related acute lung injury resulting from designated blood transfusion between mother and child: A report of two cases. Am J Clin Pathol 2004;121: 590-2. 77. Su L, Kamel H. How do we investigate and manage donors associated with a suspected case of transfusion-related acute lung injury. Transfusion 2007;47:1118-24. 78. Payne R. The development and persistence of leukoagglutinins in parous women. Blood 1962;19:411-24. 79. Densmore TL, Goodnough LT, Ali S, et al. Prevalence of HLA sensitization in female apheresis donors. Transfusion 1999;39:103-6.

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80. Bray RA, Harris SB, Josephson CD, et al. Unappreciated risk factors for transplant patients: HLA antibodies in blood components. Hum Immunol 2004;65:240-4. 81. Toy P, Hollis-Perry KM, Jun J, Nakagawa M. Recipients of blood from a donor with multiple HLA antibodies: A lookback study of transfusion-related acute lung injury. Transfusion 2004;44: 1683-8. 82. Kopko PM, Marshall CS, MacKenzie MR, et al. Transfusion-related acute lung injury: Report of a clinical look-back investigation. JAMA 2002;287:1968-71. 83. Nishimura M, Hashimoto S, Satake M, et al. Interference with TRALI-causing anti-HLA DR alloantibody induction of human pulmonary microvascular endothelial cell injury by purified soluble HLA DR. Vox Sang 2007;93:78-82. 84. Wallis JP, Dzik S. Is fresh frozen plasma overtransfused in the United States? Transfusion 2004;44:1674-5.

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85. Dzik WH. The James Blundell Award Lecture 2006: Transfusion and the treatment of haemorrhage: past, present and future. Transfus Med 2007;17:367-74. 86. Engelfriet CP, Reesink HW, Brand A, et al. Transfusion-related acute lung injury (TRALI). Vox Sang 2001;81:269-83. 87. Chapman CE, Williamson LM, Cohen H, et al. The impact of male donor plasma on haemovigilence reports of transfusion related lung injury (TRALI) in the UK (abstract). Vox Sang 2006;91(S3):227. 88. Insunza A, Romon I, Gonzalez-Ponte ML, et al. Implementation of a strategy to prevent TRALI in a regional blood centre. Transfus Med 2004;14:157-64. 89. Bux J. Transfusion-related acute lung injury (TRALI): A serious adverse event of blood transfusion. Vox Sang 2005;89:1-10. 90. Solheim BG. Plasma-induced TRALI is avoided with solvent/detergent-treated plasma (abstract). Transfus Alternat Transfus Med 2005;7(suppl):57.

V

Cell and Tissue Therapy

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57

HLA Antigens and Alleles Thomas M. Williams Professor and Chair, Department of Pathology, University of New Mexico School of Medicine, and Director, HLA and Molecular Diagnostics, TriCore Reference Laboratories, Albuquerque, New Mexico, USA

The human major histocompatibility complex (MHC) resides on the short arm of chromosome 6. The complete genomic sequence of this region contains more than 200 genes and spans 4 to 8 megabases, depending on where the telomeric boundary is drawn.1 Many of the genes in the MHC encode proteins involved in immune responses, including the Class I and Class II antigenic molecules of the HLA system; the Class III molecules comprising complement components C2 and C4, and tumor necrosis factor; and the peptide transporter proteins TAP1 and TAP2. This chapter focuses on the genetic structure of the MHC, current HLA nomenclature, tests for detecting HLA, and the biomedical significance of HLA antigens. The existence of white cell antigens independent of red cell genetic systems was suggested as early as 1952. In 1958, Jan Dausset2,3 detected, in a patient who had undergone multiple transfusions, the first leukocyte antibody [called Mac (HLAA2)]. This discovery was followed in 1958 by independent studies on similar antibodies in postpartum serum, reported by van Rood et al4 and Payne and Rolfs.5 These antibodies originally were thought to be autoimmune in nature or to be isoantibodies responsible for febrile (nonhemolytic) transfusion reactions. Early in 1960, Brunning et al6 found that these antibodies could be used to detect several diallelic genetic systems present on the cells of most tissues. Until 1964, advances in HLA typing were hampered by inconsistent and nonspecific leukoagglutination testing methods. This status changed with the development of the microlymphocytotoxicity assay by Terasaki and McClelland.7,8 In a slightly modified form, this test continued to be the standard for clinical HLA typing until the widespread adoption of nucleicacid-based methods in the 1990s. In 1964, the first International Histocompatibility Workshop was held. Since then, 13 workshops have been held at 2- to 4-year intervals. These workshops are designed to confirm scientific

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

findings, to develop unifying concepts regarding the MHC, and to upgrade existing HLA nomenclature. Table 57-1 presents an overview of the workshops, the year and their major themes. In the third workshop (1967), it was established that the HLA antigens belonged to the same genetic system,9 and the term HL-A, coined from the Hu leukocyte system of Dausset et al10 and the LA system of Payne et al,11 was assigned to identify this system. By 1970, it was established through skin grafting studies with human subjects, particularly families with HLA-identical siblings, that these antigens were important in organ transplantation.12 In 1973, it became clear from results of one-way mixed leukocyte cultures with homozygous testing cells that other genetic polymorphisms existed within the MHC.13 Also by 1973, the remarkable association of HLA with certain diseases became apparent.14,15 In the late 1970s, HLA-D (defined by mixed leukocyte culture) antigens were established,16 serologically detected antigens on B lymphocytes [HLA-DR (D-related)] were found to be closely related to the HLA-D antigenic products,17,18 and the existence of the HLA-C locus was confirmed.19 From 1980 to 1987, advances in biochemistry, molecular biology, and the development of monoclonal antibodies to the MHC antigenic product markedly expanded knowledge about the MHC. Workshops during this time confirmed the existence of the DQ and DP loci20,21 and focused on increasingly refined methods for identifying the burgeoning number of HLA alleles and correlation of the serologic, biochemical, and molecular biologic findings related to the MHC. In the 1996 and subsequent two workshops, a primary focus was the use of powerful genomic technology to identify the more than 2000 MHC alleles now known to exist in human populations around the world.

Major Histocompatibility Complex The Class I, II, and III genes of the MHC lie in a 4-megabase region of chromosome 6 (8 megabases if one includes the more telomeric Class I-related gene HFE and the butyrophilin family of genes.)22

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Table 57-1 Historical Overview of International Histocompatibility Workshops International Workshops

Year

Major Theme

I II III IV V VI VII VIII IX X XI XII XIII XIV

1964 1965 1967 1970 1972 1975 1977 1980 1984 1987 1991 1996 2002 2005

Comparison of serologic techniques Standardized techniques and nomenclature One major genetic system named HL-A International anti-sera analysis Population differences for HLA Identification of D(DR) locus by cellular techniques; HLA-C locus confirmed Serologic detection of DR antigens Compendium of HLA antigen frequencies Introduction of RFLP molecular techniques; DQ/DP loci confirmed Standardization and application of RFLP; introduction of PCR/SSOPH typing Standardization and application of PCR/SSOPH HLA Class II typing to transplantation, disease association, and population genetics Analysis of Class I and Class II alleles by means of DNA sequencing Definition of extent of Class I and II gene variation and application to typing of volunteer donors Genomics and immune responses

RFLP ⫽ restriction fragment length polymorphism; PCR ⫽ polymerase chain reaction; SSOPH ⫽ sequence-specific oligonucleotide probe hybridization.

C E Tapasin DNA DMA N T R O M E DPB1 DPA1 DMB R I C 21OH

DOB DRB3/4/5 LMP

TAP

Bf C2

DQB1 DQA1

Class II region

TNFα ⫹ β

DRB1

DRA

MICA

Class III region C4B, A

B C

E

A

HFE

H G F Class I region

The telomeric 2000 kb of the MHC contains a number of genes, including those encoding the Class I antigens (Fig 57-1). The Class I HLA-A, -B, and -C genes reside in this region, as do the nonclassical MHC-Ib genes HLA-E, -F, and -G. HLA-E, -F, and -G are expressed at lower levels than are the classical genes, do not have the extensive polymorphism of the HLA-A, -B, and -C genes, and appear to have more limited functions in the immune system. Between the HLA-B locus and the tumor necrosis factor loci described later resides the MHC Class I chain-related

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T E L O M E R I C

Figure 57-1. Selected genes in the major histocompatibility region on human chromosome 6 in the Class I, II, and III regions. Distances between loci are not drawn to scale. For exact gene locations, see http://www. sanger.ac.uk/HGP/Chr6/MHC.shtml.

genes that encode variants of Class I proteins. Loci within this family display extensive polymorphism and encode proteins that interact with T cells and participate in natural killer cell activation.23 Finally, the HFE gene responsible for genetic hemochromatosis lies approximately 4.3 megabases telomeric to HLA-A.24 Centromeric of the HLA-B locus of the Class I region is a 1-megabase segment traditionally called the Class III region (Fig 57-1).22 This region has a dense and diverse array of genes, many with as yet unknown functions. Some of these Class III

Chapter 57: HLA Antigens and Alleles

α2

α3

S

α1

S

β1

S

α1

Variable antigenic sites

α2

Constant

S

S

S

S

S

β2

β2

Microglobulin

S

S

S

S

Ig

Like

Membrane Cytoplasm C

Figure 57-2. Simplified illustration shows the biochemical structure of Class I and II antigens. Modified with permission from Strominger.27

C

C

Class I

Class II

HLA–A,B,C

HLA–DR,DQ,DP

genes encode proteins with functions in innate immunity and inflammation. This region includes the complement components C2, Bf (factor B protein of the alternate pathway of complement activation), and C4. In most persons, the C4 locus is duplicated as C4A and C4B, each encoding a functional, somewhat biochemically different, molecule. Members of the tumor necrosis family also lie within the Class III region. Other genes within the Class III region, such as those encoding 21-hydroxylase enzymes involved in steroid metabolism, have no direct link to immunity. Although linkage to specific HLA alleles may be helpful in assessing 21-hydroxlase deficiency associated with congenital adrenal hyperplasia, direct genotyping is now possible. The Class II gene loci occupy approximately 1 megabase of DNA centromeric to the Class III region (Fig 57-1).22 Within this region are 18 closely linked loci that code for the α and β chains of the Class II antigenic proteins. Proceeding from the telomeric to the centromeric boundaries of the region, the first gene cluster is composed of several β-chain loci and one α-chain locus that encode the HLA-DR antigens. The DRB1 locus is responsible for the DR1 to DR18 specificities, whereas the DRB3, DRB4, and DRB5 loci are responsible for the DR52, DR53, and DR51 specificities, respectively. Each DR specificity is formed by a heterodimer encoded by the DRB1, 3, 4, or 5 locus and the DRA locus. Centromeric to the DRB genes are the DQ genes (Fig 57-1). The DQA1 and DQB1 loci encode the HLA-DQ specificities. Near the centromeric end of the MHC, the genes DPA1 and DPB1 encode the HLA-DPw1 to DPw6 specificities. The Class II region also contains several pseudogenes related to the genes encoding the DR, DQ, and DP antigens with errors preventing successful transcription and translation. Other genes with important accessory functions to Class I and II antigen presentation are present in the Class II region. The LMP2 and LMP7 genes, the TAP1 and TAP2 genes, and the tapasin gene encode proteins that participate in protein degradation and peptide transport and loading in the Class I system.25 The HLA-DM and HLA-DO loci encode proteins involved in peptide loading in the Class II system.

(A)

α1

α2

β2m

α3

(B)

Figure 57-3. (A) Side view of Class I antigen molecule. (B) View of the antigenbinding pocket looking down toward the cell membrane. Modified with permission from Bjorkman et al.28

Class I and II Antigens and Their Function The structure, tissue distribution, and function of the glycoprotein antigenic products of the Class I and Class II genes of the MHC differ considerably. Both Class I and Class II antigens are heterodimeric, having three to four extracellular domains in addition to transmembrane and intracytoplasmic regions (Fig 57-2).26 Class I antigens are composed of a 45,000-D α heavy chain encoded by the HLA-A, -B, and -C gene loci noncovalently bound to a 12,000-D light chain, β2-microglobulin, encoded by a gene locus on chromosome 12. The α chain is a transmembrane protein, whereas β2-microglobulin is extracellular. Domains α1 and α2 contain variable amino acid sequences and thus represent the antigenic sites. The α3 domain has constant sequences homologous to the constant regions of immunoglobulin proteins. The α3 domain of the heavy chain and β2-microglobulin are noncovalently associated with each other near the cell membrane. The α1 and α2 domains sit on top of them to form peptide antigen-binding cleft with a floor of eight flat β sheets bound on the sides by two long α helixes, as illustrated in Fig 57-3. Most of the polymorphic amino acid changes

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Section V

of the Class I histocompatibility antigen differences are associated with the floor or sides of this pocket, although some map to other domains.28,29 Class II antigens are composed of a 33,000-D α chain noncovalently associated with a 28,000-D β chain (Fig 57-2). The α chain is encoded by the A gene loci and the β chain by the B gene loci in the HLA-D region of the MHC. For example, the DQ antigen α and β chains are encoded by the DQA1 and DQB1 genes, respectively. The α1 and β1 domains are variable and form an antigen-binding groove similar to that described earlier.30 The α2 and β2 domains are relatively constant with immunoglobulin-like homology. Class I antigens have a universal tissue distribution as plasma membrane proteins of all nucleated cells and platelets. Class II antigens have a limited tissue distribution primarily on antigenpresenting cells such as B lymphocytes and macrophages, although they can be induced in several cell types, including endothelial cells. Class I and II molecules acquire self and nonself peptides in their binding cleft and display them on the plasma membrane of cells for recognition by the T-cell antigen receptor.25 In the normal state, T cells that bind with high affinity to self peptides in the context of an individual’s Class I and II molecules are either deleted or suppressed by a variety of mechanisms to prevent autoimmune disorders. However, peptides derived from viruses, bacteria, and some parasites presented by the Class I and Class II molecules to T-cell antigen receptors evoke an immune response. Class I molecules primarily acquire 8- to 10-amino-acid peptides generated by means of degradation of cytoplasmic proteins in the proteosome system. Class I antigens function as MHC restriction elements in the destruction of virus-infected target cells and present peptides to CD8 cytotoxic T cells. Class II molecules bind 13- to 25-amino-acid peptides of exogenous and endogenous origin degraded within the endosomal system. Class II antigens are MHC restriction elements that augment the sensitizing limb of the immune response and present peptides to CD4

helper T cells. Activation of CD8 and CD4 T cells then results in a program of cell division and differentiation resulting in cellular and humoral immune responses.25

Nomenclature and Polymorphism of the HLA System Table 57-2 illustrates the HLA gene loci, the variable polypeptide chains, the number of related serologic specificities, and the number of alleles within each specificity. In the Class I region, the HLA-A, -B, and -C loci are all highly polymorphic with 300 to 1000 known alleles.31 Multiple alleles are present within most of the known serotypes. For example, the A2 serotype has more than 160 alleles. In the Class II region, the HLA-DRA locus is almost monomorphic, whereas the DRB genes are polymorphic, with hundreds of known alleles. The DQA1 and DQB1 genes and the DPA1 and DPB1 loci demonstrate similar but less extensive polymorphism. The DQA1, DPA1, and DPB1 allelic variation can be detected with DNA typing but not with serologic testing.32 The HLA system is the most polymorphic genetic system known to exist in humans. The number of different phenotypes possible from all combinations of these HLA alleles is greater than the global population. Fortunately, linkage disequilibrium in all human populations results in great overrepresentation of certain haplotypes, which makes finding HLA-identical individuals within a population for purposes such as unrelated marrow transplantation possible. Exons in which most of the polymorphism in the Class I (exons 2 and 3) and Class II (exon 2) genes occurs encode the α1 and α2 and the α1 and β1 domains, respectively, which interact with bound peptides.31 New alleles appear to emerge at a fairly high rate and become fixed in populations, in theory, if they provide a selective advantage in presenting peptides from infectious organisms. New alleles are generated by means of point mutation, recombination, and gene conversion-like events.31,32

Table 57-2. HLA Nomenclature Genetic Locus

Encoded Polypeptide

Antigen or Associated Specificity

Allelic Equivalent

Number of Known Alleles/ Polypeptides

HLA-A HLA-B HLA-C DRA DRB1 DQA1 DQB1 DPA1 DPB1

α α α α β1 α β1 α1 β1

A1 to A80 B7 to B81 Cw1 to Cw10

A*0101 to *8001 B*0702 to *8301 Cw*0102 to *1803 DRA*0101 to *0102 DRB1*0101 to *1612 DQA1*0101 to *0602 DQB1*0501 to *0634 DPA1*0103 to *0401 DPB1*0101 to *9901

630/495 979/833 338/264 3/2 549/462 34/25 90/66 24/15 128/114

DQ1 to DQ9 DPw1 to DPw6

The number of polypeptides known for each locus reflects unique amino acid sequences encoded by a larger number of alleles that may differ only by silent polymorphisms.

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The nomenclature of the HLA system is established by an international committee sponsored by the World Health Organization and is updated frequently.33,34 Table 57-2 shows the general scheme for designating HLA antigens and alleles. Each serologic specificity is prefixed by the genetic system designation HLA-, followed by a letter denoting the encoded antigen (eg, A, B, C, DR). This letter is followed by a digit indicating specificity.33 For example the A locus encodes the specificities HLA-A1 through A80; the B locus, HLA-B7 through B81; the C locus, HLA-Cw1 through Cw10; the DRA and DRB1 loci, HLADR1 through DR18; and the DQB1 and DQA1 loci, HLA-DQ1 through DQ9. The nomenclature of the HLA system was first established with serologic data. Molecular data were introduced later according to the precise DNA sequences of each allele. The existing nomenclature had to be modified to accommodate this information. This modification is shown in Table 57-2. For Class I alleles, because only the β chain is variable, the molecular designation is the system (HLA-), the locus (A, B, or C), an asterisk (*), and a four-digit number. The first two digits of this number correspond to the serologic specificity; for example, HLA-A*0301 is the first molecular allele of the HLA-A3 serologic specificity, and HLA-A*0302 is the second. In the Class II region, because both the α and β chains can be variable, the locus designation must include the polypeptide chain responsible for the allele. For example, Class II, HLA-DRB1*1501 is interpreted as HLA system, DR locus, β-1 polypeptide chain (B1), DR15 serologic specificity (15 after the asterisk), first molecular allele (01). For molecular alleles that have no serologic equivalents, alleles are sequentially numbered; for example, HLA-DQA1*0101 is the first molecular allele of the α1 polypeptide chain of the DQ. Fifth, sixth, and seventh digits are employed to designate alleles with silent polymorphisms or with variation occurring outside exons.

largely replaced by nucleic-acid-based techniques for the detection of Class II alleles. The application of molecular genetic technology to the field of histocompatibility has led to an unprecedented expansion in knowledge of the MHC at the molecular level. DNA sequences have been obtained for the HLA genes and alleles in a variety of populations worldwide.33 These sequence data have been useful for several reasons. 1. They have revealed the underlying DNA sequence variations among individuals (and therefore, amino acid variations) responsible for the antigenic differences in HLA molecules detectable with traditional serologic and cellular HLA testing. 2. They have made apparent the fact that results of serologic HLA tests define only broad groups of Class I and Class II alleles. Numerous subgroups of alleles distinguishable by sequence differences are present within these broad serologic groups. The result is a very large number of known alleles.38 3. Sequence data have allowed detailed definition of the genetic basis of the HLA-mediated disease associations described later. 4. Knowledge of the DNA sequences of HLA alleles has facilitated the substitution of nucleic-acid-based methods for serologic and cellular methods for clinical HLA typing. Several methods have been developed to identify Class I and II alleles. These range from restriction fragment length polymorphism assays to direct DNA sequencing (Table 57-3). Most laboratories have replaced or complemented serologic typing with DNA-based typing methods for several reasons. Precise identification of alleles not possible by serologic means is clinically important in unrelated bone transplantation, as discussed later. Second, nucleic-acid-based typing can help resolve serologic typings compromised by cross-reactivity or complicated by clinical states resulting in pancytopenia or poor expression of HLA antigens on lymphocyte membranes. Third, even in the best circumstances, serologic typing may not be accurate, especially in ethnic groups with great diversity at loci such as HLA-B.39

Identification of HLA Antigens and Alleles The detection of HLA antigens and alleles is generally accomplished with three basic methods (Table 57-3). The microlymphocytotoxicity assay developed by Terasaki and McClelland7 of peripheral blood lymphocytes isolated from whole blood has been the standard test for the serologic detection of HLA-A, -B, and -C antigens. This is a complement-dependent test in which the addition of rabbit serum to HLA antibodies fixed to the lymphocyte membrane causes cell death. The endpoint is leakage or retention of fluorescent dyes from lymphocytes with damaged or intact cell membranes, respectively. Modifications of this test, primarily an increase in incubation time and isolation of B lymphocytes from peripheral blood, allow typing for the HLA-DR and -DQ antigens.17 Variations of the mixed leukocyte reaction (Table 57-3) with homozygous testing cells, primed lymphocytes, or T-cell clones are the primary methods for detecting HLA-D, -DQ, and -DP antigens.35-37 The mixed leukocyte reaction methods have been

Table 57-3. Detection of HLA Antigens and Alleles Serologic methods ● HLA-A, -B, and -C: microlymphocytotoxicity test ● HLA-DR and DO: modified microlymphocytotoxicity test, B-cell-enriched lymphocytes Cellular methods ● HLA-D(DR) and DQ: one-way mixed lymphocyte reaction (MLR) with – Homozygous testing cells – Primed lymphocytes (PLT) – Cloned T cells ● HLA-DP: MLR in PLT or with T-cell clones Nucleic-acid-based methods for Class I and II allele identification ● Sequence-specific primer polymerase chain reaction ● Sequence-specific oligonucleotide probe hybridization analysis ● Automated DNA sequencing ● Emerging DNA sequencing techniques

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Sequence-Specific Oligonucleotide Probe Hybridization Relevant regions of the Class I and Class II genes are amplified from genomic DNA by means of the polymerase chain reaction (PCR) with two oligonucleotide primers that anneal to 5⬘ and 3⬘ flanking regions that are conserved (are identical) among individuals. Care must be taken in choosing primers to find ones that are locus-specific (amplify HLA-A but not the related HLA-B locus, for example), that amplify all known alleles at a locus, and that result in roughly equal amplification of the two alleles in a heterozygous individual. After the PCR, the amplified DNA is generally interrogated with allele-specific DNA probes attached to solid supports. Identification of the individual alleles is accomplished by means of hybridization of the PCR products with a series of fluorescently labeled sequencespecific oligonucleotide probes.40,41 Probes are chosen to anneal to amplified regions of the Class I and II genes that vary from allele to allele. Probes with nucleotide sequences perfectly complementary to the amplified DNA hybridize specifically to the PCR product target and can be detected via fluorescence. With careful control of stringency conditions of hybridization and washing, these probes can detect single-base-pair differences, which result in singleamino-acid substitutions in the expressed proteins. Alleles are assigned on the basis of panels of positive and negative hybridization reactions with oligonucleotides specific for a particular allele or sequence. Sequence-specific oligonucleotide probe hybridization (SSOPH) typing is complex because it requires a substantial number of oligonucleotide probes to detect and differentiate among the large number of known Class I and II alleles. However, the development of multiplexed methods for SSOPH employing fluorescent microspheres with attached allele-specific probes has greatly improved the ease of use and efficiency of this method and made it a leading means for HLA typing in many laboratories.42

There is no need for a series of detecting oligonucleotide probes for each allele. Polymerase chain reaction products are prepared that will encompass the relevant polymorphic positions within the Class I or II gene to be sequenced. Sequencing primers are annealed to the PCR products to generate sequencing ladders that are separated on a high-resolution gel. Any known or previously unknown allele in the population can be identified by means of inspection of the DNA sequence or electropherogram. Electropherograms must be of high quality for accurate detection of heterozygous positions in the sequenced PCR products. The development of high-throughput automated fluorescent DNAsequencing machines, analysis software, and capillary electrophoresis systems has made this method less cumbersome. Direct sequencing for HLA typing yields unparalleled precision in allele identification. As the number of known Class I and II alleles continues to escalate, the SSOPH and sequence-specific PCR methods have become increasingly complex, making direct sequencing an increasingly attractive method for laboratories.44-46

Choice of HLA Typing Method Several factors influence a decision to use one HLA typing method over another. Sequence-specific PCR and reverse SSOPH can be performed in a time (4 to 5 hours) appropriate for clinical situations such as cadaveric renal transplantation. DNA sequencing is a reasonable approach for situations such as unrelated marrow transplantation in which allele-level HLA matching of donors and recipients is desired and turnaround times of a few days to a week are acceptable.

Genotypes, Phenotypes, and Haplotypes Sequence-Specific Primer Polymerase Chain Reaction Polymerase chain reaction primers are designed so that their 3⬘-most one or two nucleotides are complementary to base positions within Class I and II genes that differ for different alleles.43 Sequence-specific primers with 3⬘ ends that anneal perfectly to sequences present in a particular allele or allele subgroup result in productive DNA amplification. Other alleles or allele subgroups are not amplified. Gel electrophoresis can then be used to detect the presence or absence of PCR products of the appropriate size, and hybridization assays are avoided. Sequencespecific PCR is most useful for medium-resolution HLA typing and for initial quick identification of broad antigen groups, such as DR52. Then, SSOPH or DNA sequencing can be used to define microvariants of these specificities, if necessary. Sequence-specific PCR requires 100 or more simultaneously performed PCR assays per patient to identify HLA-A, -B, and -DRB allele groups at serologically equivalent resolution. The method requires optimization to avoid failed amplification reactions in each run.

Sequence-Based Typing Sequence-based typing has the advantage of requiring only a limited number of primers for the PCR for each HLA locus.

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HLA antigens are expressed codominantly. The phenotype of expressed antigens for any given person is not equivalent to the genotype. Because HLA genes are closely linked on chromosome 6, they are inherited in units known as haplotypes. Each sibling of a family inherits one of two HLA haplotypes from each parent. Figure 57-4 illustrates the segregation of haplotypes in a family of seven, focusing on the A, B, Cw, and DR serologic specificities. Analysis of the data for a single individual does not allow assignment of the serologic types present to a specific paternally or maternally derived chromosome; that is, the typing data are unphased. Thus, one cannot be certain whether the DR7 in sibling 3 is present on a chromosome carrying B8 or B13. However, analysis of the typing data for the entire family and the transmission of specificities to the children make it possible to phase each of the pairs of parental chromosomes and to determine haplotypes. For example, one of the paternal chromosome-6 pairs, haplotype [a], contains HLA-A1, Cw4, B35, and DR1; the other paternal chromosome, haplotype [b], includes HLA-A3, Cw7, B8, and DR7. Similarly, the maternal chromosome-6 pairs can be separated as haplotypes [c] and [d]. Each child in this family inherits [a] or [b] from the father and [c] or [d] from

Chapter 57: HLA Antigens and Alleles

HLA –

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Haplotype

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Figure 57-4. HLA-A, -B, -C, and -DR antigens segregating as haplotypes in a family. The data reflect the genotypes of each family member as well as the parental haplotypes.

Mother 2

24

Sibling 2

Sibling 3

Sibling 4

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the mother. Each child differs from each parent by one HLA haplotype. Because there can be only four parental haplotypes, the chances are 1 in 4 that the siblings will have same paternalmaternal haplotypes (are HLA-identical), 1 in 2 that siblings will differ by one haplotype, and 1 in 4 that they will differ by two haplotypes. For example, siblings 4 and 5 are HLA-identical, and sibling 4 shares one haplotype with siblings 2 and 3 but no haplotypes with sibling 1. Because siblings 4 and 5 are genetically identical with regard to the MHC, their lymphocytes will not react in mixed leukocyte culture. The difference between phenotypes and genotypes can be clarified even further with sibling 4 (Fig 57-4) as an example. If family data were not available, this sibling’s phenotype would be written as HLA-A3, A24; Cw2, Cw7; B8, B27; DR4, DR7. This implies lack of family data that would allow assignment of the A, C, B, and DR combinations to the appropriate haplotypes. With family data available, however, the genotype is written HLA-A3, Cw7, B8, DR7/A24, Cw2, B27, DR4. The slash in this statement clearly defines the two haplotypes inherited by this child. Although HLA gene loci are closely linked, there is a low frequency of recombination during meiosis. The crossover rate is approximately 0.8% between the A and B loci and approximately 0.5% between the B and DR loci. For example, 1 of every 200 families exhibits recombination between the B and DR genes. HLA allele and haplotype frequencies exhibit ethnic variation. Some alleles and hapotypes are widely distributed around the globe, and others are almost exclusively within a particular ethnic group. In population studies, the phenomenon of linkage disequilibrium is apparent in all groups. This term indicates that in randomly mating populations, the haplotype frequency for

HLA identical; MLR–Negative

two or more linked gene loci is significantly higher than would be expected by chance alone. The expected frequency is obtained from the product of the gene frequencies of the involved genes. For example, among persons of European ancestry, the observed A1, B8 haplotype frequency (7.0%) is approximately 4.4 times greater than the expected haplotype frequency (1.6%). These excess haplotype frequencies may exist for a variety of reasons, including high prevalence in the founders of a population and selective pressure from infectious organisms.47

Medical and Biologic Significance of HLA The HLA system plays a role in several areas of biomedical significance including the following: ● Antigen presentation ● Association with certain diseases ● Organ and stem cell transplantation ● Platelet transfusion ● Population genetics and anthropology. Tracing the appearance of novel HLA alleles in populations and comparing the frequencies of alleles among populations to gain information about human origins and migration in anthropologic and evolutionary studies is a fascinating field of inquiry but beyond the scope of this chapter.48 The extensive polymorphism of the HLA loci has been exploited for many years in identity testing; however, the use of microsatellite loci for this purpose has supplanted the use of HLA loci. The role of HLA Class I typing to support platelet transfusion is described in Chapter 11.

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Transplantation Transplantation poses a special problem in medicine because the extreme polymorphism of the HLA loci make it unlikely that an unrelated donor of cells, tissues, or organs will be matched at an allele level or at a serologic level with a recipient without a concerted effort to identify matched donors and recipients. One solution is to identify an HLA-identical sibling, but many potential transplant recipients do not have access to these donors. For organ transplantation, powerful immunosuppressant drugs have made transplantation feasible with mismatched living and cadaveric donors and recipients. However, better HLA-A, -B, and -DR matching at a serologic level generally increases the half-life of the transplanted organ and decreases overall morbidity.49 Thus cadaveric donor kidneys are shared on a national basis with waiting recipients who have end-stage renal failure and no mismatches.50 Finding well-matched donors of hematopoietic stem cells if there are no HLA-identical siblings is facilitated by resources such as the National Marrow Donor Program, which has enrolled several million potential donors with known HLA types.51 The goal for unrelated stem cell donation is increasingly allele-level matches for HLA-A, -B, -C, and -DRB1, although the level of mismatching permissable is incompletely understood. Subtle allelic mismatches between stem cell donors and recipients appear to increase the risk of severe graft-vs-host disease and decrease overall survival.52,53 The use of umbilical cord blood stem cells and the depletion of T cells from transplanted cells may allow greater donor-recipient mismatches.54,55 Retrospective studies are continuing to assess in a definitive manner the level of matching necessary for successful stem cell transplantation.56 Hematopoietic stem cell transplantation is discussed in Chapters 33 through 36. There are two major mechanisms by which Class I and II donor-recipient mismatches may adversely affect transplantation—production of HLA antibodies and cell-mediated rejection. Transplant recipients become sensitized or produce antibodies directed against Class I and Class II specificities through several routes. Exposure to fetal HLA molecules encoded by paternal haplotypes during pregnancy, especially multiple pregnancies, can lead to the presence of long-lasting, high-titer HLA antibodies. Similarly, mismatched HLA antigens from previous organ donors and donors of transfused blood components can lead to sensitization. The presence of recipient preformed HLA antibodies may lead to hyperacute rejection of a donor organ bearing the relevant HLA specificities.26 Therefore, laboratories maintain serum screening programs to detect and identify HLA antibodies in waiting recipients so that inappropriate donors can be avoided. Immediately before transplantation, crossmatches to detect donor-directed HLA antibodies are performed between donor lymphocytes and recipient serum by means of the microcytotoxicity assays described earlier or more sensitive flow cytometric technology.57 Donor HLA antigens mismatched with recipient HLA antigens can elicit a strong cell-mediated immune response. As many as 10% of a recipient’s T cells are able to recognize and respond to donor-mismatched HLA antigen.58 These responses

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contribute to acute rejection in organ transplantation and graftvs-host disease in stem cell transplantation. HLA matching and immunosuppressive therapy help to mitigate this problem.

Disease Association The association of certain HLA alleles and their encoded antigens with particular diseases, especially autoimmune disorders, has been known for some time. In more than 40 disorders, there is significant deviation in the frequency of HLA antigens from that of healthy controls (Table 57-4).59 In general, HLA-associated diseases have certain common features. They are known or suspected to have an inherited component, usually have autoimmune features, and display a clinical course often featuring repeated acute relapses followed by remission. For most HLA-associated diseases, the etiologic factor is unknown and the pathophysiologic mechanism is incompletely understood. One hypothesis for the link between HLA antigens and disease suggests that a necessary but not sufficient requirement for disease development is the differential ability of a Class I or II heterodimer encoded by a specific allele to present autoantigens to the T-cell receptor. Two of the strongest HLA associations are the DQB1*0602 allele with narcolepsy60 and B*27 alleles with ankylosing spondylitis.59 Almost all patients with narcolepsy have the associated alleles; approximately 90% of patients with ankylosing spondylitis have the associated alleles. In populations without these disorders, the frequencies of DQB1*0602 (25% to 30%) and of the B*27 group (5% to 10%) are substantial but do not approach the frequencies among persons with these disorders. Thus the antigens involved are not unique to narcolepsy and ankylosing spondylitis but are overrepresented among affected persons. Approximately 3% of persons with a B*27 allele have ankylosing spondylitis, a risk approximately 100-fold greater than that among persons without B*27. Results of HLA testing that show the absence of DQB1*0602 or B*27 alleles is useful to help rule out a diagnosis of narcolepsy or ankylosing spondylitis for a patient. Results that show the presence of these alleles are less useful because of the prevalence of these alleles in the general population. The association of HLA antigens with most other autoimmune disorders usually does not carry the high relative risk that narcolepsy and ankylosing spondylitis do. Although these HLA associations are generally less useful in diagnosis, they provide important insights into the pathophysiologic mechanism of these diseases and may help assess prognosis. For example, approximately 95% of patients with type 1 diabetes mellitus (formerly known as insulin-dependent diabetes mellitus) have either the DRB1*03 or the DRB1*04 allele or both.61 Patients heterozygous for DRB1*03/*04 have a relative risk for type 1 diabetes mellitus three to six times greater than that of patients with DRB1*03 or DRB1*04 alone or in combination with another DRB1 allele. Susceptibility to type 1 diabetes mellitus appears to be linked to haplotypes containing DRB1*03 or DRB1*04 with DQB1*02 and DQB1*03 alleles. Many persons with rheumatoid arthritis have inherited DRB1*04 alleles encoding a common epitope. Heterozygosity

Chapter 57: HLA Antigens and Alleles

Table 57-4. Selected Diseases Associated with HLA Alleles Disorder

HLA Linkage

Comments

Ankylosing spondylitis

B*27 alleles

Narcolepsy

DQB1*0602

Type 1 diabetes mellitus Rheumatoid arthritis

DRB1*03/*04, DQB1*02, and *03 alleles DRB1*04 alleles sharing epitope encoded by codons 67-74 Multiple loci and alleles

Absence of B*27 may be useful in ruling out diagnosis of ankylosing spondylitis DQB1 locus or linked loci and alterations in hypocretin or orexin peptides/receptors contribute to narcolepsy Presence of DRB1*03/*04 confers a 25-fold relative risk May be most useful in predicting severity of disease

Class I and II Associations

Latency period before onset of AIDS

Abacavir hypersensitivity B*5701 Mutation of Nonimmune Response Genes within the MHC 21-Hydroxlase deficiency Complement component 2 (C2) deficiency Hemochromatosis

Diagnosis by direct CYP21 genotyping Diagnosis by assessment of complement levels Diagnosis by direct HFE genotyping

and homozygosity for alleles encoding this epitope may be predictive of a more severe course of arthritis. Thus allele identification may be helpful in assessing prognosis.62 A number of studies have been performed in an attempt to define specific HLA alleles that confer susceptibility or resistance to AIDS (Table 57-4).63,64 There appear to be HLA alleles that interact with other genetic and environmental factors to influence the outcome of this multifactorial disease. However, results may vary somewhat among studies given the complexity of AIDS, differences in the ethnicity of the populations studied, and the subtypes of the human immunodeficiency virus type 1 involved. Recently, hypersensitivity to the antiretroviral drug abacavir was linked to the presence of HLAB*5701 in treated patients.65,66 Near complete elimination of hypersensitivity morbidity was achieved by first screening patients to exclude the 5% to 10% with B*5701 from exposure to this agent. In this example, HLA allele identification serves as a pharmacogenomic test to stratify a patient population for targeted therapy. Because the MHC contains genes with no obvious role in immune system function, some diseases caused by point mutation or deletion of genes in this region cause disorders not directly related to immunity. However, because of linkage disequilibrium, overrepresentation of specific Class I or Class II antigens may occur in these disorders. Congenital adrenal hyperplasia is caused by mutations in the MHC Class III region genes encoding 21-hydroxylase.67 Although linkage to specific HLA alleles is present in persons with congenital adrenal hyperplasia, direct genotyping is generally preferable for diagnostic purposes. Similarly, genetic hemochromatosis is caused by mutations in the HFE gene telomeric to the HLA-A locus.24 Although the presence of A*03 confers several-fold excess risk of hemochromatosis, the most direct route to genetic diagnosis is HFE genotyping.

Latency period shortened by homozygosity at loci and modified by multiple susceptibility and resistance alleles Abacavir hypersensitivity largely a result of inheritance of B*5701 Linked to cosegregating HLA haplotypes in families HFE is a Class-I-related gene at the telomeric end of the MHC

Summary The HLA loci on chromosome 6 are composed of a series of Class I and II genes that display a degree of naturally occurring polymorphism unmatched within the rest of the human genome. This polymorphism appears to be maintained to ensure appropriate responses to the infectious organisms encountered by global human populations. The crucial role of the Class I and II molecules is to bind to the degradation products of proteins and present them to T-cell antigen receptors for recognition as endogenous or foreign peptides. MHC polymorphism has several consequences in medicine and biology beyond the normal immune response: 1) MHC genetic variation can be exploited as a tool for anthropologic study of human migration and development. 2) Specific HLA alleles are associated with a propensity to development of particular disease entities, especially autoimmune disorders. 3) The challenge of finding HLA-matched donors and recipients presents special problems in stem cell and organ transplantation.

Disclaimer The author has disclosed no conflicts of interest.

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49. 50.

51.

52.

53.

54.

to serological DR typing in clinical practice including donorrecipient matching in cadaveric transplantation. Tissue Antigens 1992;39:225-35. Wu J, Griffith BB, Bassinger S, et al. Strategies for unambiguous detection of allelic heterozygosity via direct DNA sequencing of PCR products: Application to the HLA DRB1 locus. Mol Diagn 1996;1:89-98. Versluis LF, Rozumuller EH, Duran K, et al. Ambiguous DPB1 allele combinations resolved by direct sequencing of selectively amplified alleles. Tissue Antigens 1995;46:345-9. Turner S, Ellexson ME, Hickman HD, et al. Sequence-based typing provides a new look at HLA-C diversity. J Immunol 1998;161: 1406-13. Alper CA, Awdeh Z, Yunis EJ. Conserved, extended MHC haplotypes. Exp Clin Immunogenet 1992;9:58-71. Mack SJ, Erlich HA. HLA Class II polymorphism in the Ticuna of Brazil: Evolutionary implications of the DRB1*0807 allele. Tissue Antigens 1998;51:41-50. Suthanthiran M, Strom TB. Renal transplantation. N Engl J Med 1994;331:365-76. Takemoto SK, Terasaki PI, Gjertson DW, et al. Twelve years’ experience with national sharing of HLA-matched cadaveric kidneys for transplantation. N Engl J Med 2000;343:1078-84. Kernan NA, Bartsch G, Ash RC, et al. Analysis of 462 transplantations from unrelated donors facilitated by the National Marrow Donor Program. N Engl J Med 1993;328:593-602. Flomenberg N, Baxter-Lowe LA, Confer D, et al. Impact of class I and class II high resolution matching on outcomes of unrelated donor bone marrow transplantation: HLA-C mismatching is associated with a strong adverse effect on transplant outcome. Blood 2004;104:1923-30. Lee SJ, Klein J, Haagenson M, et al. High resolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplantation. Blood 2007; 110:4576-83. Kurtzberg J, Laughlin M, Graham ML, et al. Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. N Engl J Med 1996;335:157-66.

55. Aversa F, Tabilio A, Velardi A. Treatment of high-risk acute leukemia with T-cell-depleted stem cells from related donors with one fully mismatched HLA haplotype. N Engl J Med 1998;339: 1186-93. 56. Hurley CK, Fernandez-Vina M, Hildebrand WH, et al. A high degree of HLA disparity arises from limited allelic diversity: Analysis of 1775 unrelated bone marrow transplant donor-recipient pairs. Hum Immunol 2007;68:30-40. 57. Gebel HM, Bray RA. Sensitization and sensitivity: Defining the unsensitized patient. Transplantation 2000;69:1370-4. 58. Murphy KM, Travers P, Walport M. Antigen presentation to T lymphocytes. In: Janeway’s immunobiology. 7th ed. New York: Garland Science, 2007:181-218. 59. Lechler R, ed. HLA and disease. London: Academic Press, 1994. 60. Pelin Z, Guilleminault C, Risch N, et al. HLA-DQB1*0602 homozygosity increases relative risk for narcolepsy but not disease severity in two ethnic groups. US Modafinil in Narcolepsy Multicenter Study Group. Tissue Antigens 1998;51:96-100. 61. Aitman TJ, Todd JA. Molecular genetics of diabetes mellitus. Baillieres Clin Endocrinol Metab 1995;9:631-56. 62. Weyand CM, Hicok KC, Conn DL, et al. The influence of HLADRB1 genes on disease severity in rheumatoid arthritis. Ann Intern Med 1992;117:801-6. 63. Carrington M, Nelson GW, Martin MP, et al. HLA and HIV-1: Heterozygote advantage and B*35-Cw*04 disadvantage. Science 1999;283:1748-52. 64. Hendel H, Caillat-Zucman S, Lebuanec H, et al. New Class I and II HLA alleles strongly associated with opposite patterns of progression to AIDS. J Immunol 1999;162:6942-6. 65. Mallal S, Nolan D, Witt C, et al. Association between presence of HLA-B*5701, HLA-DR7, and HLA-DQ3 and hypersensitivity reactions to HIV-1 reverse transcriptase inhibitor abacavir. Lancet 2002;359:727-32. 66. Mallal S, Phillips E, Carosi G, et al. HLA-B*5701 screening for hypersensitivity to abacavir. N Engl J Med 2008;358:568-79. 67. White PC, Speiser PW. Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr Rev 2000;21:245-91.

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58

Tissue Banking Jeanne V. Linden,1 William F. Zaloga,2 & A. Bradley Eisenbrey3 1

Director, Blood and Tissue Resources, Wadsworth Center, New York State Department of Health, Clinical Associate Professor, Albany Medical College, and Adjunct Associate Professor, School of Public Health, State University of New York at Albany, Albany, New York, USA 2 Associate Director, Blood and Tissue Resources, Wadsworth Center, New York State Department of Health, and Adjunct Assistant Professor, Albany Medical College, Albany, New York, USA 3 Laboratory Director, Gift of Life Michigan, Ann Arbor, Michigan, and Assistant Professor of Pathology, Wayne State University School of Medicine, Detroit, Michigan, USA

In the United States, organ and tissue procurement has been organized in such a way so as to maximize availability in an efficient fashion that is fair to both donor and recipient. Most of the organ procurement efforts have become systematized and organized under federal mandate; there is, however, a growing cooperation between organ procurement organizations (OPOs) and tissue recovery organizations to facilitate meeting the requirements of both organ and tissue procurement. Successful tissue preservation has encouraged the use of bone and soft tissue grafts, cardiovascular tissue, and skin in the clinical setting. Semen cryobanking has been practiced for many years, and the demand for embryo banking services has greatly increased recently as assisted reproductive procedures have become more diverse and much more widely available. As technology in this area continues to expand, blood banks may play an increasingly important role in coordinating, supporting, and directing transplantation and transplant-related interventions.

Growth of Tissue Banking Each year, hundreds of thousands of patients benefit from donated bone, cartilage, and tendon used to reconstruct and rehabilitate joints and other bony structures, corneas to restore sight, skin to treat burns, veins and great vessels to restore blood flow, heart valves to restore cardiac function, and reproductive tissue to treat infertility. In addition to whole grafts and sections, bone, in particular, can be machined into specialized products to conform with manufactured support constructs. It can also be ground into demineralized bone matrix (DBM), which is available as a ground powder or as combination forms such as gels, pastes, strips, and even moldable grafts designed to avoid migration in specific applications. Myriad types of tissues are transplanted in a variety of medical and dental specialties for

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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diverse clinical applications (Table 58-1).1 The most commonly transplanted allografts are bone and cornea (Table 58-2). The total number and range of tissues collected, processed, and transplanted in the United States are difficult to determine, because there is no national reporting system. In 1999, tissue banks accredited by the American Association of Tissue Banks (AATB) distributed approximately 750,000 allografts for transplantation. By 2003, the figure had more than doubled. The American Orthopaedic Society for Sports Medicine reported that its members used more than 60,000 allografts in knee surgery alone, in 2005.2 The quantity of musculoskeletal tissue allografts used annually is approximately 1,500,000. Cornea transplants numbered ⬎45,000 in 2006.2 Skin is distributed at the rate of about 17,000 square feet annually. Transplantation of heart valves is now estimated at ⬎2500 annually. These procedures require annual donation of cardiovascular tissue, musculoskeletal tissue, and skin from ⬎40,000 deceased donors (New York State Department of Health, unpublished data, 2005), and donation of eyes from ⬎38,500 deceased donors.3 In addition to such tissues from deceased donors, many tissues for clinical use are derived from living donors, including semen and oocytes for use in artificial insemination and assisted reproductive technology procedures. In 2005, New York State-licensed semen banks located throughout the United States processed 27,118 ejaculates from 962 semen donors. In addition, semen from 16,347 client depositors was collected and cryopreserved for later use by the client depositor’s wife or other “intimate partner” (New York State Department of Health, unpublished data, 2005). Donor oocytes were used in approximately 12% of all assisted reproductive technology cycles carried out in the United States in 2005.4 Tissue from living donors is also considered by some to include human milk to nourish low-birthweight, premature infants. The Human Milk Banking Association of North America reports the existence of 11 active member banks.5 The Food and Drug Administration (FDA), however, does not consider human milk to be subject to regulations that apply to human allograft tissue. Because no comprehensive national usage figures are available, it is difficult to determine the rate of increase in the demand for

Chapter 58: Tissue Banking

Table 58-1. Some Common Human Allograft Applications, by Specialty1 Specialty

Procedure/Application

Allograft Types Used

General orthopedics

Trauma/fracture repair; osseous defect repair; acetabular repair; total joint revision/arthroplasty

Femoral head; femoral condyle; whole, proximal, or distal bone shaft (femur, tibia, humerus); hemi-pelvis; cancellous bone; corticocancellous bone; cortical strut/screw/pin; bi-cortical strip; tri-cortical wedge; whole joint* (knee, ankle, shoulder, elbow); osteoarticular graft*; osteochondral graft*; DBM; osteobiologics

Sports medicine

Tendon, anterior cruciate ligament, posterior cruciate ligament, other knee ligament repair; meniscus repair/ replacement; osteochondral defects; rotator cuff repair; ankle/tendon ligament repair

Patellar ligament; Achilles tendon; tibialis tendon; semitendinosus tendon; gracillis tendon; peroneous longus tendon; fascia lata; rotator cuff; meniscus; meniscus w/tibial plateau; osteochondral plug*; femoral hemi-condyle*; acellular dermal matrix

Craniofacial/maxillofacial

Cranial reconstruction; maxillary/mandibular reconstruction; facial palsy repair

Mandible; dura mater*; pericardium; fascia lata; acellular dermal matrix; bi-cortical strip; tri-cortical wedge; DBM; osteobiologics; sclera

Dental

Alveolar ridge augmentation for dental implant placement; onlay grafting; sinus elevations/augmentation; socket/ridge preservation; intrabony defect repair

DBM; osteobiologics; sclera; acellular dermal matrix

Ophthalmology

Postcataract corneal edema repair; Fuchs dystrophy repair; glaucoma drainage valve implantation; corneo-scleral fistula repair; keratoconus correction; phaco burn repair; orbital reconstruction following enucleation; eyelid ectropion repair; eyelid reconstruction

Cornea*; sclera*; pericardium

Neurosurgical

Cervical/lumbar interbody fusion; intermedullary rod placement; dura replacement

Dura mater*; fascia lata; pericardium; cancellous bone; corticocancellous bone; cortical strut; bi-cortical strip; various machined and constructed proprietary bone forms*; amniotic membrane*; acellular dermal matrix

Burn treatment

Wound covering†

Fresh skin*; cryopreserved skin*; freeze-dried skin*; acellular dermal matrix; amniotic membrane*

General surgery

Urologic incontinence procedure; pelvic floor reconstruction; herniorrhaphy

Fascia lata; pericardium; lyophilized skin*; acellular dermal matrix

Cardiac

Congenital anomaly repair (both valve and outflow tract major vessel repair/replacement†); cardiac valve and vessel repair/ replacement; major vessel blood “shunting” procedures

Aortic valve*; pulmonary valve*; various conduit-use-only grafts* from the ascending aorta or thoracic aorta or from the pulmonary artery trunk or its branches

Vascular

Vaso-occlusive disease (peripheral, abdominal, thoracic); cardiac artery bypass grafting; arteriovenous shunt insertion; muscle-flap or organ transplant vascular bed extensions; replacement of infected prosthetic devices

Greater saphenous vein*; aorto-iliac artery*; iliac vein*; iliac artery*

*

Cannot be sterilized. Can be life saving. DBM ⫽ dimineralized bone matrix.

†

tissues. However, novel uses for tissue in transplantation have emerged that had not been envisioned even a few years ago. Amniotic membrane is transplanted to correct epithelial eye defects; bioengineered skin products are used for wound healing; and nerve tissue is transplanted to serve as a conduit for nerve regeneration in damaged limbs. In late 1998, the medical world was fascinated by the first hand transplant surgery; there have been at least 18 worldwide since, and the world’s first face transplant procedure was performed in November 2005. In the future, human pluripotent progenitor cells from embryos may be used in the treatment of Parkinson disease, diabetes, and spinal cord injuries.

Tissue Donation Living Donors The tissue most commonly donated by living donors is blood, the primary subject of this book, but living donors also provide other tissues. Tissue donation by a living person generally is limited to renewable tissue, such as gametes, extraembryonic tissue, and milk. Except for autografts, which can be expanded by culturing for use on burned patients, skin is usually recovered from deceased donors. However, some tissue banks have

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Table 58-2. Allogeneic Tissue Distributed for Clinical Use Annually in the United States Tissue Deceased Donor Tissue Musculoskeletal tissue* Cornea† Pericardium* Tendon* Skin (sq ft)* Fascia* Heart valves‡ Vessels‡ Living Donor Tissue*‡ Semen (vials)‡ Oocyte/embryos§ Amniotic membrane‡

Units (Estimated)

1,500,000 47,067 11,304 32,441 16,597 16,754 2,750 3,000 ⬎100,000 35,185 2,198

*American Association of Tissue Banks. Annual survey of accredited tissue banks. McLean, VA: American Association of Tissue Banks, 2003. † Eye Bank Association of America. 2006 Eye banking statistical report. Washington, DC: Eye Bank Association of America, 2007. ‡ New York State Department of Health (NYSDOH). Unpublished projected data based on 74,366 semen vials/straws distributed by New York State-licensed tissue banks in 2005. (NYSDOH licenses semen banks located nationwide.) § Centers for Disease Control and Prevention; American Society for Reproductive Medicine; Society for Assisted Reproductive Technology. 2005 Assisted reproductive technology success rates: National summary and fertility clinic reports. Atlanta, GA: Centers for Disease Control and Prevention; 2007.

developed processes to promote living donor skin donation following extensive weight loss (⬎300 such donations per year in the United States). Cartilage can also be cultured for autologous transplant in knee repair. Bone can be obtained from living donors in the form of femoral heads and tibial plateaus that are removed and would otherwise be discarded during surgical procedures (total hip and knee replacements with prostheses). Outside the United States, such surgical bone banking remains a significant source of bone grafts in many countries.

Deceased Donors In contrast to organ donors, tissue donors (excluding living donors, of course) do not need to have functioning circulation. Tissues such as bone, eyes, and skin can generally be collected up to 24 hours after cessation of the donor’s cardiac and respiratory functions, depending on the temperature and environment in which the donor body is stored. Tissues such as bone and skin can be donated by deceased organ donors (a standard criterion for organ donors in the United States is brain death), but more commonly, tissues are donated by other hospitalized patients who have been judged deceased by both cardiorespiratory and neurologic criteria. Recently, tissue procurement practices have become more effective as a result of increased cooperation between tissue banks and OPOs, through routine referral requirements6 and statutes (eg, New York State Public Health

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Law §4351). Referrals for eye banks are also made through OPOs. However, many tissue banks, especially eye banks, obtain a significant portion of their referrals from medical examiners or coroners who have access to large numbers of young, healthy individuals who died suddenly and never reached a hospital.3

Routine Referral for Donation The availability of tissues for transplantation depends on strong public support, the presence of laws that clear legal barriers to donation and that authorize consent before death (through such means as driver’s licenses, donor registries, and advance directives), and the availability of health professionals trained in how to approach the next of kin on the subject of donation. (The next of kin is/are a deceased person’s closest relative(s), as determined by a hierarchy specified in state law.) Key to donation in the United States is a spirit of altruism and volunteerism, coupled with autonomous choice being valued. Consent to donate is informed and voluntary with few exceptions. About one-third of eye banks are located in states with medical examiner laws that allow removal of corneas for transplantation under the following circumstances: 1) the body is under the medical examiner’s jurisdiction; 2) autopsy is required by law; 3) there is no known objection by the next of kin; and 4) the removal will not disturb the body’s appearance.3,7,8 These state laws presume consent and permit removal of eyes without an interview of the next of kin concerning the deceased’s medical and social history, including human immunodeficiency virus (HIV) and hepatitis risk behaviors. Maryland expanded the law to cover non-medicalexaminer cases when a next of kin cannot be found.9 In 2006, ⬎34,000 corneas were provided for transplantation; about 10% of these were obtained through use of a state’s medical examiner law. Nationwide the number of corneas obtained under medical examiner laws has been declining, mainly because of ethical concerns, but also because of increased FDA regulation that requires specific screening for relevant communicable diseases, which must include a donor medical and social history interview with a knowledgeable historian.3,7 In the late 1980s, most states enacted “required request” laws that sought to increase the supply of organs and tissues by requiring that hospital personnel request permission of the next of kin for organ and tissue donation at the time of a patient’s death (if the prospective donor was medically eligible). Following findings that families were more likely to consent to donation if approached by requesters who had received training in the most effective ways to present donation opportunities, some states amended these laws to require referral of all deaths to an OPO or designated tissue bank, whose specially trained requesters would then ask for consent. Federal routine referral requirements were implemented by the Health Care Financing Administration (now the Centers for Medicare and Medicaid Services) in Hospital Conditions of Participation for Organ, Tissues, and Eye Donation, which became effective August 21, 1998.6 These rules mandate, as a condition of Medicaid and Medicare reimbursement, that all hospitals establish formal relationships with an

Chapter 58: Tissue Banking

OPO in the service area, with a tissue bank, and with an eye bank. Further, the rules require that hospital personnel notify OPOs of all deaths and imminent deaths so that potential donors are identified, and so that the next of kin will be approached about donation, when appropriate. The effect of these routine referral laws is not clear. In New York, where such a law went into effect on January 1, 1998, mixed results have been reported. Although a small increase in tissue donation has been realized statewide, certain metropolitan areas have actually reported a decline in eye donation. It is also not clear whether the increase in tissue donation in recent years is the result of required referral or of enhanced advertising campaigns and increased public awareness of the need for organ and tissue donation. Under routine referral, the costs associated with around-the-clock reporting of deaths and imminent deaths are proving to be high in certain areas, although the cost to OPOs is shared by Medicare and the transplanting hospitals through “standard acquisition charges” applied to individual organs transplanted. Uniform Anatomical Gift Laws were developed to standardize state requirements for first party consent, requesting personnel, and recovery. A 2006 revised version, which expanded definitions, clarified roles, and enhanced a focus on personal autonomy, had been enacted in 20 states and had been introduced in 15 more by early 2008 (see http://www.anatomicalgiftact.org).

Organization of Tissue Banking in the United States Unlike the organ procurement and sharing system, tissue banking is not formally organized based on geography. There are approximately 15 independent tissue banks that process conventional musculoskeletal tissue, four that process cardiovascular tissue, and 13 that process skin. Although most are not for profit, a few for-profit companies process human tissue for transplantation. These processors usually independently perform donor eligibility assessment, including laboratory testing, even if the tissue bank that recovered the tissue had already performed such an assessment. An unpublished AATB survey of 2003 activity found that donor eligibility had been determined by a tissue bank accredited by AATB for about 95% of the bone grafts distributed (Scott Brubaker, personal communication, August 17, 2007). There are approximately 89 not-for-profit eye banks accredited by the Eye Bank Association of America (EBAA; see http://www.restoresight.org). Almost all cornea allografts are provided by these eye banks. Blood centers with expertise in donor recruitment, donor eligibility determination, cell cryopreservation, and compliance with standards and regulations are good candidates to undertake tissue banking services. Some blood centers have already taken on the challenge of providing comprehensive tissue bank services, and they recover, process, store, and distribute tissues. Procedures to achieve allograft safety and efficacy are guided by national professional standards set by such organizations as AATB,10 EBAA,11 and the American Society for Reproductive

Medicine.12 The FDA and some states, including New York, Florida, and California, have regulatory and licensure requirements (see section on oversight of tissue banks below).

Public Attitudes Public altruism and the willingness of people to be organ and tissue donors form the foundation of the transplant system. A 1986 Gallup survey assessing the public’s attitudes found that people were more willing to consent to donation if they were provided with relevant information. Effective public education is thus a key component of successful organ and tissue donation programs. A major impediment to the procurement of greater numbers of transplantable organs and tissues has been the failure of health-care professionals to adequately present the next of kin the option of organ and tissue donation.9 Grieving relatives of a potential donor may not spontaneously consider donation, but they should gently be offered the opportunity by persons trained to make organ and/or tissue donation presentations (“donation approach”). The donation approach is much more effective when “decoupled” from the care of the dying or deceased by having a person separate from the patient care team make the approach. This decoupling also reduces the risk of or the perception of a conflict of interest between patient care and organ and tissue recovery. The situation has improved gradually through professional education and encouragement by federal and state legislation and initiatives. The Department of Health and Human Services Office of the Inspector General (OIG) conducted an investigation of some tissue bank practices. In a 2001 report, the OIG recommended that tissue banks make their finances public and enhance consent processes by describing potential uses of donated tissue and disclosing the bank’s relationships with other companies.13 The OIG also recommended that the FDA expedite the publication of its proposed regulatory agenda for tissue banks. Media attention to organ and tissue transplantation has increased the recognition and credibility of OPOs, as well as the public and professional support for donation. Some media inquiries, such as the Orange County Register’s April 2000 report entitled “The Body Brokers,” and a number of 2006 reports regarding alleged theft and forgery in tissue donation consent and recovery, may have served to shake the public’s confidence that donated tissues are handled with respect and do not result in excessive corporate profits. Such articles triggered a national debate on the ethics of procuring and processing tissue for profit. Questions were raised about the propriety of financial gain from tissue donation, possible diversion of tissue needed for lifesaving purposes to cosmetic uses, weaknesses of the consent process, and the extent to which tissue banks were monitored. There is evidence that the industry responded to such concerns. For example, one criticism voiced was that skin needed for medically necessary reconstruction was sometimes diverted for cosmetic surgical uses, potentially creating shortages of fresh

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Table 58-3. Infectious Diseases Reported to Have Been Transmitted by Deceased Donor Allografts Allografts

Infectious Disease

Bone

Hepatitis C Hepatitis, unspecified type Human immunodeficiency virus, type 1 Bacteria Tuberculosis Fungus

Cornea

Hepatitis B Rabies Creutzfeldt-Jakob disease Cytomegalovirus (?) Bacteria Fungus

Dura mater

Creutzfeldt-Jakob disease

Heart valve

Hepatitis B Tuberculosis Fungus

Skin

Bacteria Cytomegalovirus (?) Human immunodeficiency virus, type 1 (?)

Pericardium

Creutzfeldt-Jakob disease Bacteria

Pancreatic islet

Bacteria

Adapted from Eastlund.14,15

skin for burn patients. Today donated human skin is routinely recovered using a thin and thick method (thin for burn patients and thick to provide collagen), which is balanced to ensure that availability of skin for burn patients is not compromised. Additionally, research has resulted in development of clinically useful synthetic products that fill most of the needs for dermal components of products for use in cosmetic procedures.

Tissue Transplant-Transmissible Diseases and Prevention Transmissible Diseases Despite a careful donor selection process, the risk of donorto-recipient transmission of viral, bacterial, fungal, and prion diseases cannot be eliminated (Table 58-3).14-16 In one wellpublicized case, 48 organ or tissue recipients received an organ or tissue from a single donor who, although he had no risk for HIV infection found by history, proved to have been recently infected with HIV and in the window period before HIV-1 antibody could be detected by the assays in use at the time (October 1985).17 All four organ recipients became infected with HIV, but the majority of tissue recipients did not. Whole unprocessed frozen bone did transmit HIV to three recipients, while bone from which the marrow had been removed did not; transplanted

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corneas, lyophilized soft tissue, and γ-irradiated dura mater also did not transmit the virus. This case additionally served to highlight vulnerabilities associated with inadequate disposition records, given that six recipients could not be identified from hospital records. In 2002, before routine implementation of hepatitis C virus (HCV) nucleic acid testing (NAT) for tissue donors, tissue from a man with no identifiable infectious disease risk by history or physical examination, and a negative test for antiHCV, was found to have transmitted HCV to recipients.18 All organ recipients who could subsequently be tested were found to be infected with HCV. Among 32 tissue recipients, five probable cases occurred: one of two saphenous vein recipients, one of three tendon recipients, and one of three recipients of tendon with bone allografts. All eight recipients whose infection was linked to the transplant were determined to be infected with the same HCV genotype. The current risk of viral transmission is thought to be exceedingly low.19,20 In addition to donor selection and testing strategies, processing methods now in use further reduce the risk for tissues not requiring viable cells. West Nile virus transmission linked to breastfeeding of a woman’s own infant, to organ transplantation, and to blood transfusion have been reported,21 but transmission through donor tissue has not been documented. Chagas’ disease has been linked to transplantation of organs but transmission of parasitic diseases through vascular tissue allografts (considered likely to be possible) has not been reported.22,23 Although vessel grafts associated with organ transplantation, in which arteries or veins from a different donor may be used if the donor and/or recipient vessels are damaged or insufficient, are not considered tissue by the FDA, such grafts caused documented transmission of rabies to two recipients in 2004.24 Transmission of malignancy via tissue transplantation has not been reported, but is thought to be possible. Infectious diseases can also be transmitted through transplantation of tissue from living donors. Cases are well documented in the semen banking arena (Table 58-4),25 as well as in the use of surgical discard bone, such as a femoral head collected during hip arthroplasty.15 Transmission of bacteria is a known risk of tissue transplantation. Bacteria can be present in the donor, either as a normal occurrence or as the result of a disease process or medical intervention.16 Resuscitation efforts can increase the dispersion of such organisms. In addition, agonal bacteremia is a well-known process whereby endogenous bacteria, such as normal intestinal flora, begin to disperse throughout the body after cessation of cardiopulmonary functions, as the putrefaction process begins. This process is accelerated in persons with sepsis, rhabdomyolysis, or a cocaine overdose before death.39 Bacteria can be introduced during tissue recovery; during processing, whether through cross contamination, insufficient aseptic technique, or contaminated chemicals or solutions; or even during packaging. Some tissues, including ocular tissue, skin, and semen, are inherently not sterile even if collected aseptically; fortunately, the contaminants are primarily skin contaminants with low virulence in most recipients, and are susceptible to standard antibiotics.

Chapter 58: Tissue Banking

Table 58-4. Transmission of Infectious Diseases to Semen Recipients Reference

Agents and Diseases

No. of Reported Cases/ No. of Women Exposed*

Transmissibility

Stewart et al26 Chiasson et al27 Rekart28 Mascola et al29

HIV-1 (donor) HIV-1 (donor) HIV-1 (donor) HIV-1 (donor)

CDC30 Berry et al31

HIV-1 (husband-wife) Hepatitis B virus Hepatitis C virus Gonorrhea Gonorrhea? Ureaplasma urealyticum Mycoplasma hominis Trichomonas vaginalis HSV-2 Chlamydia trachomatis Group B streptococcus Cytomegalovirus Human papilloma virus HTLV-I Syphilis

4/8 1/134 2/24 1/89 2/46 1/10 1/1 1/1 0 1/1 3/unknown 1/1 1/1 2/2 1/2 1/1 2/2 0 0 0 0

Yes Yes Yes Yes Yes Yes Yes Yes Likely Yes Yes Yes Yes Yes Yes Yes Yes Possible Possible Likely Possible

Fiumara32 Hansen et al33 Barwin34 Caspi et al35 Kleegman36 Moore et al37 Nagel et al38 Kleegman36

*

Number tested, where applicable.

Used with permission from Linden and Crister.25 HIV-1 ⫽ human immunodeficiency virus, type 1; HSV-2 ⫽ herpes simplex virus, type 2; HTLV-I ⫽ human T-cell lymphotropic virus, type I.

At particular risk for transmission of bacteria are the articular cartilage allografts used in knee surgery, because these cannot be subjected to extensive processing if their mechanical properties and chondrocyte viability are to be preserved (note, however, that the latter has not been proven to be essential). In 2001, a 23-yearold man was found to have died from Clostridium sordellii sepsis following receipt of a femoral condyle allograft. An extensive investigation identified 14 patients who had received allografts between 1998 and 2002 and who developed postoperative infections with Clostridium species.40 The tissues were derived from nine donors, but all were prepared by the same processor, and investigation identified several factors that could have contributed to the infections. Although the tissues were cultured, they had already been suspended in antimicrobial solutions, likely leading to false-negative results. Additionally, for two of the donors, including the donor of the tissue that was implicated in the fatal case, the interval between death and refrigeration of the body (19 hours for the donor in the fatal case) exceeded the industry’s voluntary standards at the time, likely permitting excessive bacterial proliferation. Tissue was processed aseptically without the use of disinfection or terminal sterilization, and the processing methods used had not been validated. Human error can also lead to release of contaminated tissues. In one case, a technician failed to follow standard procedures, resulting in release of tissue labeled as

having been subjected to irradiation when, in fact, no irradiation had occurred.41 However, a wound infection in a tissue recipient, or even disseminated bacteremia, does not necessarily implicate the donor tissue. Site infections are a well-known risk of many surgical procedures and can result from the use of contaminated solutions or equipment insufficiently sterilized between procedures.42

Risk Reduction Procedures After death has been declared and consent for donation has been given, the process of determining the suitability of the potential deceased donor begins. The risk of disease transmission is minimized by collection and careful review of 1) information obtained during interviews with family member(s) or other knowledgeable historian(s) and health-care providers; 2) available medical records; 3) findings of a physical assessment; 4) results of an autopsy, if performed; and 5) results of blood tests for infectious disease markers. Procedures for processing and, in some cases, sterilizing tissues also contribute to tissue safety.

Donor History Screening Evaluation of the health and behavioral risk history of the prospective donor is an important step in establishing the suitability

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of tissues for transplantation and preventing the transmission of infectious diseases. Family members may be able to provide reliable medical and behavior information, but friends and associates may provide relevant information not known to family members, especially the legal next of kin. Medical personnel may also be aware of significant medical and risk information. General donor eligibility criteria include the absence of systemic infection or any infectious or malignant disease transmissible by tissues and of behavioral risks for HIV infection or viral hepatitis. Malignancy generally disqualifies the donor, unless the malignancy is nonmetastatic or not known to metastasize to the tissue to be recovered (eg, virtually no cancer is known to metastasize to the eye or skin), and there is no suspicion of direct regional spread. The donor history review is specifically designed to reject those at high risk for viral hepatitis or HIV (eg, nonmedical injected drug users; men who have had sex with other men; persons who exchanged sex for money or drugs; persons with hemophilia or related clotting disorders; and persons with recent significant incarceration or with symptoms suggestive of a current viral infection).10 Beyond the general selection criteria for donors, there are specific eligibility criteria for each tissue type, to facilitate selection of tissues that will function adequately, as well as those that will not transmit disease. For bone and soft tissue, these include such factors as: donor age, for bone that is intended to be used for weight-bearing functions; no evidence of significant metabolic bone disease or a connective tissue disorder; and no exposure to toxic substances that could accumulate in the tissue to be recovered. Donors of cardiac tissues are screened for a history of significant valvular disease or cardiac infection, and vascular donors may be determined ineligible if there is a history of diabetes, vasculitis, varicose veins, or significant atherosclerosis. Cardiac and vascular tissue donations are also limited by age restrictions per AATB standards and individual tissue bank policy. When skin is recovered, areas of skin exhibiting signs of a skin infection, or where a rash, nevus, or tattoo is present, are avoided. Cornea donors cannot have a history of refractive corneal procedures, such as radial keratotomy. Also per EBAA and AATB standards, tissue recovery sites in deceased donors are evaluated for trauma and tissue is not recovered from sites found to be damaged. Donors of reproductive cells or tissues are screened for evidence or risk of inheritable diseases, and there are age restrictions. Extraembryonic tissue such as amnion and umbilical vein require the delivery to be full term; meconium staining of amniotic fluid is not acceptable, and there can be no current pelvic or vaginal infection in the mother. Consent is obtained from both parents. In an effort to improve the efficacy and uniformity of the donor history acquisition process, a multiorganizational project team was established to develop a standardized donor history questionnaire and materials, modeled after the acceptable full-length donor history questionnaire and accompanying materials for blood donors. The project team includes members from the AATB, AABB, Association of Organ Procurement

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Organizations, EBAA, NATCO (formerly the North American Transplant Coordinators Organization), Centers for Disease Control and Prevention (CDC), FDA, Health Resources Services Administration (HRSA), United Network for Organ Sharing (UNOS), and Health Canada. The goal is to eliminate questions that do not contribute significant information, and to develop simple and concise questions that employ terminology easily understood by the lay public. The ongoing effort is intended to produce a standardized form, with an accompanying standard operating procedure, to serve as a guide for donor history information collection by tissue banks. Use of the form is not intended to be mandatory, but it will be designed to facilitate effective history collection, in compliance with regulatory and professional standards requirements.

Donor Physical Assessment The physical assessment seeks evidence that is consistent with risk for infectious diseases that would disqualify the donor. Such findings include unexplained jaundice or icterus; enlarged lymph nodes; unexplained white lesions in the oral cavity; blue or purple spots on the skin (possible Kaposi sarcoma); signs of nonmedical injected drug use; unexplained hepatomegaly; genital lesions consistent with a sexually transmissible disease; signs of systemic infection (generalized rash or petechiae); tattoos, piercing, or other body art that appears recent; trauma to intended retrieval sites; corneal scarring; and a rash, a scab, or a lesion that is suspicious for vaccinia. The physical assessment is fully documented so that it can be reviewed, along with medical/social/ behavioral history, medical records, and records of an autopsy (if performed). AATB offers a sample tissue donor physical assessment form and instructions for its use (see http://www.aatb. timberlakepublishing.com/files/guidancedocument1v2.pdf), to facilitate the performance and documentation of this assessment in a thorough manner by tissue banks. Tissue Recovery Tissues are collected aseptically in an operating room, in an autopsy room, or other suitable location where aseptic procedures can be performed. AATB standards require control of recovery site parameters including size/space, location, traffic, lighting, plumbing/drainage, ventilation, cleanliness of the room and furniture surfaces, presence or absence of pests, absence of other activities occurring simultaneously, sources of contamination, and capacity to permit proper handling of contaminated equipment and disposal of biohazardous waste. In addition, all working surfaces must be disinfected. Following a surgical scrub, the person performing the recovery dons proper attire (gown and gloves). Body preparation, including shaving body hair; cleansing the skin with antimicrobial agent(s); and surgical draping of the body is performed, consistent with aseptic technique. Using aseptic technique, recovery of tissue is sequenced, and well-defined zone recovery methods are employed. These recovery methods are described in AATB’s Guidance Document 2, “Prevention of

Chapter 58: Tissue Banking

Contamination and Cross-contamination at Recovery: Practices and Culture Results” (see http://www.aatb.timberlakepublishing. com/files/bulletin46attachment1.pdf). Generally, recovered tissues are not processed at the time of recovery. Tissues are often cultured at recovery, then individually packaged in sterile wraps, labeled with a unique donor identifier, placed on wet ice, and sent without delay to tissue banks that will process them. Following donation, the donor body is usually reconstructed to permit normal funeral arrangements and viewing. The organ/tissue donor coordinator’s responsibilities continue after the transplant procedure, in that a letter of appreciation may be sent to the next of kin, giving general information about the use of the donated organs and tissues. This communication serves as a liaison between the donor’s family and the organ/tissue procurement agency. Later, contact may be reestablished to assist in the amelioration of grief and bereavement. However, some donor families specifically request that there be no postdonation communication. AATB standards require formal establishment of donor family services. AATB has published a guidance document, “Providing Service to Tissue Donor Families,” which describes support that should be considered (see http://www.aatb. timberlakepublishing.com/files/2007bulletin34attachment.pdf).

Infectious Disease Testing Infectious disease marker testing includes HIV-1 and HIV-2 antibodies and nucleic acid, HCV antibody and nucleic acid, hepatitis B surface antigen (HBsAg), human T-cell lymphotropic virus, types I and II (HTLV-I/II) antibody, and syphilis. Tissue banks also screen donors for antibodies to hepatitis B core antigen (anti-HBc), which, although a confirmatory test is not available, may indicate HBV infection even when HBsAg is not detectable. Whenever possible, infectious disease marker testing should be performed on pretransfusion/preinfusion blood samples to avoid false-negative results caused by dilution in the event that transfusions and/or infusions had been given shortly before the time of death.43 In the case of posttransfusion/infusion specimens, algorithms for determining the extent of plasma dilution are available.10 Testing of postmortem (postasystole) blood specimens may be complicated by hemolysis or the presence of sediment, which can cause false-positive results in some HBsAg enzyme immunoassays (EIAs) and false-negative results in some HIV, HBV, and HCV NAT assays.19 There are tests on the market for HIV and hepatitis that have been validated for use with postmortem specimens and approved by the FDA for use in tissue donor screening (see http://www.fda.gov/cber/tissue/prod. htm). When practical, tissue from living donors is preserved and quarantined before the donor is restested for HIV and hepatitis viruses in order to rule out seroconversion during the period of storage. Specific regulatory requirements for quarantine apply to donor semen. Additionally, both semen and oocyte donors must be found negative for Neisseria gonorrhea and Chlamydia trachomatis and are usually tested for carriage of at least one genetic disorder, as indicated by donor racial and ethnic

background. Donors may also undergo specific testing for rare genetic disorders if the recipient couple seeks a donor known to be negative for a particular gene mutation.

Tissue Sterilization Tissue sterilization is defined as the killing or elimination of all microorganisms, whereas disinfection refers to the removal of microbial contamination, from allograft tissue. The Association for the Advancement of Medical Instrumentation (AAMI), a standard-setting organization for the medical manufacturing industry, defines Sterility Assurance Level (SAL) as the probability that an individual device, dose, or unit is nonsterile (ie, one or more viable microorganisms being present) after it has been exposed to a validated sterilization process. While absolute sterility theoretically would represent an absence of any pathogen, SAL is generally applied only to the level of possible contamination with bacteria or parasites. In contrast to log reduction of viruses determined in assessments of virus reduction methods, SAL is an absolute determined by the ability of the method to eradicate or reduce microorganisms, the susceptibility of organisms that may be present to the sterilization method applied, and the maximal bioburden that could occur in the initial material. Thus, a process validated to achieve a specific SAL must include a step to assess the initial bioburden present and its susceptibility in order to qualify a specific level of assurance. For example, an SAL of 10⫺6 means that there is less than a 1 in 1,000,000 chance of a viable microorganism remaining after the sterilization procedure. The FDA requires that medical devices be sterilized using a method validated to achieve an SAL of 10⫺6. A medical device derived from or that includes a biological product component must also meet an SAL of 10⫺6 if it is to be labeled sterile. An SAL of 10⫺3, or a 1 in 1000 chance of a viable microorganism being present, is a more achievable goal selected by some processors for aseptically processed tissues unable to withstand the harsh treatment needed to achieve a more restrictive SAL without an impairment of tissue function. Such tissues may not then be labeled as sterile. The complex physical structures and density of musculoskeletal tissues pose challenges for adequate penetration of antimicrobial agents and eradication of microorganisms. Allografts will not tolerate methods usually applied to metal and plastic medical devices because such treatment would impair the mechanical and biologic properties necessary for clinical utility. As an alternative, sterilization of tissues has been accomplished by several methods, including heat, chemicals, ethylene oxide gas, and irradiation. However, not all sterilants have adequate tissue penetration. This is particularly the case for gases and liquids. The initial bioburden, which may be high in some tissues, must be considered. Some tissues are treated with antibiotics in vitro before storage, but this treatment decontaminates only the surface and may be effective against bacteria only. A variety of methods, including chemical treatments and irradiation, have been used to reduce or eliminate pathogens in

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tissue intended for transplantation. The introduction of bone sterilization by ethylene oxide gas simplified bone processing and facilitated the widespread use of sterilized air-dried and lyophilized bone products.44 The effects of ethylene oxide treatment on the biomechanical and osteoinductive capacity of bone allografts have been questioned, although animal studies have yielded inconsistent results.45-47 These concerns, combined with those regarding the carcinogenic potential of ethylene oxide and its breakdown products, have largely led to abandonment of this method in the United States and the United Kingdom. First introduced over 30 years ago, γ-irradiation of bone is still used widely, usually employing a cobalt-60 source. The γ-rays penetrate bone effectively and work by generating free radicals, which have adverse effects on collagen and limit utility in soft tissues unless performed in a controlled dose fashion at ultra low temperature. The minimal bacteriocidal level of γ-irradiation is 10 to 20 kGy (1 kGy ⫽ 100,000 rad). Uncontrolled human studies have shown irradiated, calcified, and demineralized bone grafts to be clinically effective.48,49 Numerous studies have shown that mineralized bone allografts irradiated at 25 to 30 kGy are also clinically effective, with success rates of 85% to 91% reported.50,51 In controlled studies, the clinical effectiveness of bone allografts subjected to 25 kGy irradiation was comparable to that of nonirradiated bone grafts,52 although doses exceeding 25 kGy for cortical bone and 60 kGy for cancellous bone have been found to induce cross-linking of collagen and to impair mechanical function in a dose-dependent fashion.53 There is in-vitro evidence that irradiation reduces osteoclast activity and increases osteoblast apoptosis, and that residual bacterial products induce inflammatory bone resorption following macrophage inactivation.53 However, the clinical significance of these findings has not been established. Newer processes employing radioprotectants have preserved bone allograft integrity when doses ⬎25 kGy are applied and controlled-dose methods permit successful irradiation at lower doses (see proprietary chemical sterilization methods to follow). Irradiated demineralized bone has active osteoinductive activity and has been effective in nonstructural clinical applications. Concerns about pathogen transmission and the limitations of irradiation, especially for soft tissues, have prompted improvements in sterilization methods and in the validation of these methods. A number of proprietary chemical-based processing methods have been developed with aims of effectively penetrating tissues and reducing, killing, or inactivating microorganisms and viruses without unacceptable adverse effects on the tissue’s biomechanical properties. Additionally, for use in transplantation, the agents must either be able to be effectively removed or be nontoxic. All methods in current use are applied only to tissue from donors who have met stringent criteria for medical history and behavioral risk assessment as well as negative results on infectious disease marker testing. Some popular methods used are described below. The Tutoplast process (Tutogen Medical, Gainesville, FL) was the first comprehensive method to sterilize and preserve

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tissue without compromising its biological or mechanical properties. The process has been in use for over 30 years, for a variety of hard and soft tissues, including bone, fascia lata, pericardium, dermis, amniotic membrane, and sclera. Initially, lipids are removed in an ultrasonic acetone bath that also inactivates enveloped viruses and reduces prion activity. Bacteria are destroyed using alternating hyperosmotic saline and purified waterbaths that also wash out cellular debris. Soluble proteins, nonenveloped viruses, and bacterial spores are destroyed in multiple hydrogen peroxide baths, and a 1N sodium hydroxide treatment further reduces prion infectivity by 6 logs. A final acetone wash removes any residual prions and inactivates any remaining enveloped viruses. Vacuum extraction dehydrates the tissue before the grafts are shaped and then double-barrier packaged. Terminal sterilization using low-dose γ-irradiation yields an SAL of 10⫺6. The Allowash XG process (LifeNet Health, Virginia Beach, VA) employs six steps, including bioburden control and assessment; minimization of contamination during processing; rigorous cleaning and disinfection steps, which employ detergents, antibiotics, alcohols, and hydrogen peroxide; and a final step of low-temperature, controlled-dose γ-irradiation. The process has been validated to achieve an SAL of 10⫺6. The BioCleanse process (Regeneration Technologies, Alachua, FL) employs low-temperature addition of chemical sterilants, such as hydrogen peroxide and isopropyl alcohol, which permeate the tissue’s inner matrix, followed by pressure variations intended to drive the sterilants into and out of the tissue. Regeneration Technologies reports an SAL of 10⫺6 for soft tissues without adverse effects on the initial allograft mechanical properties. The Clearant process (Clearant, Los Angeles, CA) avoids the negative effects of γ-irradiation through addition of free radical scavengers, employing dimethylsulfoxide (DMSO) and propylene glycol as pretreatment radioprotectants. Although the process subjects tissue to 50 kGy of radiation and achieves an SAL of 10⫺6 for bacteria, fungi, yeast, and spores, the tissue’s biomechanical properties are retained. The Musculoskeletal Transplant Foundation (Edison, NJ) uses a series of chemicals, including nonionic detergents, hydrogen peroxide, and alcohol, to treat cortical and cancellous bone grafts. For soft tissues, such as bone-patellar tendon allografts, an antibiotic mixture containing gentamicin, amphotericin B, and primaxin is added, and then washed out to a nondetectable concentration. The Musculoskeletal Transplant Foundation claims an SAL of 10⫺3 for its products. Incoming tissues whose bioburden exceeds prescribed parameters are pretreated with low-dose γ-irradiation. NovaSterilis (Lansing, NY) has developed a sterilization technique that uses supercritical carbon dioxide at low temperatures and relatively low pressures, resulting in transient acidification, which is lethal to bacteria and viruses, with good penetration reported. However, this technique is still in development, and data on retention of allograft mechanical properties are limited.

Chapter 58: Tissue Banking

General Principles of Tissue Preservation and Clinical Use Except for bone, which is usually lyophilized (freeze dried), the most common method of storing recovered tissues is at cold temperatures, either by refrigeration or by freezing (Table 58-5). For short-term storage, refrigeration at about 4ºC often suffices, whereas long-term storage usually requires a frozen state. Several types of tissues can be preserved by multiple methods. Bone, dura mater, and amnion can be effectively cryopreserved or lyophilized. Much of the lyophilized and cryopreserved human tissue used in transplantation is intended to serve a structural purpose and maintenance of cell viability is not necessary. Tissues of this type, which include dermis, are composed of an extracellular matrix (such as collagen) with few or no viable cells present to support the matrix after transplantation, although they can contribute growth factors to facilitate remodeling. Even when the processing method used is intended to preserve cell viability, the donor cells will die following transplantation. The extracellular matrix, whether transplanted containing viable cells or devoid of them, is repopulated through the ingrowth of metabolically active recipient cells. More gradually, depending in part on the size and type of the allograft implanted, remodeling occurs, and the transplanted structure may eventually be entirely replaced by host cells.55 The transplantation of allograft heart valves and cardiac conduit tissues provided in an acellular matrix form is being studied to determine the rate and extent of repopulation with recipient cells.56 In some tissues, such as cornea, a single layer of viable donor cells is important, and this requirement necessitates maintenance of the tissue in culture medium at refrigerated temperature.57

Other human tissues, such as marrow, skin, and gametes, are stored by either refrigeration or cryopreservation. In the latter, a controlled-rate freezing process and a cryoprotectant remove water from the cells while maintaining viability. The usefulness of tissues requiring posttransplantation cell viability depends on their maintenance of not only metabolic activity, but also capacity to synthesize protein, proliferate, or differentiate.

Bone Bone allografts have many uses, including provision of acetabular and proximal femoral support for replacement of failed prosthetic hip joints, packing of benign bone cysts, fusion of the cervical or lumbar spine to correct disk disease or scoliosis, restoration of alveolar bone in periodontal pockets, reconstruction of maxillofacial deficits, and replacement of bone that has been resected because of a bone malignancy, such as osteosarcoma (see Table 58-1). The last procedure is accomplished with large osteochondral allografts that permit tumor resection and achievement of a cure without limb amputation. Historically, bone allografts were prepared by cutting a larger graft using simple techniques. Today’s technology allows the cutting, machining, and piecing together of allografts via precision instrumentation, and has resulted in stronger and more versatile grafts that can withstand the challenges of new surgical techniques. Linear grooves, notches, or crosshatchings may be incised into bone surfaces to make the bone graft less likely to slip or become dislodged after placement. Many bone allografts, especially those used in neurosurgical applications, are now placed using precision instrumentation that not only ensures exact placement, but also enhances stability. Allografts can be cut or shaped

Table 58-5. Recommended Human Tissue Storage Conditions and Durations Tissue

Storage Condition

Maximal Storage Duration

Bone

⫺40ºC or colder ⫺40 to ⫺20ºC 1-10ºC

5 years* 6 months 5 days

Tendon Fascia lata

⫺40ºC or colder Lyophilized, ambient temperature ⫺40ºC or colder

5 years* 5 years* 5 years*

Articular cartilage

⫺40ºC or colder 1-10ºC

5 years* 5 days

Skin

⫺40ºC or colder Lyophilized, ambient temperature

Not defined Not defined

Cornea

2-8ºC

14 days

Semen

Liquid nitrogen, immersed Liquid nitrogen, vapor phase

Not defined Not defined

Cardiac, vascular tissue

⫺100ºC or colder

Not defined

Dura mater

Lyophilized, ambient temperature

Not defined

*Or as validated by the processor. Adapted with permission from Pearson et al10 and Eastlund.54

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to precise angles that accommodate, for instance, lordosis of the cervical and lumbar spine. Advanced processing methods have resulted in improvements in availability and also in graft biocompatibility and functioning, in such processes as osteoinduction, osteoconduction, bioresorption, and tissue regeneration.1 Fresh autograft can be taken from the patient’s own iliac crest during surgery, but this practice is becoming less common.55 Fresh bone autograft is preferred by some surgeons, but preserved allografts are practical and accepted alternatives that approximate the results obtained with fresh bone autograft.58 In some patients, an autograft is not an option because sufficient bone is not available. In addition, the use of bone allografts reduces operating room time and the number of operative sites, leading to reduced morbidity and lower cost. The use of bone allograft does carry the risk of donor-to-recipient transmission of infectious disease,15 although this risk is minimized through careful donor selection and testing, and disinfection and sterilization of tissue during processing.

detect donors who had been infected with HBV shortly before donating but who no longer have detectable HBsAg. Generally, culturing for bacteria is performed on each bone to be fresh frozen or on each batch/lot of cryopreserved bone. Allograft bone is further prepared by removal of extraneous tissue; it is then packaged, and immediately either shipped to the processing tissue bank on wet ice or sent to temporary freezer storage at ⫺40ºC or colder. Frozen bone allografts are available as whole bones or cut into usable shapes and sizes. Frozen bone can generally be stored up to 5 years at ⫺40ºC or colder, but the maximal storage duration and expiration date may vary based on processing and storage methods, as validated by the tissue bank. There is no evidence that the biomechanical or osteoinductive properties decline during frozen storage. However, in the absence of cryopreservation, frozen bone does not maintain cellular viability. Thus, frozen bone is used for structural support that depends on an intact calcified extracellular matrix or is used as filler to promote new bone formation.

Frozen Bone Frozen bone, collected under aseptic conditions and then frozen or cryopreserved, is available in a wide variety of shapes and sizes from deceased donors, or as femoral heads or tibial plateaus obtained from living donors undergoing total joint replacement in an operating room (now an uncommon practice in the United States, but continuing in Canada, Australia, and Europe). This frozen bone is largely free of bacteria, but it does carry the risk of viral transmission. Diseases known to have been transmitted by unprocessed bone include AIDS, tuberculosis, and hepatitis.15 Frozen bone can cause alloimmunization from exposure to antigens on the attached connective tissues, marrow, and blood, although such alloimmunization apparently does not affect the graft’s efficacy. Detailed reviews addressing the role of histocompatibility and the immune response in bone allograft transplantation have been published.59,60 Antibodies to histocompatibility antigens,59,60 blood group antigens,61,62 and bone matrix proteins have been induced by transplanted frozen bone. In order to avoid Rh alloimmunization, bone from an Rh-negative donor is usually selected when using bone that has not been processed to remove red cells and the recipient is an Rh-negative female of childbearing potential. Bone that otherwise would be discarded can be collected from living donors during surgical procedures (total hip or knee replacements with prostheses). The eligibility of a volunteer living donor is determined by a careful health history review, a directed physical examination if indicated, and donor testing for anti-HIV-1 and -2, HIV-1 NAT, HBsAg, anti-HBc (total), anti-HTLV-I/II, anti-HCV, HCV NAT, and syphilis.10 In some countries, if NAT assays are not performed, the donated tissue is quarantined, and the living donor is retested 6 months later for infectious disease markers. This quarantine and retesting process is intended to eliminate donors who were in the early stage of viral infection but were antibody negative at the time of donation (in the window period). A follow-up anti-HBc test may

Lyophilized Bone Following aseptic recovery, deceased donor bone can be placed on ice for transport to temporary storage in a freezer and maintained frozen at ⫺40ºC or colder, and then can later be sent to a tissue processor with dry ice as a refrigerant. Alternatively, immediately after recovery, the tissue can be placed on wet ice and expedited directly to the processing tissue bank where, within 72 hours of recovery, it is frozen at ⫺40ºC or colder until processing. Such processing includes removal of surface tissues and internal fat, blood, and marrow by means of mechanical agitation, high-pressure water jets, and alcohol soaks. It can also include detergents and other solutions as part of a proprietary process. Then the bone is milled into clinically useful shapes and sizes. This may include computer-guided milling and use of assemblies that result in complex mechanical structures. Conventional allografts include corticocancellous strips, wedges, and dowels; cortical struts and rings; and cancellous and corticocancellous cubes and chips. Bone can also be ground into a powder and be available as DBM and products that include it. DBM, which is also known as demineralized freeze-dried bone allograft, is derived from cortical bone and is available in combination products in the form of gels, pastes, putties, and flexible strips or sheets. DBM itself may be obtained in specific granule or particle sizes, as a powder, or in entangled, twisted fiber configurations. DBM primarily provides growth factors, but accompanying collagen can help play a structural role as a scaffold for future bone growth. The combining of DBM with approved polymer carriers results in moldable grafts that are user friendly for the surgeon, do not migrate after placement, and whose bone content does not dissolve following transplantation. Such grafts can readily be applied to completely fill bony defects and to act as a scaffold for ingrowth of the recipient’s own cells, or they can be used to enhance other structural repair devices, such as dental implants, vertebral body spacers and cages, or support devices such as rods, screws, and plates.1

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The bone allografts are lyophilized to a residual moisture content of ⬍6% or 8% (depending on measurement method) and packaged into peel packs or “boat” packaging. Routine quality control testing of bone is designed to monitor safety, rather than potency or efficacy. Sterility is assessed by the testing of samples of each batch, or by another acceptable method. If a robust validated sterilization process is used, sterility may need to be assessed only at established intervals, such as quarterly. Potency can be evaluated by using assays for osteoinductive capacity and biomechanical properties, but these analyses usually are conducted only when there is a change in the production process. Lyophilized bone is brittle unless fully rehydrated before use. Lyophilized bone can usually be stored at ambient temperature for up to 5 years if the graft’s package integrity and its vacuum are maintained, depending on validation performed by the tissue processor. Bone collected aseptically in an operating room and processed aseptically can be lyophilized without use of a sterilant. Because the bone, theoretically, is bacteria-free, final sterilization with γ-irradiation may not be necessary. Although the bone should be free of bacteria, it still has the potential to transmit disease. Despite this risk, some physicians have preferred aseptically processed, nonsterilized lyophilized bone because it was thought to have better osteoinductive capacity than sterilized bone. However, controlled-dose low-temperature radiation has been found to have no significant effect on osteoinductive capacity.63

Ear Ossicles Ear ossicles are used as a special kind of bone graft to correct selected cases of deafness in which the patient’s own ossicles have suffered congenital, traumatic, or postinfectious damage.64 Ear ossicles are procured by removal of the temporal bone en bloc or as a core with a bone-plug cutter. The temporal bone can be stored temporarily, for months if frozen, or up to 2 weeks if preserved in formalin; the tympanic membrane and ossicular chain are then dissected. Ossicles have been stored for up to 2 months in cialit (an organomercuric compound), and for up to 1 year at room temperature in buffered formaldehyde. Alternatively, ossicles are dissected at the time of collection, lyophilized, and then sterilized by γ-irradiation. Lyophilized ossicles can be stored at ambient temperature for up to 5 years.

Connective Tissue Cartilage and Meniscus Human cartilage can be transplanted at weight-bearing or nonweight-bearing sites. For non-weight-bearing uses such as nasal reconstruction and mandibular or orbital rim augmentation, the graft provides structural support and need not be viable. Costal cartilage can be recovered for this use. The cartilage can be sterilized by γ-irradiation and stored in saline at refrigerated temperatures, or it can be lyophilized and stored at ambient temperature. Articular cartilage can be transplanted to weight-bearing articular surfaces to replace focal cartilage defects caused by

trauma or degenerative disease, particularly in the knee. Cartilage in an osteochondral or osteoarticular allograft can be obtained as a femoral hemicondyle, a tibial plateau or fragment, or a measured segment removed with a template cutter that can be pressfitted into a similarly cut area in the recipient. Osteochondral allografts avoid autograft harvest site morbidity and are advantageous when the focal articular cartilage defects being repaired are large (⬎2.5 cm).65 It has been assumed that, in weight-bearing applications, chondrocytes must survive the collection and preservation process and remain viable, producing normal cartilage matrix to maintain mechanical properties. It appears that chondrocytes deep within the cartilage matrix resist cell-mediated immune responses by the recipient and, if kept viable during storage, are able to survive after transplantation. Cartilage grafts from histoincompatible donors, stored ⬍24 hours at 4ºC, have survived for as long as 7 years after transplantation, if the grafts developed a sound union and if conditions for correct biomechanical functioning were present.66 Articular cartilage collected in a sterile manner can be stored at 4ºC in saline or electrolyte solutions with or without 10% fetal calf serum and antibiotics.67 An FDA rule proposed in January 2007 would prohibit the use of certain cattle materials. This rule indicates that the FDA considers cow-to-calf transmission of bovine spongiform encephalopathy unproven by current evidence, and the agency believes that procedures could provide assurance that contamination has been prevented.68 Osteoarticular and osteochondral allografts can be stored refrigerated for up to 28 days with successful clinical outcomes. If they have been cryopreserved, expiry for these allografts may be extended to 1 year. Cartilage cryopreserved with or without DMSO maintains viability with variable degrees of success.69 The use of a large osteochondral allograft, such as the femur with the articular cartilage attached, is thought to require preservation of cartilage viability in order to maintain biomechanical properties. To accomplish this, grafts have been stored at refrigerated temperatures in electrolyte solutions for up to 1 month, or have been frozen in 10% glycerol or 15% DMSO and stored at ⫺70ºC or colder.70 Following transplantation in humans and animals, the surface of the articular cartilage allograft undergoes degenerative changes within a few years. These grafts have the same risk of disease transmission as other fresh tissue allografts. Menisci are C-shaped disks of fibrocartilage interposed between the femoral condyle and tibia. The presence and integrity of the meniscus are essential for knee mechanics and biochemical functions. Loss or disruption of the meniscus is associated with pain, joint laxity, and degenerative arthritis. Meniscal transplantation has been proposed as a method of providing a biologically and biomechanically acceptable structure to replace a damaged or removed meniscus, with a goal of relieving pain, decreasing stress on the anterior cruciate ligament, and preventing late arthritis, although evidence of allograft tissue being chondroprotective is lacking. Although there have been unpublished reports of successful transplantation of menisci stored ⬍24 hours at 4ºC, fresh menisci are not usually

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available. Cryopreserved menisci are used successfully, with good outcomes (including reduced pain and increased knee function) reported.71,72

Tendon and Ligament The knee is the joint most frequently involved in sports-related injury. Arthroscopic methods for replacing the anterior or posterior cruciate ligaments with autografts, allografts, or artificial tendons and ligaments are frequently used. Despite the attendant need for sacrifice or weakening of normal structures, the use of autografts appears to have the highest success rate and the lowest incidence of complications. However, allografts may be indicated for multiple ligament knee injuries, anterior cruciate ligament revisions, or posterior cruciate ligament reconstruction, and when extensor mechanisms are impaired (as with previous tendon tears). It is sometimes preferable to avoid the morbidity associated with autograft harvesting. In addition, there are occasions when sources of adequate autograft tissue are not available.73 Allografts used to replace the injured anterior cruciate ligament are usually derived from deceased donor patellar ligaments, tendons of the leg (eg, tibialis, semitendinosus, gracilis, and peroneus longus), or Achilles tendons. Ligament and tendon allografts are usually stored frozen, but some are stored lyophilized. In-vitro biomechanical properties of tendons do not seem to be greatly affected by freezing, lyophilizing, or ethylene oxide sterilization.74 However, many surgeons eschew lyophilized tendon allografts because of experiences with clinical failure. Frozen tendon allografts are commonly sterilized by γ-irradiation, although this can reduce their mechanical strength, particularly if the dose exceeds 20 kGy.75 There is no evidence that maintenance of cellular viability during processing and storage is important to clinical effectiveness. The effect of irradiation on the biomechanical properties of human tissue has been explored extensively, with inconsistent results. This is probably because the studies failed to use uniform irradiation methods and comparable study designs. A key study found a difference in average stress at failure between nonirradiated and γ-irradiated tendons; that difference is likely a consequence of the free radicals generated, which can cause minor crosslinking of collagen fibers and alteration of the tendon’s material properties.76 In order to eliminate the potential for elongation of irradiated grafts after implantation, the authors encouraged pretensioning of grafts before insertion. Fascia Lata The fascia lata is a broad fibrous membrane surrounding the thigh muscles. The thick lateral portion acts as a flattened tendon, and its muscular insertions helping to maintain the trunk in an erect posture. Fascia lata can be removed and transplanted as an autograft or allograft. As an allograft, fascia lata has been used to suspend the upper eyelid to correct ptosis, to replace injured anterior cruciate ligaments, to provide support for bladder suspension, and to repair ankle, hip, and shoulder suspensions (ie, repair of a ruptured shoulder rotator cuff). Fascia lata usually is preserved by lyophilization, resulting in a residual moisture of ⬍6% or 8% (depending on measurement method), the graft

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is then sterilized by γ-irradiation and stored for up to 5 years at ambient temperature. After rehydration, the graft’s biomechanical properties equal those of fresh frozen fascia lata. The use of fascia lata has become less popular because of the availability of alternative products, such as decellularized skin.

Dura Mater Dura mater is the outermost, toughest, and most fibrous of the three meningeal membranes covering the brain and spinal cord. The intracranial portion is collected, processed, stored, and distributed for several clinical applications; the most common use is the closure of dural defects caused by resection of tumor or the repair of traumatic injury. Human dura allograft most commonly is preserved by lyophilization. Ethylene oxide and γ-irradiation are effective in preventing transmission of viruses and bacteria; however, Creutzfeldt-Jakob disease (CJD) has been transmitted by dura mater treated by these methods. Following findings by Brown and coworkers,77,78 in 1986 The Committee on Health Care Issues of the American Neurological Association recommended using 1N NaOH for 1 hour or steam autoclaving for 1 hour at 132ºC as standard sterilization procedures for CJD-infected tissue or contaminated materials. Donors with a history of clinical dementia or other central nervous system disorders are not accepted as dura mater donors. Lyophilization and sterilization treatments do not lessen the effectiveness of dura mater allografts. Reconstituted freeze-dried dura mater is thick and strong, holds suture well, and is incorporated into normal surrounding tissue without rejection. Because of the risks and resulting decreased demand, dura mater is currently processed in the US by only one tissue bank.

Skin Human skin allograft is the dressing of choice for temporary grafting onto deep burn wounds whenever sufficient amounts of autograft skin are unavailable. Early excision of burned tissue and covering of the wound with deceased donor skin allograft has shortened hospitalization and decreased mortality more than has any other treatment.79 A skin allograft provides temporary coverage and acts as a barrier against loss of water, electrolytes, protein, and heat. It reduces opportunities for the invasion of bacteria and speeds re-epithelialization. Skin allografts are replaced periodically until the patient’s vascular bed is reestablished. Skin allografts also are used for unhealed skin defects (decubitus ulcers, autograft skin donor sites, pedicle flap donor sites, and traumatically denuded areas). While skin has historically been used only as a covering, decellularized (mechanically and chemically treated) skin offers the opportunities for use of a collagen matrix that can be implanted and be remodeled within the site with the recipient’s own cells. It is used for such applications as bladder suspension surgery and repair of large defects, such as postoperative hernias and dehisced wounds. In an analogous fashion, Taylor and colleagues56 have used perfusiondecellularized cardiac tissue matrix in an attempt to develop a bioartificial heart.

Chapter 58: Tissue Banking

After collection, fresh skin can be stored in medium at 1 to 10ºC for up to 14 days,10 but fresh skin is seldom used today. Skin also can be frozen using a method that retains cell viability, in order to improve availability. Because cell viability declines during refrigerated storage, results are best when cryopreservation is performed within 2 to 3 days after recovery. Cryopreserved skin can be prepared as strips (often 3-inch by 8-inch sections), either unmeshed or meshed (most commonly with a 1:1.5 expansion ratio, which triples the area that can be covered). The skin then covered in fine-mesh gauze and laid flat, is packaged, and is then cryopreserved, with glycerol or DMSO at a concentration of 10% or 15% as a cryoprotectant. Cryogenic damage is minimized by controlling the rate of freezing to between ⫺1 and ⫺5ºC/minute. Many tissue banks use a “heat sink” freezing method, rather than one that employs computer-controlled freezing chambers. Heat sinks involve aluminum plates combined with styrofoam-insulated boxes; these are placed directly into a ⫺70ºC mechanical freezer. This simple process provides a slow, controlled freezing rate that is acceptable for skin and that also maintains cellular viability.80 AATB standards permit frozen storage in a mechanical freezer at ⫺40ºC or colder, in the vapor phase of liquid nitrogen, or submerged in liquid nitrogen.10 The maximal allowable storage period in the frozen state during which viability and structural integrity are maintained has not been determined. Cryopreserved skin allograft usually is transported from the tissue bank to the hospital with dry ice in order to maintain a frozen environment until use. Skin for use in burn applications generally is not preserved by lyophilization because this method decreases clinical efficacy. However, lyophilized skin is sometimes used by oral surgeons to cover oral mucous membrane defects and to speed re-epithelialization. Lyophilized acellular dermal matrix is also available, in two thicknesses for different applications, and can serve as a natural biological matrix for soft tissue augmentation in soft tissue defects and in periodontal peri-implant soft tissue management. Following hydration, lyophilized skin has multidirectional strength and can adapt to surface contours, and it then is resorbed over 4 to 6 months, depending on the site, defect size, patient age and health status, and the biomechanical load on the graft. Depending on processing method and packaging configuration, lyophylized skin can be stored as long as 5 years at ambient temperature or it may require refrigeration.

Ocular Tissue Cornea is one of the most frequently transplanted tissues; ⬎34,000 corneas were transplanted in the United States in 2006. Corneal transplantation has become highly effective because of improvements in suture materials, surgical instruments, and topical antiinflammatory medications to control rejection. It is now considered a standard therapy for a variety of conditions. However, early in this century demand decreased because of improvements in cataract surgery techniques compared with those in use in the 1990s, during which time use for complications of cataract surgery had supplanted keratoconus as the

leading indication. More recently, development of a new technique that uses an air bubble to hold the allograft in place, obviating the need for sutures and retaining the normal topography, has resulted in faster recovery and improved visual acuity, as well as a reduction in the occurrence of adverse effects. As a consequence, corneal transplantation for Fuchs epithelial dystrophy is becoming an accepted therapy at much earlier stages of the disease, resulting in a resurgence of demand for cornea allografts. Currently the most common indications for corneal transplantation are postcataract surgery corneal edema, keratoconus, Fuchs dystrophy, and corneal regrafting. Donor cells in the avascular full-thickness cornea graft enjoy long-term survival without the aid of histocompatibility matching because the recipient site is also almost completely avascular. Some experts believe that the failure rate of 5% to 10% might be improved by HLA matching; recipients known to be sensitized to HLA antigens have rejection rates higher than nonsensitized recipients.81 The possibility of alloimunization is of particular concern in patients who are undergoing repeat grafting procedures because of graft failure or who have ocular infections, as the corneal rim may become quite vascularized. Systemic immunosuppressants are not used, but topical corticosteroids are used routinely. Sclera may be used in the repair of ocular deficits, in orbital reconstruction following enucleation, and in some dental applications.82 Ocular tissue can be recovered by enucleation or by in situ excision of the cornea, with a rim of sclera. It is preferable that recovery be performed within 6 hours after death. The oldest method of storage for whole globes was at 4ºC in a moist chamber; this method appeared to maintain viable endothelial cells sufficient for graft efficacy for as long as 48 hours after recovery. Because it yields improved viability, a more common method today is storage of the cornea, with attached rim of sclera, at 4ºC in a modified tissue culture medium, based on that developed in 1974 by McCarey and Kaufman.83 One example commonly used is Optisol-GS (Bausch & Lomb, Irvine, CA), which contains dextran (as an osmotic agent), chondroitin sulfate, gentamicin, and streptomycin. Storage of corneas in the medium, at 2 to 8ºC, can maintain endothelial viability for as long as 14 days, and can maintain functional integrity for eutopic graft applications not requiring visual acuity for even longer storage periods.84 Grafts are usually used within 7 days. Although they have been treated with antibiotics, allograft corneas are not considered sterile. An alternative culture technique, used at a few eye banks, has maintained viable corneas for as long as 5 weeks in tissue culture medium at 34ºC.85 Rarely, corneas are frozen with cryoprotectants. Sclera is usually preserved in ⭓70% ethyl alcohol; such a method yields a shelf-life as long as 2 years.

Cardiovascular Tissue Cardiovascular tissue includes cardiac tissues (heart valves) and vessels that can be used as conduits. Donor medical history requirements differ, so AATB has established separate standards for cardiac tissues and for vascular tissues.10 Since their introduction 4 decades ago, human heart valve allografts have been

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shown to be a superior alternative for patients needing heart valve replacement and for whom mechanical and porcine valves are contraindicated. Human heart valve allografts do not require recipient anticoagulation, have a lower incidence of thromboembolism, and appear relatively resistant to infection. After valve allograft transplantation, donor endothelium is not maintained, but donor fibroblasts remain for an undetermined period. Because anticoagulation is unnecessary, human valve allografts are the graft of choice for children, females of childbearing potential, and patients with infection in the aortic root. The use of allograft valves has been slowed, however, because implantation is more difficult technically and because their availability is limited. Additionally, clinical results with transplantation of xenograft tissue valves have improved, although these are not available in the small sizes required by many pediatric patients. Technical impediments have made it impossible to successfully produce man-made (either completely artificial or modified porcine) replacement heart valves for use in neonates and other pediatric patients who require very small grafts. Only donated human heart valves from newborns or small children offer unobstructed blood flow through such a small annulus. Also, the tissue’s pliability renders human allografts adaptable to the ingenuity of cardiothoracic surgeons who repair congenital defects by using allografts to replace underdeveloped or otherwise defective valves or outflow tracts, or to construct valves and tracts that may be absent.86 Complex repairs may need to be staged over many years or may be only palliative. Clinical use of nonvalved conduit sections of cardiac allografts, mostly from the main pulmonary artery and/or its branches, has reached a level equivalent to that of the two semilunar valve allografts. On a global scale, availability of cryopreserved pediatric allograft heart valves has historically been low and unable to meet demands. To obtain valve allografts, hearts are recovered aseptically, immersed in a sterile isotonic solution within a sterile container, placed on wet ice, and transported expeditiously to a tissue processing facility. The pulmonic and aortic valves, along with their intact outflow tracts and/or small pieces of these conduits, are dissected free of the heart within 48 hours of donor asystole, and then placed in tissue culture medium amended with a low-dose antibiotic cocktail. Initially, human heart valves were stored up until use at 5ºC in tissue culture medium containing antibiotics. However, although their cell viability was lost under these conditions after 48 hours, such grafts could be used successfully after a few weeks in such refrigerated storage. Subsequently, studies demonstrated that cryopreservation of heart valves allowed successful banking of valves of various sizes and types. Additionally, such allograft valves were found to be superior, based on their retaining the cellular matrix and having a low clinical incidence of valve degeneration, rupture, leaflet perforation, and valve-related death. For these reasons, human heart valves generally are cryopreserved,87 with a method that includes an initial exposure to antibiotic solutions for 12 to 24 hours. Cryopreservation then follows, using a 10% DMSO solution tissue culture medium that often is amended with 10% fetal

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calf serum. Freezing is accomplished using a computer-assisted controlled rate of ⫺1ºC/minute to ⫺40ºC. Valves generally are stored in the vapor phase of liquid nitrogen. Heart valves can be stored indefinitely in liquid nitrogen, although the nature of any deterioration during storage is not well characterized. The viability of cryopreserved connective tissue matrix cells is maintained, but at a lower level than that of fresh valves,88 and endothelial viability is lost.89 The aorta and iliac arteries can be preserved using the same methods applied to heart valves. Frequently, the aortic arch is preserved with the aortic valve intact; such grafts are intended for transplantation as a valved conduit. Preservation and storage methods are similar to those for valves. Synthetic grafts are the primary graft of choice, but such grafts may be less effective in an infected field. Aortoiliac arteries are used successfully as conduits in mycotic aneurysm repairs, when synthetic grafts have become infected, and for aortoenteric fistulas in an infected field.90 Arterial or venous segments of vascular organs may be recovered in order to provide a source of vascular “conduits” for use in organ transplants when the organ’s attached vessels are damaged or inadequate. Vascular “conduits” that have been recovered and transplanted under these conditions are not considered tissues under the FDA’s human cells, tissues, or cellular or tissue-based products (HCT/Ps) rules, but they are regulated as organs under 42 CFR Part 121. Donor screening and testing, as well as labeling and storage requirements, are identical to those for donor organs specified in a federal contract with the Organ Procurement and Transplantation Network. Autograft veins are used in cardiac and peripheral vascular bypass graft procedures whenever it is possible, but veins from deceased donors that have been recovered under aseptic conditions may be used for revascularization when autologous mammary or saphenous vein grafts are not available. These veins are more fragile than are the great vessels, and they are more easily damaged during recovery and during thawing and preparation for implantation. Cryopreservation of veins is similar to that of cardiac allografts. Arterial grafts are seldom used for this purpose because their layers tend to separate during thawing. Wellestablished tissue bank procedures are designed to retain venous endothelial cells during recovery, processing, and preservation, but these cells are rapidly sloughed off the lumen after the vein is transplanted into the high-pressure arterial system. Retention of endothelial cells during recovery and processing does aid, however, in reduction of the risk of thrombosis or failure after implantation through protection of the integrity of the vessel’s basement membrane.86 Although not proven to be necessary for successful clinical outcome or to prevent alloimmunization, ABO- and Rh-compatible allograft valves and vessel conduits are usually requested. Cryopreservation is known to alter the expression of antigens on donated cardiovascular tissue, but the mechanism of this effect is not well understood. Some studies have shown that the use of these tissue allografts may carry a risk of HLA antigen sensitization.91

Chapter 58: Tissue Banking

Peripheral Nerve

Reproductive Tissue

Fresh autografts of peripheral sensory nerves are used in nerve repair, but this practice is hampered by donor site morbidity and resulting limitations on the amount of autologous nerve tissue that can be made available. Although allografts ideally might repair peripheral nerve defects without requiring the sacrifice of the patient’s own nerve, frozen, irradiated, and lyophilized allografts have not functioned well. New animal studies using nerve allografts cold-preserved for 7 weeks have shown promising results, as have cultured Schwann cells added into synthetic conduits.92 Axogen, Inc. (Alachua, FL) has developed a thermally acellularized nerve allograft scaffold called Avance that is treated with chondroitinase in order to degrade chondroitin sulfate proteoglycan. Such grafts have been shown to inhibit both aberrant growth and retrograde regeneration in the absence of any immunosuppressive therapy. Animal studies employing such an approach demonstrated enhancement of nerve regeneration. The first human Avance nerve allograft was implanted in July 2007 into a 38-year-old man who had suffered a traumatic facial injury; a single nerve allograft was used to connect the severed nerve root to three nerve branches. The surgeons informally reported that the graft’s handling characteristics were superior to those of autograft tissue, but it is too early to assess the extent and character of nerve regeneration.

Semen Assisted reproductive technology procedures and artificial insemination of a female with her partner’s previously stored semen or with donor semen are established therapies for the clinical management of infertility or when a woman does not have a male partner. Cryopreserved semen is used in both forms of artificial insemination. Cryopreserved semen can be stored by a man, termed a “client depositor,” who may become sterile as a consequence of therapy for testicular malignancy or for another reason, for later use with his wife or other “intimate partner,” or even with a surrogate mother. Sperm can even be collected postmortem, but such a practice poses ethical issues regarding the lack of consent. According to the FDA, there are approximately 110 semen banks in the United States. In 2005, semen banks licensed by New York State processed 27,118 ejaculates from 962 donors into 145,751 vials/straws in inventory. These banks distributed 74,366 vials/straws of semen for clinical use (New York State Department of Health, unpublished data, 2005). Semen banks usually offer a library of donors from whom donated frozen semen specimens are available. The offering of a selection of donors facilitates the matching of donor’s hair and eye color, race, and other genetically determined characteristics with those of the intended father or co-parent or with those of both parents. These donors are usually “anonymous”; such a donor’s identity is known only to a few of the semen bank staff. However, some semen banks offer a program through which some donors have agreed to disclose their identities when offspring reach the age of 18 or, sometimes, even before use of the semen. Sexually transmitted diseases, including HIV infection, can be transmitted by donor semen to women undergoing artificial insemination.25,97,98 Cryopreservation permits extended storage and the retesting of donors at least 6 months after the donation of specimens to be released. This process is intended to prevent use of semen donated by a recently infected man, before development of detectable antibody or viral nucleic acid (during the “window period”). Other diseases and organisms transmissible by donor semen include hepatitis B, gonorrhea, Ureaplasma urealyticum, Mycoplasma hominis, Trichomonas vaginalis, and Chlamydia trachomatis. Transmission of HTLV-I, syphilis, HCV, and human papillomavirus may also be possible (see Table 58-4).25 The basic practices and techniques of semen cryopreservation have changed little since the cryopreservative glycerol was discovered accidentally by Polge and coworkers99 in 1949. Glycerol remains the standard cryoprotectant, and liquid nitrogen remains in use for storage. Freezing methods in use have been designed to control the rate of temperature decline, and to prevent thermal shock initially by cooling the semen slowly in air or in a waterbath to 5ºC before initiation of the actual freezing process, which takes place in the vapor phase of liquid nitrogen, or in a programmable controlled-rate freezing device. After freezing, semen can be stored in the liquid phase of liquid nitrogen indefinitely.

Parathyroid Hypercalcemia, kidney stones, and other complications associated with hyperparathyroidism can be treated by surgical removal of the parathyroids. Hyperparathyroidism often is caused by a single parathyroid adenoma, but in 10% of cases, generalized parathyroid hyperplasia is found, rendering the removal of all four parathyroids necessary. Postoperatively, the lack of parathyroid hormone can result in permanent hypocalcemia in 5% of patients. To prevent this outcome, autotransplantation of a small amount of parathyroid tissue is performed during total parathyroidectomy in order to provide a controlled source of parathyroid hormone.93 The parathyroid tissue is placed in the sternocleidomastoid muscle, flexor muscle groups, or subcutaneous tissue of the forearm. The remaining parathyroid tissue can be divided, placed in vials containing chilled tissue culture medium, and then cryopreserved using 10% autologous serum and 10% DMSO. The excess tissue then can be frozen under controlled conditions and stored in liquid nitrogen at ⫺196ºC.94 Frozen parathyroid autograft can be retrieved for subsequent use if the tissue implanted at time that the parathyroid was removed proves to be insufficient, fails to function, or becomes infected. Cryopreservation of parathyroid tissue with DMSO maintains cell viability and graft function. This is illustrated by postimplant amelioration of hypocalcemia and sustained elevation of parathyroid hormone in the venous effluent of the grafted forearm compared with that of the nongrafted forearm.95 Postthaw viability can also be demonstrated in vitro by the suppression of parathyroid hormone secretion by the addition of increasing calcium concentrations.96 The maximal duration of cryopreserved parathyroid tissue storage has not been determined.

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While a defective pregnancy as the result of sperm injury during the freezing-thawing process is a theoretic concern, such an effect has not been demonstrated. Cryopreservation of semen does not influence the frequency of abortions or multiple births, or the infant’s gender, body size, or intelligence.100 In fact, there is some evidence that indicates that the safety of cryopreserved semen actually might exceed that of fresh semen. One study reported finding birth defects in 0.7% of offspring and spontaneous abortion of 7.7% of pregnancies achieved using cryopreserved semen,101 whereas in the general population the frequency of birth defects is 6% and the frequency of spontaneous abortion is 10% to 18% of pregnancies.

Oocytes and Embryos Since the birth of Louise Brown, the world’s first “test tube baby,” on July 25, 1978, there has been an explosion in the use of assisted reproductive technologies, such that several techniques have been accepted as standard medical therapy. There are at least 422 assisted reproductive technology programs in the United States.4 While the technology to freeze unfertilized oocytes reliably is still in development, embryos are routinely cryopreserved. In 2005, ⬎32,000 embryos, 13% of which were created using donor oocytes and/or donor semen, were transferred into patients in New York. In addition, ⬎46,000 embryos were in storage in New York alone (New York State Department of Health, unpublished data, 2005). Many of those embryos were created using donor semen and/or donor oocytes. Donor oocytes were used in approximately 12% of all assisted reproductive technology cycles carried out in the United States in 2005.4 Many of these were embryos created for recipients ⬎35 years of age, using an oocyte donor who was much younger than the recipient. Embryo transfer success rates have been shown to be influenced far more by the age of the oocyte source than by the age of the uterus into which embryos are transferred. Medical history and infectious disease testing requirements similar to those for semen donors apply, although a quarantine period and retesting are not required.

Extraembryonic Tissue Preservation and Transplantation Extraembryonic tissues that have been used occasionally for transplantation include the amnion and the umbilical vein. Fetal amnion, which is the smooth, slippery, glistening membrane lining the fluid-filled space surrounding the fetus, has been used as a covering for nonhealing chronic leg ulcers, burns, and raw surfaces following mastectomy, and in major oral cavity reconstruction and vaginoplasty. Amnion also has been used as a pelvic peritoneum substitute following pelvic exenteration and as a source of replacement enzymes for infants with inborn errors of metabolism.102 Most of the fetal amnion is covered on the maternal side by the chorion, a slightly roughened membrane. Amnion is sterilely collected during cesarean section. The amnion’s epithelium and basement membrane can be separated by blunt dissection from the underlying chorion immediately after collection or after temporary storage. The amnion is then cryopreserved or lyophilized. Human umbilical vein allografts previously were used occasionally

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as vascular substitutes to provide venous access for hemodialysis or as an arterial bypass graft, but such allografts proved to be inferior to saphenous vein autografts. Such umbilical vein grafts are no longer available, following application of FDA device manufacturing requirements to their recovery and processing.

Donor-Recipient Matching For most tissues, donor-recipient HLA matching is not necessary and is rarely done. Tissues such as bone, fascia, tendon, cartilage, and dura mater are not preserved or transplanted in a viable state; rather, they serve as a support or matrix that the recipient’s own cells can enter and gradually replace. Immunologic rejection, therefore, is not a significant concern, and matching of blood group or HLA antigens is considered unnecessary. There are exceptions, however. Immunologic rejection can occur in patients who have received a repeat cornea graft; therefore, efforts are made to use HLA-matched corneas in these patients.103 HLA sensitization has also been reported in recipients of vascular allografts or allograft heart valves.104 The ABO antigens are a significant consideration in transplantation because they constitute very strong histocompatibility antigens. Because they are expressed on vascular endothelium, major ABO mismatching can cause rapid graft rejection resulting from endothelial damage by ABO antibodies and subsequent widespread thrombosis within the graft. Therefore, ABO matching is important to the success of vascularized organ grafts (ie, kidney, heart, liver, and pancreas). ABO matching is not important for a successful outcome when using most tissue grafts (ie, fascia, bone, heart valves, skin, and cornea). However, hypersensitivity to antigens expressed by fresh or cryopreserved donor tissue is a rare occurrence and appears to be dependent on an undetermined unusual immune response by the recipient. Alloimmunization to Rh(D), Fy(a), and Jk(b) red cell antigens following transplantation of frozen unprocessed bone has been reported.61,62 Consequently, frozen unprocessed bone allografts usually are matched with the donor for the D antigen if the recipient is a female of childbearing potential, in addition to being matched for ABO group.

Transfusion Service Support of Tissue Transplantation Hospital transfusion services have been greatly affected by transplantation, and have encountered new and increased demands for services. They are involved in transplantation in several ways, including: 1) providing traditional blood components, 2) providing new or special blood components, 3) taking responsibility for tissue acquisition, storage, distribution, and tracking, and 4) providing specialized services. For organ transplants, the major demand is for traditional blood components, although special preparation may be required (see Chapter 40).

Chapter 58: Tissue Banking

FDA regulations for tissue (see below) cover donor selection and testing, tissue recovery, processing, storage, labeling, and distribution to the “consignee” (21 CFR Part 1271). The consignee can be a distributor, a surgeon in a hospital operating room, a dentist in his or her office, or a designated individual or department in a hospital or other health-care institution. Tissue “banks” that are located in hospitals are not regulated by the FDA if they serve only to store and dispense tissues provided by comprehensive tissue banks or distributors. A hospital tissue service can be centralized in a support area for the operating rooms, hospital central supply services, or the hospital transfusion service. Alternatively, tissues can be handled using a decentralized system and be ordered, received, and stored by each functional area of the hospital in which they are used. Currently, most human deceased donor tissue (bone, skin, heart valves, veins, and tendons) is recovered by an organ and tissue procurement agency or regional tissue bank, sent for graft processing and packaging, and then distributed directly to the operating room or to other hospital sections in which tissue transplantation occurs. However, in the absence of centralization, records of storage and recipient identification may be inadequate. In one case involving an HIV-infected donor, the recipients of six of the tissues could not be identified from hospital records.16 The Joint Commission standard on record-keeping and traceability of tissues (PC 17.20), College of American Pathologists transfusion medicine checklist (TRM.45250, Phase II), and AABB Standards105 (Standards 5.1.6.2, 5.19.6.1, and 6.2.3) require that the organization’s records permit tracing of any tissue from the donor or source facility to all recipients or other final tissue disposition. However, New York State is the only government regulatory agency that requires centralized tracking of tissues to the recipients. The hospital transfusion service has the capacity, experience, and skills to act as a central depot and distribution point for all human tissue and to ensure that storage, issuance, and disposition records are maintained. Functions include allograft selection; vendor qualification and price negotiation; receipt of tissues, including inspection and accessioning; proper storage; inventory control; issuance; and record-keeping, which must ensure traceability to recipient or other disposition.106 Recordkeeping, especially if barcodes are used in the laboratory, is complicated by the fact that barcodes are not yet standardized among tissue banks, although adoption of ISBT code 128 labeling standards has been suggested.107 An additional challenge is that some tissues are produced in lots or batches, so each individual unit may not carry a unique identifier as is the case with blood components. Effective development of such tissue storage and distribution services takes time and relies on good relationships and communications with both operating room staff who will handle tissues and the surgeons who use them.106 A transfusion service operating as a central tissue repository and dispensing service may also be called upon to manage autologous tissues, such as calvaria (skull bone flaps), bone, skin, and parathyroid gland. Such tissues may require preparation and packaging before storage. Testing is not required, but careful

labeling and record-keeping are essential. Such tissues may ultimately be reimplanted in the original location (such as calvaria) or in a heterotopic location (such as limbs for parathyroid gland). It is prudent to establish time limits for storage either on an individual basis as specified by the surgeon or on a generalized basis, because stored tissues may not be claimed if the tissue was not needed because the patient died or for other reasons. The tissue dispensing service may also be called upon to package tissues in an appropriate, qualified, properly labeled transport container for transport to another institution.106

Reimbursement Reimbursement for tissue transplantation is similar to that for blood transfusion. The tissue bank recovers expenses through a service fee (per tissue) billed to the hospital. This service fee includes such costs as services rendered by the organ/tissue recovery agency; recovery supplies and logistical support of the recovery agent that may be provided by the tissue processor; the tissue processor’s operating costs associated with processing, storage, and distribution, as well as research and development; and overhead costs incurred with support of all operations. Health-care insurance carriers reimburse hospitals for most tissue service fees. Current procedural terminology codes specific to allograft transplantation procedures are available and used routinely.

Oversight With the rapid growth of all areas of tissue banking, there has been an increasing need for accountability and for measures that ensure that safe, quality tissues are available for clinical use. Quality improvement can be effected through voluntary standards, and most tissue banks have incorporated the achievement of high standards into their goals. The AATB has established comprehensive standards for donor screening, recovery and processing of musculoskeletal, cardiac, vascular, and skin tissues, and reproductive cells.10 Additionally, the standards contain: institutional requirements; descriptions of required functional components of a tissue bank; requirements for construction and management of records and development of procedures; requirements for informed consent, tissue labeling, storage, and release; expectations for handling adverse outcomes, investigations, and tissue recalls; requirements for establishment of a quality program; specifications for equipment and facilities; and guidelines for tissue dispensing services and tissue distribution intermediaries. AATB’s Standards for Tissue Banking are referenced not only by tissue bankers, but also by end-user health-care facilities, other standard-setting organizations, and regulators worldwide. In 2008, 104 tissue banks in North America held AATB accreditation. AABB Standards for Blood Banks and Transfusion Services105 address tissue inspection (Standards 4.3 and 5.10), handling (Standard 5.1.5), storage (Standard 5.1.8), preparation and

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dispensing (Standard 5.15.3), handling adverse events (Standard 7.4.4), and record-keeping, which must provide traceability to each recipient or other disposition (Standards 5.19.6.1, 5.1.6.2, and 6.2.3). The Joint Commission implemented standards for storage and issuance of tissue for hospitals, ambulatory surgery centers, effective July 1, 2005 (Standards PC.17.10, PC.17.20, PC.17.30). These standards apply to bone, tendon, fascia, and cartilage, as well as other cellular tissues of both human and animal (xenograft) origin. The standards address key functions, including the need to develop procedures for tissue acquisition and storage, record-keeping and tracking, and follow-up of adverse events and suspected allograft-caused infections, which must be reported to the tissue bank from which the tissue was obtained. Similar to federal regulations and AATB Standards, the minimal record retention period is specified to be 10 years from the date of transplantation, distribution, other disposition, or expiration, whichever is latest. The College of American Pathologists’ laboratory accreditation program’s transfusion medicine checklist includes several questions on storage and issuance of tissues, including accountability; procedures for proper storage, handling, in accordance with the source facility’s directions; procedures for investigating recipient infections and adverse events, and handling look-back notifications from a supplier; and record-keeping, which allow for tracking from donor to recipient and vice versa (TRM.45050 - TRM.45250). Until recently, large segments of the tissue banking industry have been unregulated by government agencies. A few states, specifically New York, Florida, and California, adopted licensure requirements in the early 1990s. The New York State Department of Health has the most comprehensive oversight program. Beginning in 1991, New York established administrative, licensure, and technical requirements for all entities that collect, process, store, and/or distribute tissue in New York. Currently, 817 tissue banks, including 79 banks that recover and/or process cardiovascular, musculoskeletal, and/or skin tissue from deceased donors; 18 eye banks; 64 semen banks; 39 oocyte-embryo banks; 63 hematopoietic progenitor cell banks; and 84 nontransplant anatomic banks (banks providing bodies, body parts, and tissues for health professional education and medical research), are licensed to operate in New York. New York State regulations extend to the clinical use of tissue, such as physicians’ offices where semen is used in artificial insemination and hospitals and ambulatory surgery centers that transplant tissue. Such transplantation services constitute the remainder of licensed tissue banks. A 2001 federal report found, however, that 44% of tissue banks held neither accreditation by a professional organization nor licensure by a state agency.13 FDA authority to create and “enforce regulations necessary to prevent the introduction, transmission, or spread of communicable diseases between the States or from foreign countries into the States” under section 361(a) of the US Public Health Service Act (42 USC 264) applies to human tissue intended for transplantation. Formal enforcement policy and regulations did not exist until December 14, 1993 (codified in 21 CFR Parts 16

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and 1270), when the “Interim Rule: Human Tissue Intended for Transplantation,” which required donor screening, infectious disease testing and record-keeping “to prevent transmission of infectious diseases through human tissue used in transplantation,” was adopted in response to reports of HIV transmission by human tissue and of potentially unsafe bone imported into the United States.108 Reproductive tissue, human milk, and marrow were specifically excluded. These regulations were supplanted by a series of federal regulations, published in stages, first announced in the Proposed Approach to the Regulation of Cellular and Tissue-Based Products in March 1997.109 A final rule, “Human Cells, Tissues, and Cellular and Tissue-Based Products: Establishment Registration and Listing,” published in January 2001, required organizations that are engaged in tissue recovery, donor qualification, tissue processing, and/or tissue-related laboratory testing to register as a tissue establishment with the FDA. The rule (21 CFR Part 1271) became effective for all tissue banks on March 29, 2004. A final rule, “Eligibility Determination for Donors of Human Cells, Tissues, and Cellular and Tissue-Based Products,” published May 25, 2004, set forth donor eligibility requirements, including health history screening and laboratory testing. Another final rule, “Current Good Tissue Practice for Human Cell, Tissue and Cellular and Tissue-Based Product Establishments; Inspection and Enforcement,” published on November 24, 2004, established elements of good tissue practice, analogous to good manufacturing practice for blood banks. Both rules became effective May 25, 2005 (see 21 CFR Parts 1270 and 1271). An interim final rule, published on May 25, 2005 for immediate implementation, made a few technical corrections regarding donor screening and testing and product labeling. Following consideration of comments regarding the interim final rule, the final rule was published on June 19, 2007, but was unchanged from the May 25, 2005 rule (see 21 CFR Parts 1270 and 1271). In addition to requirements for establishment registration, donor eligibility screening and testing, and good tissue practice, the regulations set forth requirements for adverse reaction reporting and also define inspection and recall authority. To improve tissue safety and surveillance, the FDA Current Good Tissue Practice Rule, effective May 25, 2005, requires that tissue establishments report infectious adverse events after allograft transplantation to the FDA through its MedWatch adverse event reporting system. In fiscal year 2006, the first full year of the requirement, 70 reports were filed by tissue banks; 44 of these reports were flagged to indicate possible recall of tissue(s). Of these flagged reports, the majority (26) pertained to acceptance of ineligible donors for whom one or more components of the donor qualification process was not performed or was insufficiently documented. The FDA also encourages healthcare professionals, patients, and consumers to voluntary report tissue adverse reactions through the MedWatch system (see http://69.20.19.211/CBER/ tissue/hctadverse.htm). The CDC is currently funding efforts to develop the Transplantation Transmission Sentinel Network, which is intended to facilitate

Chapter 58: Tissue Banking

recognition of adverse events associated with transplanted allografts. The system is being developed by UNOS, in collaboration with several stakeholders, including the AATB, EBAA, Association of Organ Procurement Organizations, American Academy of Orthopaedic Surgeons, American Orthopaedic Society for Sports Medicine, Society of Thoracic Surgeons, HRSA, and FDA.

Acknowledgments The authors thank Scott Brubaker, Lisa Eisenlohr, Patricia Dahl, and Perry Lange for their technical advice and Marcia Kolakoski for her assistance with manuscript preparation.

Disclaimer The authors have disclosed no conflicts of interest.

References 1. Eisenbrey AB, Gottschall JL, eds. Hospital tissue management: A practitioner’s handbook. Bethesda, MD: AABB, 2008. 2. McAllister DR, Joyce MJ, Mann BJ, Vangsness CT Jr. Allograft update: The current status of tissue regulation, procurement, processing, and sterilization. Am J Sports Med 2007;35:2148-58. 3. Eye Bank Association of America. 2006 Eye banking statistical report. Washington, DC: Eye Bank Association of America, 2007. 4. Centers for Disease Control and Prevention; American Society for Reproductive Medicine; Society for Assisted Reproductive Technology. 2005 Assisted reproductive technology success rates: National summary and fertility clinic reports. Atlanta, GA: Centers for Disease Control and Prevention, 2007. 5. Milk bank locations. Raleigh, NC: Human Milk Banking Association of North America, 2008. [Available at: http://www.hmbana.org/ index.php;mode⫽locations (accessed January 15, 2008)]. 6. Health Care Financing Administration. 42 CFR Part 482. Final rule. Medicare and Medicaid programs: Hospital conditions of participation: Identification of potential organ, tissue and eye donors and transplant hospitals provision of transplant-related data. Fed Regist 1998;63:33856-75. 7. Haurwitz RKM. Corneas removed without permission. Austin American Statesman, August 18, 2002. [Available at http://www.austin360.com/statesman/editions/sunday/news_1.html]. 8. Lee PP, Stark WJ, Yang JC. Cornea donation laws in the United States. Arch Ophthalmol 1989;107:1585-9. 9. Prottas J, Batten HL. Health professionals and hospital administrators in organ procurement: Attitudes, reservations, and their resolutions. Am J Public Health 1988;78:642-5. 10. Pearson K, Dock N, Brubaker S, eds. Standards for tissue banking. 12th ed. McLean, VA: American Association of Tissue Banks, 2008. 11. Eye Bank Association of America. Medical standards. Washington, DC: Eye Bank Association of America, 2007. 12. The American Society for Reproductive Medicine. Guidelines for gamete and embryo donation. Birmingham, AL: The American Society for Reproductive Medicine, 2006.

13. Department of Health and Human Services Office of Inspector General. Informed consent in tissue donation: Expectations and realities. Boston, MA: Office of Inspector General, 2001. 14. Eastlund T. Infectious disease transmission through cell, tissue, and organ transplantation: Reducing the risk through donor selection. Cell Transplant 1995;4:455-77. 15. Eastlund T. Viral infections transmitted through tissue transplantation. In: Kennedy JF, Phillips GO, Williams PA, eds. Sterilization of tissues using ionizing radiations. Boca Raton, FL: CRC Press, 2005:255-78. 16. Eastlund T. Bacterial infection transmitted by human tissue allograft transplantation. Cell Tissue Bank 2006;7:147-66. 17. Simonds RJ, Holmberg SD, Hurwitz RL, et al. Transmission of human immunodeficiency virus type 1 from a seronegative organ and tissue donor. N Engl J Med 1992;326:726-32. 18. Tugwell BD, Patel PR, Williams IT, et al. Transmission of hepatitis C virus to several organ and tissue recipients from an antibodynegative donor. Ann Intern Med 2005;143:648-54. 19. Zou S, Dodd RY, Stramer SL, Strong DM. Tissue Safety Study Group. Probability of viremia with HBV, HCV, HIV, and HTLV among tissue donors in the United States. N Engl J Med 2004;351:751-9. 20. Strong DM, Nelson K, Pierce M, Stramer SL. Preventing disease transmission by deceased tissue donors by testing blood for viral nucleic acid. Cell Tissue Bank 2005;6:255-62. 21. Centers for Disease Control and Prevention. Possible West Nile virus transmission to an infant through breast-feeding—Michigan, 2002. MMWR Morb Mortal Wkly Rep 2002;51:877-8. 22. Centers for Disease Control and Prevention. Chagas disease after organ transplantation—United States, 2001. MMWR Morb Mortal Wkly Rep 2002;51:210-2. 23. Centers for Disease Control and Prevention. Chagas disease after organ transplantation—Los Angeles, California, 2006. MMWR Morb Mortal Wkly Rep 2006;55:798-800. 24. Srinivasan A, Burton EC, Kuehnert MJ, et al. Rabies in Transplant Recipients Investigation Team. Transmission of rabies virus from an organ donor to four transplant recipients. N Engl J Med 2005;352:1103-11. 25. Linden JV, Critser JK. Therapeutic insemination by donor II: A review of its known risks. Reprod Med Rev 1995;4:19-29. 26. Stewart GJ, Tyler JP, Cunningham AL, et al. Transmission of human T-cell lymphotropic virus type III (HTLV-III) by artificial insemination by donor. Lancet 1985;ii:581-5. 27. Chiasson MA, Stoneburner RL, Joseph SC. Human immunodeficiency virus transmission through artificial insemination. J Acquir Immune Defic Syndr 1990;3:69-72. 28. Rekart ML. HIV transmission by artificial insemination (abstract). IVth International Conference on AIDS. Washington, DC: BIODATA Publishers, 1993:266. 29. Mascola L, Araneta M, Eller A, et al. Risk of HIV infection following artificial insemination (abstract). IXth International Conference on AIDS in affiliation with the IVth STD World Congress. London: Sage Publications, 1993:647. 30. Centers for Disease Control. Epidemiologic notes and reports HIV-1 infection and artificial insemination with processed semen. MMWR Morb Mortal Wkly Rep 1990;39:249, 255-6. 31. Berry WR, Gottesfeld RL, Alter HJ, Vierling JM. Transmission of hepatitis B virus by artificial insemination. JAMA 1987; 257:1079-81. 32. Fiumara NJ. Transmission of gonorrhoea by artificial insemination. Br J Vener Dis 1972;48:308-9.

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33. Hansen KB, Nielsen NC, Rebbe H. Artificial insemination in Denmark by frozen donor semen supplied from a central bank. Br J Obstet Gynaecol 1979;86:384-6. 34. Barwin BN. Transmission of Ureaplasma urealyticum by artificial insemination. Fertil Steril 1984;41:326-7. 35. Caspi E, Herczeg E, Solomon F, Sompolinsky D. Amnionitis and T strain mycoplasmemia. Am J Obstet Gynecol 1971;111:1102-6. 36. Kleegman SJ. Therapeutic donor insemination. Conn Med 1967;31:705-13. 37. Moore DE, Ashley RL, Zarutskie PW, et al. Transmission of genital herpes by donor insemination. JAMA 1989;261:3441-3. 38. Nagel TC, Tagatz GE, Campbell BF. Transmission of Chlamydia trachomatis by artificial insemination. Fertil Steril 1986;46:959-60. 39. Perper JA. Time of death and changes after death, Part 1: Anatomical considerations. In: Spitz WU, ed. Spitz and Fisher’s medicolegal investigation of death: Guidelines for the application of pathology to crime investigation. 3rd ed. Springfield, IL: Charles C. Thomas, 1993:14-49. 40. Kainer MA, Linden JV, Whaley DN, et al. Clostridium infections associated with musculoskeletal-tissue allografts. N Engl J Med 2004;350:2564-71. 41. Centers for Disease Control. Septic arthritis following anterior cruciate ligament reconstruction using tendon allografts—Florida and Louisiana, 2000. MMWR Morb Mortal Wkly Rep 2001;50: 1081-3. 42. Centers for Disease Control and Prevention. Update: Allograftassociated bacterial infections—United States, 2002. MMWR Morb Mortal Wkly Rep 2002;51:207-10. 43. Bowen PA II, Lobel SA, Caruana RJ, et al. Transmission of human immunodeficiency virus (HIV) by transplantation: Clinical aspects and time course analysis of viral antigenemia and antibody production. Ann Intern Med 1988;108:46-8. 44. Cloward RB. Gas-sterilized cadaver bone grafts for spinal fusion operations. A simplified bone bank. Spine 1980;5:4-10. 45. Janovec M, Dvorak K. Autolyzed antigen-extracted allogeneic bone for bridging segmented diaphyseal bone defects in rabbits. Clin Orthop Relat Res 1988;229:249-56. 46. Cornell C, Lane JM, Nottebaert M, et al. Effect of ethylene oxide sterilisation upon bone inductive properties of demineralised bone matrix. Transfus Med 1987;15:165-74. 47. Sherman P, Hollinger P. Bone implant sterilization-ethylene oxide versus cobalt 60 irradiation. Presentation at the annual meeting of the American Association of Oral and Maxillofacial Surgery, Boston, MA, September 29-30, 1988. 48. Glowacki J, Kaban LB, Murray JE, et al. Application of the biological principle of induced osteogenesis for craniofacial defects. Lancet 1981;1:959-62. 49. DeVries PH, Badgley CE, Hartman JT. Radiation sterilization of homogenous bone transplants utilizing radioactive cobalt. J Bone Joint Surg 1985;40:187-203. 50. Zasacki W. The efficacy of application of lyophilized, radiationsterilized bone graft in orthopedic surgery. Clin Orthop Relat Res 1991;272:82-7. 51. Komender J, Malczewska H, Komender A. Therapeutic effects of transplantation of lyophilized and radiation-sterilized, allogeneic bone. Clin Orthop Relat Res 1991;272:38-49. 52. Loty B, Courpied JP, Tomeno B, et al. Bone allografts sterilised by irradiation. Biological properties, procurement and results of 150 massive allografts. Int Orthop 1990;14:237-42.

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53. Nguyen H, Morgan DAF, Forwood MR. Sterilization of allograft bone: Effects of gamma irradiation on allograft biology and biomechanics. Cell Tissue Bank 2007;8:93-105. 54. Eastlund T. Tissue and organ transplantation and the hospital tissue transplantation service. In: Roback JD, Combs MR, Grossman BJ, Hillyer CD, eds. Technical manual, 16th edition. Bethesda, MD: AABB, 2008:833-64. 55. Woll JE, Smith DM. Bone and connective tissue. Clin Lab Med 2005;25:499-518. 56. Ott HC, Matthiesen TS, Goh SK, et al. Perfusion-decellularized matrix: Using nature’s platform to engineer a bioartificial heart. Nat Med 2008;14:213-21. 57. Mian S, Kamyar R, Sugar A, et al. Regulation of eye banking and uses of ocular tissue for transplantation. Clin Lab Med 2005;25: 607-24. 58. Eastlund T. Bone transplantation and bone banking. In: Lonstein JE, Bradford DS, Winter RB, Ogilvie J, eds. Moe’s textbook of scoliosis and other spinal deformities. 3rd ed. Philadelphia, PA: WB Saunders, 1995:581-94. 59. Stevenson S, Horowitz M. The response to bone allografts. J Bone Joint Surg Am 1992;74:939-50. 60. Friedlaender GE, Horowitz MC. Immune responses to osteochondral allografts: Nature and significance. Orthopedics 1992;15: 1171-5. 61. Musclow CE, Dietz G, Bell RS, et al. Alloimmunization by blood group antigens from bone allografts. Immunohematol 1992;9:102-4. 62. Cheek RF, Harmon JV, Stowell CP. Red cell alloimmunization after a bone allograft. Transfusion 1995;35:507-9. 63. Dziedzic-Goclawska A, Ostrowski K, Stachowicz W, et al. Effect of radiation sterilization on the osteoinductive properties and the rate of remodeling of bone implants preserved by lyophilization and deep-freezing. Clin Orthop Relat Res 1991;272:30-7. 64. Lang J, Kerr AG, Smyth GD. Long-term viability of transplanted ossicles. J Laryngol Otol 1986;100:741-7. 65. Gross AE. Repair of cartilage defects in the knee. J Knee Surg 2002;15:167-9. 66. Oakeshott RD, Farine I, Pritzker KP, et al. A clinical and histologic analysis of failed fresh osteochondral allografts. Clin Orthop Relat Res 1988;233:283-94. 67. Schachar NS, Cucheran DI, Frank CB. Viability of intact articular cartilage at various times after donor death. Trans Orthop Res Soc 1988;12:436. 68. 21 CFR Parts 211, 226, 300, 500, 530, 600, 895, and 1271. Use of materials derived from cattle in medical products intended for use in humans and drugs intended for use in ruminants; proposed rule. Fed Regist 2007;72:1581-619. 69. Tomford WW, Mankin HJ, Friedlaender GE, et al. Methods of banking bone and cartilage for allograft transplantation. Orthop Clin North Am 1987;18:241-7. 70. Malinin TI, Mnaymneh W, Lo HK, Hinkle DK. Cryopreservation of articular cartilage. Ultrastructural observations and long-term results of experimental distal femoral transplantation. Clin Orthop Relat Res 1994;303:18-32. 71. Noyes FR, Barber-Westin SD, Rankin M. Meniscal transplantation in symptomatic patients less than fifty years old. J Bone Joint Surg Am 2004;86-A:1392-404. 72. von Lewinski G, Milachowski KA, Weismeier K, et al. Twentyyear results of combined meniscal allograft transplantation,

Chapter 58: Tissue Banking

73. 74.

75.

76. 77.

78.

79. 80.

81. 82. 83.

84. 85.

86. 87.

88.

89.

90.

anterior cruciate ligament reconstruction and advancement of the medial collateral ligament. Knee Surg Sports Traumatol Arthrosc 2007;15:1072-82. Tom JA, Rodeo SA. Soft tissue allografts for knee reconstruction in sports medicine. Clin Orthop Relat Res 2002;402:135-56. Bechtold JE, Eastlund DT, Butts MK, et al. The effects of freezedrying and ethylene oxide sterilization on the mechanical properties of human patellar tendon. Am J Sports Med 1994;22:562-6. Gibbons JI, Butler DL, Grood ES, et al. Dose-dependent effect of γ-irradiation on the material properties of frozen bone-patellar tendon-bone allografts. Trans Orthop Res Soc 1989;14:513. De Deyne P, Haut RC. Some effects of gamma irradiation on patellar tendon allografts. Connect Tissue Res 1991;27:51-62. Brown P, Rowher RG, Gajdusek DC. Sodium hydroxide decontamination of Creutzfeldt-Jakob disease virus (letter). N Engl J Med 1984;310:727. Brown P, Rowher RG, Gajdusek DC. Newer data on the inactivation of scrapie virus or Creutzfeldt-Jakob disease virus in brain tissue. J Infect Dis 1986;153:1145-8. Muller MJ, Herndon DN. The challenge of burns. Lancet 1994;343:216-20. Konstantinow A, Muhlbauer W, Hartinger A, von Donnersmarck GG. Skin banking: A simple method for cryopreservation of splitthickness skin and cultured human epidermal keratinocytes. Ann Plast Surg 1991;26:89-97. Smolin G, Goodman D. Corneal graft reaction. Int Ophthalmol Clin 1988;28:30-6. Nathan R. Combined pedicle and scleral grafts for alveolar ridge augmentation. CDA J 1983;11:44-5. Bourne WM. Corneal preservation. In: Kaufman HE, McDonald MB, Barron BA, et al, eds. The cornea. New York: Churchill Livingstone, 1988:713-24. Doughman DJ. Corneal tissue preservation. Int Ophthalmol Clin 1988;28:50-6. Doughman DJ, Harris JE, Mindrup E, et al. Prolonged donor corneal preservation in organ culture: Long-term clinical evaluation. Cornea 1992;1:7-20. Hopkins RA. Cardiac reconstructions with allograft tissues. New York: Springer, 2005. O’Brien MF, Stafford EG, Gardner MAH, et al. Cryopreserved viable allograft aortic valves. In: Yankah AC, Hetzer R, Miller DC, et al, eds. Cardiac valve allografts 1972-1987. New York: Springer-Verlag, 1988:311-21. Messier RH Jr, Domkowski PW, Aly HM, et al. High energy phosphate depletion in leaflet matrix cells during processing of cryopreserved cardiac valves. J Surg Res 1992;52:483-8. Loose R, Markant S, Sievers HH, Bernhard A. Fate of endothelial cells during transport, cryopreservation, and thawing of heart valve allografts. Transplant Proc 1993;25:3247-50. Vogt PR, Brunner-La Rocca HP, Carrel T, et al. Cryopreserved arterial allografts in the treatment of major vascular infection: A comparison with conventional surgical techniques. J Thorac Cardiovasc Surg 1998;116:965-72.

91. Gandhi MJ, Strong DM. Cardiovascular tissues for transplantation. Clin Lab Med 2005;25:571-85. 92. Fox IK, Jaramillo A, Hunter DA, et al. Prolonged cold preservation of nerve allografts. Muscle Nerve 2005;31:59-69. 93. Walker RP, Paloyan E, Kelley TF, et al. Parathyroid autotransplantation in patients undergoing a total thyroidectomy: A review of 261 patients. Otolaryngol Head Neck Surg 1994;111:258-64. 94. Herrera MF, Grant CS, van Heerden JA, et al. The effect of cryopreservation on cell viability and hormone secretion in human parathyroid tissue. Surgery 1992;112:1096-101. 95. Rothmund M, Wagner PK. Assessment of parathyroid graft function after autotransplantation of fresh and cryopreserved tissue. World J Surg 1984;8:527-33. 96. Wagner PK, Rumpelt HJ, Krause U, Rothmund M. The effect of cryopreservation on hormone secretion in vitro and morphology of human parathyroid tissue. Surgery 1986;99:257-64. 97. Mascola L, Guinan ME. Screening to reduce transmission of sexually transmitted diseases in semen used for artificial insemination. N Engl J Med 1986;314:1354-9. 98. Araneta MR, Mascola L, Eller A, et al. HIV transmission through donor artificial insemination. JAMA 1995;273:854-8. 99. Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 1949;164:666-9. 100. Karow AM. Human gametes. In: Karow AM, Pegg DE, eds. Organ preservation for transplantation. New York: Marcel Dekker, 1981:377-409. 101. Sherman JK. History of artificial insemination and the development of human semen banking. In: LaSalle B, Rinfret AP, eds. The integrity of frozen spermatozoa. Washington, DC: National Academy of Sciences, 1978:201-7. 102. Scaggiante B, Pineschi A, Sustersich M, et al. Successful therapy of Niemann-Pick disease by implantation of human amniotic membrane. Transplantation 1987;44:59-61. 103. Forstot SL, Binder PS. Corneal transplantation. In: Chatterjee SN, ed. Organ transplantation. Boston, MA: John Wright, 1982:557-88. 104. Balzer KM, Luther B, Sandmann W, Wassmuth R. Donor-specific sensitization by cadaveric venous allografts used for arterial reconstruction in peripheral arterial occlusive vascular disease. Tissue Antigens 2004;64:13-17. 105. Price TH. Standards for blood banks and transfusion services. 25th ed. Bethesda, MD: AABB, 2008. 106. Eisenbrey AB, Strong DM. Tissue banking in the hospital setting. In: Hillyer CD, Silberstein LE, Ness PM, et al, eds. Blood banking and transfusion medicine—basic principles and practice. 2nd ed. Philadelphia, PA: Churchill Livingstone, 2007:853-9. 107. Fehily D, Ashford P, Poniatowski S. Traceability of human tissues for transplantation: The development and implementation of a coding system using ISBT 128. Organs Tissues Cells 2004;2:83-8. 108. 21 CFR Parts 16 and 1270. Human tissue intended for transplantation; interim rule. Fed Regist 1993;58:65514-21. 109. Food and Drug Administration. Proposed approach to regulation of cellular and tissue-based products. Fed Regist 1997;62:9721-2.

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59

Adoptive Immunotherapy Catherine M. Bollard1 & Helen E. Heslop2 1

Associate Professor of Pediatrics, Immunology and Medicine, Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children’s Hospital and The Methodist Hospital, Houston, Texas, USA 2 Professor of Medicine and Pediatrics and Dan L. Duncan Chair, Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children’s Hospital and The Methodist Hospital, Houston, Texas, USA

Adoptive immunotherapy can be defined as the transfer of cellular products with the goal of increasing immunity against cancer or infections. A number of populations have been evaluated in clinical trials including donor leukocyte infusions (DLI) from allogeneic hematopoietic stem cell transplantation (HSCT) donors, activated T-cell population cells, natural killer (NK) cells, and antigen-specific T cells. At present, there is a defined role for adoptive immunotherapy using T-cell approaches in treating relapse of certain malignancies and some types of infection after allogeneic HSCT.1-5 Extending this modality of treatment to patients with malignancies outside the HSCT setting is theoretically attractive, but successful applications are currently limited to settings where the tumors express immunogenic antigens such as viral antigens. This chapter reviews the current status of adoptive immunotherapy strategies for malignant and infectious diseases in the clinic and current areas of research to improve and extend the applicability of this approach.

Targets for Immunotherapy for Cancer A necessary prerequisite for T-cell immunotherapy approaches for cancer is identification of target antigens on the tumor cells. Advances in genomics have facilitated the identification of putative tumor antigens through the use of technologies such as serologic analysis of recombinant cDNA expression libraries and bioinformatics tools to deduce epitopes from candidate genes. However, for a tumor to be suceptible to immunotherapy approaches it must not only contain unique proteins to provide epitopes for targeting by specific immune responses, but must also present these peptides frequently enough and for sufficient duration to engage responder T cells. In addition, either the tumor cell or a specialized antigen-presenting cell through the

Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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process of cross-priming must express major histocompatibility complex (MHC) antigens and express costimulatory molecules such as CD28 to induce T-cell activation. Potential antigens for targeting tumor cells fall into several major categories (Table 59-1).

Alloantigens In the setting of allogeneic HSCT, alloantigens that differ between donor and recipient are targets for T-cell recognition. Alloantigens include human MHC molecules (in HLA-mismatched transplants) and minor histocompatibility antigens, which are naturally processed peptides derived from normal cellular proteins where different polymorphisms are present in donor and recipient.18,19 A number of minor histocompatibility antigens have been identified, and recent data showing that responses to donor lymphocyte infusions correlate with increase in immune responses directed against these antigens confirm their importance.20,21 In some of these cases, alloreactivity also results in graft-vs-host disease (GVHD), but in cases where the minor antigen targeted is selectively expressed on hematopoietic cells, a graft-vs-leukemia (GVL) effect can occur in the absence of GVHD.

Virus-Associated Tumor Antigens Some cancers (eg, subsets of lymphomas and nasopharyngeal carcinomas) are associated with viruses that present unique epitopes that are usually highly immunogenic as targets for a T-cell response. Latent Epstein-Barr virus (EBV) infection is associated with a heterogeneous group of malignant diseases (Fig 59-1) including posttransplant lymphoproliferative disease (PTLD), Hodgkin disease, nasopharyngeal carcinoma, and gastric carcinoma.4,22,23 The role of EBV in tumorigenesis is well established in the EBV-LPDs that arise in immunosuppressed individuals but is less clear in other EBV-associated malignancies. Nevertheless, the presence of EBV in tumor cells offers a target for immunotherapy approaches. Human herpesvirus-8 (HHV-8) is primarily associated with Kaposi’s sarcoma and primary effusion lymphoma in patients with AIDS, but is also found in some solid tumors.24 Unlike most malignant cells, which

Chapter 59: Adoptive Immunotherapy

Table 59-1. Potential Targets for Immunotherapy of Cancer Target

Examples

Malignancies

Viral antigens

Epstein-Barr virus

SV40

Non-Hodgkin lymphoma6 Hodgkin disease7 Nasopharyngeal carcinoma Non-Hodgkin lymphoma8

Differentiation antigen

CD19 or CD20 CD30

Leukemias and lymphomas9 Hodgkin disease and non-Hodgkin lymphoma10

Cancer testis antigens

MAGE antigens MART-1 NY-ESO SSX antigens

Testicular, melanoma, many carcinomas, and many hematologic malignancies11-13

Minor antigens

HA1, HA2

After transplantation for leukemia and lymphoma14

Tumor-specific antigens

Telomerase Idiotype

Many types of malignancies15 Non-Hodgkin lymphoma16,17

High Immunogenicity

Low

LMP 1

Figure 59-1. Type of Epstein-Barr-virus (EBV) latency. The three types of EBV latency are defined by the number and type of latent protein expressed on EBV-infected B cells and EBV-positive tumor cells.

LMP2

LMP 1

LMP2

EBNA1 EBERs

EBNA1 EBERs

EBNA1-6 EBERs

Type 1 Latency

Latency II Type 2 Latency

Type 3 Latency

Hodgkin lymphoma Non-Hodgkin lymphoma Nasopharyngeal carcinoma

Posttransplant lymphoma HIV-associated lymphoma Lymphoblastoid cell lines

Burkitt lymphoma Gastric adenocarcinoma

probably express small quantities of a single modified peptide as a cytotoxic T lymphocyte (CTL) target, virus-transformed cells may express a range of viral antigens in high copy number. These cells are, therefore, potentially much more susceptible to T-lymphocyte-mediated immunotherapy than tumor cells transformed by other mechanisms.

Lineage-Specific Antigens Differentiation antigens selectively expressed in tumor cells provide another potential target. In the context of hematologic malignancies, antigens such as CD19 and CD20 (B-lineage), CD33 (myeloid), and CD30 (expressed on Hodgkin’s Reed Sternberg cells) have been successfully targeted with antibody therapy, and preclinical studies have identified peptides that may be targets for T-cell recognition.25,26

Universal Antigens Several antigens have been identified that are overexpressed in a number of different tumor cells including telomerase and

cytochrome P450 1B1.15,27 Antigens that are crucial for tumor cell function such as cyclin or other cell cycle genes are other potential targets.28

Cancer Testis Antigens The cancer testis antigens (CTAs) comprise, to date, 44 families of genes or isoforms. Among these, 19 families are purely testisrestricted, while others are expressed in one or more somatic tissues.11 The physiologic expression of CTAs within the testis prevents their effective presentation to the immune system; hence, for those expressed only in testis, T-cell tolerance does not appear to be established. Expression of the MAGE-1 CTA was first shown in melanoma, but subsequently, expression of multiple CTAs was demonstrated in a variety of tumors.11-13 The mechanisms underlying the expression of CTAs are only partially resolved. Epigenetic regulation seems critical and demethylation of CTA promoter sequences has been observed in malignant cells.11 CTA-specific T cells able to destroy tumor cells were first identified in patients with melanoma, but subsequently were

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isolated from patients with a range of tumors29,30 and have the potential to be used as a target for many cancer immunotherapies.31,32 For example, NY-ESO-1 is frequently expressed in melanoma and poor prognosis B-cell malignancies and is capable of eliciting spontaneous humoral and T-cell immunity.30 However, it remains a challenge to expand CTLs specific for such tumor antigens in vitro to the numbers required for clinical use.

Targets for T-Cell Immunotherapy for Infections In the setting of immunotherapy for viral or fungal infection, it is also crucial to know which antigens are most important for viral/ fungal persistence and thus the best targets for immunotherapy strategies. The viral antigens expressed at different stages of viral infection have been well characterized for some viruses such as cytomegalovirus (CMV) and more recently adenovirus, but are less well defined for other viruses that cause morbidity such as BK virus.33,34 Most published studies designed to assess; the immune response to fungal antigens (eg, Aspergillus fumigatus) use crude extracts from mycelia and spores. Numerous recombinant A. fumigatus allergens associated with allergic bronchopulmonary aspergillosis have been characterized but most elicit humoral immune responses and others are toxic to peripheral blood mononuclear cells (PBMCs), are strongly homologous to human proteins, or only prime a Th2 T cell response.35 Therefore it is critical to identify allergens (eg, Asp f2 and Asp f16) that have been shown to stimulate both T- and B-cell responses from patients. Over the past few years, however, antigen identification has been simplified by the availability of tools that have enabled peptide mapping of specific viral and fungal epitopes recognized by CD4
and CD8
T cells.34,36,37

Types of Adoptive Immunotherapy Immunotherapeutic approaches that have been evaluated in the clinic can be broadly divided into studies using 1) nonspecific T cells, 2) antigen-specific T cells, 3) genetically modified T cells, and 4) NK cells.

DLI alone. Although the etiology is unclear, this could be caused by lack of antigenic expression, downregulation of T-cell recognition molecules, and/or overall tumor burden at the time of treatment. Although treatment with DLI can induce remissions in patients with disease after HSCT, unmanipulated cells also contain alloreactive T cells and can induce GVHD. The incidence of GVHD ranges from 55% to 90% and is associated with a 20% treatment-related mortality rate.5 Nevertheless, studies evaluating chronic myelogenous leukemia (CML) responses to DLI have found that up to 55% of patients who do not experience GVHD may have a disease response,43,44 demonstrating that GVL effect can be separated from GVHD. Therefore, one strategy to reduce the risk of GVHD is to infuse escalating doses of T cells in an attempt to obtain the GVL effect without GVHD. Peggs et al45 conducted a dose escalation study using DLI after HSCT for patients with hematologic malignancies and showed that in sibling transplantation, higher DLI doses were associated with a significantly increased risk of GVHD. In addition GVHD was more common, occurred at lower T-cell doses, and was more severe in the unrelated donor cohort, likely reflecting the increased alloreactivity between donor and recipient. As another approach to separate GVHD and GVL effect with DLI, investigators have evaluated specific subsets of lymphocytes, such as CD4-selected cells,46 CD8-depleted cells,47 or functionally defined subsets such as Th2 cells.48 In animal models, the culture of functionally defined T-cell subsets, such as Tc2 or Th2 cells, has allowed antitumor effects to be produced in the absence of alloreactivity. A recent Phase I clinical trial49 at the National Institute of Health (NIH) evaluated the feasibility of infusing donor CD4 Th2 cells generated ex vivo using CD3/ CD28 costimulation in the presence of interleukin (IL)-4 and IL-2. The patients had hematologic malignancies and underwent reduced-intensity conditioning regimens before HSCT. The cells were from a matched from a sibling donor who had undergone granulocyte colony-stimulating factor (G-CSF) mobilization. Nineteen patients received no additional cells, and the remaining 28 received these Th2-like cells in a dose-escalation fashion (5, 25, or 125  106 cells/kg). The Th2 cohort had accelerated lymphocyte reconstitution with acute GVHD and overall survival similar in both the Th2 and non-Th2 cohorts.49

Nonspecific T Cells Unmanipulated Allogeneic T Cells (Donor Lymphocyte Infusion) Perhaps the most successful T-cell immunotherapy to date has been the use of unmanipulated allogeneic DLI following allogeneic HSCT. DLI has been most successful in patients with chronic myeloid leukemia who relapse after transplantation (70%-80% cytogenetic remission rate)5 or with BBV-LPD (up to 90%).4,38 Moderate success has been seen when DLI was used after relapse in other malignancies such as: acute myeloid leukemia (15%-40%), low-grade lymphomas (⬃60%), and metastatic multiple myeloma (40%-60%).39-42 Less than 5% of patients with relapsed acute lymphoblastic leukemia (ALL) respond to

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Allodepleted T Cells Another approach to overcome the problem of alloreactivity is to selectively deplete the T-cell product of alloreactive cells expressing activation markers in response to alloantigen. Alloreactive cells express activation markers after exposure to alloantigen, including CD69, CD147, and the IL-2 receptor CD25, which is expressed within the first 24 hours of T-cell activation. This approach has been evaluated in three clinical trials using a CD25 immunotoxin to selectively deplete alloreactivity, two involving haploidentical CD34 selected grafts in predominantly pediatric patients and one involving HLA-identical sibling transplants in adults.50-52 In all studies the approach was feasible and safe

Chapter 59: Adoptive Immunotherapy

and was able to promote immune reconstitution and recovery of virus-specific immunity without significant GVHD. Further studies to evaluate if there is an antitumor effect are continuing.

Regulatory T Cells An alternative option to prevent GvHD is to actively downregulate alloreactive T cells by recruiting regulatory T cells (Treg cells). Treg cells suppress the proliferation of CD4
and CD8
T cells after polyclonal or antigenic stimulation in a cell contact-dependent and cytokine-independent manner.53 If CD4
, CD25
, FoxP3
, Treg cells, which are naturally present in the transplant product, are depleted before infusion into irradiated allogeneic recipient mice, then GVHD is significantly accelerated. Conversely, GVHD can be prevented by the addition of freshly isolated Treg cells to the donor product and of note the GVL effect is spared.54 Supporting the importance of this effect, studies in humans have shown that a higher frequency of Treg cells in donor cells correlates with a lower risk for developing GVHD. Several groups are now investigating the value of infusing ex-vivo expanded donor Treg cells following HSCT. Many obstacles to the effective implementation of this approach remain, such as the difficulty of using cell sorting to obtain a pure Treg-cell population for infusion, given that FoxP3, the best Treg-cell marker to date, is intracellular. Ongoing clinical trials using Tregs include the transfer of IL10-anergized TR1 cells to prevent GVHD in patients after HLAhaploidentical HSCT. In one trial,48 donor PBMCs are thawed and cultured ex vivo in the presence of irradiated host PBMCs (obtained before HSCT) and IL-10. These IL-10-anergized donor T cells are then infused at increasing doses starting from 105 per kg.55 The first clinical trial with Treg cells is ongoing in Regensburg, Germany.56,57 In this study, cancer patients with a high risk of relapse after allogeneic HSCT receive a preemptive DLI after stopping immune suppressive therapy and then 8 to 10 weeks later receive between 1  106 and 5  106 freshly isolated donor CD4
/CD25
Treg cells/ kg of body weight followed by DLI of equal T-cell numbers. In the first five patients sufficient numbers of donor Treg cells were obtained and no toxicities (eg, GVHD, infection, and relapse) were seen.55 This trial and others that are planned will provide critical information regarding the ex-vivo expansion of Treg cells and on the feasibility and safety using unmanipulated Treg cells for adoptive immunotherapy. Autologous Nonspecifically Expanded T Cells Over the past few years several studies have correlated faster recovery of lymphocyte counts with improved outcome after autologous transplant for some malignancies.58,59 To determine if T-cell recovery after autografting can be accelerated, June and colleagues have administered T cells expanded ex vivo using anti-CD3 and anti-CD28 to patients with myeloma and non-Hodgkin lymphoma (NHL) after autografting and showed rapid reconstitution of lymphocytes.60,61 In the myeloma study,61 immunotherapy was combined with vaccination and a single, early, posttransplant infusion of in-vivo vaccine-primed and

ex-vivo-costimulated autologous T cells. This treatment was followed by posttransplant booster immunizations, which led to the rapid induction of clinically relevant immunity as well as improved T-cell proliferation in response to antigens that were not contained in the vaccine.61 These results show that adoptive transfer of autologous CD3/CD28-stimulated T cells is feasible and safe and that the combination of vaccine therapy and adoptive T-cell transfer leads to enhanced memory T-cell responses. Follow-up studies are evaluating effects on tumor responses.62 Another autologous ex-vivo expanded immunotherapy product that has been evaluated in the clinic is cytokine-induced killer (CIK) cells, a unique population of CTLs with a characteristic CD3
CD56
phenotype. In a clinical study, patients with relapsed NHL or Hodgkin disease were treated with escalating doses of autologous CIK cells generated by culturing apheresis products with interferon-γ, OKT3, and IL-2.63 No significant toxicity was seen and there were partial responses and disease stabilizations.63 Because these were heavily pretreated patients who had all relapsed after transplantation, this approach may have greater activity in a setting of minimal residual disease.

Antigen-Specific T-Cell Therapies The use of antigen-specific CTLs may overcome the problem of alloreactivity and may also allow expansion of low-frequency cells. To generate these cells ex vivo requires a defined antigen and an antigen-presenting cell that can effectively present the antigen to T cells with appropriate costimulatory signals. Therefore, it is a significant challenge to generate T cells specific for tumor antigens if the malignant cell presents antigen poorly, and the putative target antigens are weak. However, proof of principle has been shown by studies administering donorderived CTLs specific for viral antigens to reconstitute immunity following HSCT.

EBV-Specific Cytotoxic T Cells Immunotherapeutic strategies aimed at targeting EBV have now been used for over 15 years. All EBV-associated malignancies are associated with the virus’ latent cycle, and three distinct types of EBV latency have been characterized64,65 with varying expression of EBV latent proteins (Fig 59-1). Latency Type III is expressed in lymphoblastoid cell lines (LCLs), which can be readily produced by infecting B cells in vitro with EBV and is characterized by expression of the entire array of nine EBV latency proteins. This pattern of EBV gene expression characterizes the EBV-LPD that occur in individuals severely immunocompromised by solidorgan or stem cell transplantation, congenital immunodeficiency or human immunodeficiency virus (HIV) infection. Latency Type II is the hallmark of EBV-positive Hodgkin disease where a more restricted array of proteins including EBNA1, BARF0, LMP1, and LMP2 are expressed. In latency Type I, found in EBV-positive Burkitt lymphoma only EBNA1 and BARF0 are expressed. Because EBNA1 is not well processed by the Class I processing machinery,66 tumor cells expressing Type I latency may not be a good target for immunotherapy approaches. However,

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Step 1: LCL generation 4-6 weeks B95-8 EBV

EBV-transformed Lymphoblastoid B cell line

PBMC ⴙIL-2

Irradiated E:T ratio 40:1

E:T ratio 4:1

E:T ratio 4:1 7 days

10 days

Step 3: CTL expansion 4-7 weeks Weekly stimulations with irradiated LCL IL-2 given 2x/week starting ~day 12

Step 2: CTL initiation

Step 4: QA/QC Sterility HLA type Phenotype Cytotoxicity

EBV-specific CTL

Figure 59-2. Generation of Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes. Step 1: Peripheral blood mononuclear cells (PBMCs) are infected with a laboratory strain of EBV (B95.8) to generate an EBV-transformed B lymphoblastoid cell line (LCL). Step 2: Irradiated LCLs are antigen-presenting cells to stimulate T cells specific for EBV at an effector to target (E:T) ratio of 40:1. Step 3: The EBV-specific cytotoxic T lymphocytes (CTLs) are stimulated with irradiated LCL at weekly intervals at an E:T ratio of 4:1 starting from Day 10. Interleukin (IL)-2 at 50-100 U/mL is added twice weekly to the cultures from Day 12. The stimulations with LCL and IL-2 is continued until sufficient numbers of T cells have been expanded. Step 4: CTLs are frozen and undergo extensive quality assurance/quality control (QA/QC) including HLA typing, phenotyping, assessment of HLA type, phenotype, and cytolytic function before being released for clinical use.

immunotherapy approaches targeting EBV antigens do have potential for treating Type II and Type III latency EBV tumors. EBV-Associated Lymphoma after Transplantation Risk factors for development of EBV-PTLD include the degree of mismatch between donor and recipient, manipulation of the graft to deplete T cells, and the degree of immunosuppression used to prevent GVHD.4 EBV-associated PTLD arising after marrow transplantation is an ideal model to evaluate the ability of T cells to target EBV-positive tumor cells because these tumors are highly immunogenic expressing all nine latent cycle EBV antigens, including the immunodominant EBNA3 antigens. Furthermore, the outgrowing EBV-infected B-cell tumors have the same phenotype and pattern of EBV gene expression as do LCLs, which are an excellent antigen-presenting cell (Fig 59-1). EBV-specific T cells can be generated in vitro using donor lymphocytes infected with a laboratory strain of EBV to initiate an LCL line that is an excellent antigen-presenting cell for presenting viral antigens. Irradiated LCLs are used to stimulate PBMCs and expand EBV-specific CTLs (Fig 59-2). EBV-specific T-cell lines have been used as prophylaxis for EBV-induced lymphoma in over 60 patients after HSCT who underwent a

924

T-cell-depleted HSCT or who were transplanted for an EBVassociated malignancy4,6,67 and none of the patients treated with this approach developed PTLD, compared with an incidence of 11.5% in a historical nontreated control group.6 These results have been confirmed by another group.68 (See Table 59-2.) Immunotherapy using EBV-CTL has also been used to treat patients with overt lymphoma. This strategy was successful in five of six patients and showed by gene marking that CTL accumulated at sites of disease.6 Other groups have also obtained encouraging response rates38,72 including one study in patients who had recurrent PTLD after initial therapy with rituximab.75 In another study, one patient who received CTL as treatment died with progressive disease 24 days after infusion.76 In this patient, the cytolytic activity of the generated CTL line was directed mainly against two HLA-11-restricted epitopes in the EBNA3b gene. However, after CTL infusion only virus with an EBNA3b deletion could be detected, allowing the tumor to evade the immune response.76 This experience suggests that it is preferable to infuse a product with broad specificity. In recipients of solid-organ transplants, the severe impairment of T-cell function as result of the required immunosuppressive regimen after transplantation also places these patients at risk

Chapter 59: Adoptive Immunotherapy

Table 59-2. Clinical Trials on Use of EBV-Cytotoxic T Cells as Prophylaxis or Treatment for Posttransplant Lymphoproliferative Disease (PTLD) after Marrow Transplantation Study

No. of Patients

Type of Transplant

Pathologic Evidence of PTLD

Cytotoxic T-cell (CTL) Lines

Results

Rooney et al,6 Heslop69

54

T-cell-depleted HSCT (mismatch related donor or matched unrelated donor)

No—prophylaxis study

Allogeneic (donor-derived) EBV-CTL

No patients developed PTLD compared with 11.5% controls

Rooney et al,6 Gottschalk et al4

6

T-cell-depleted HSCT

Yes

Allogeneic (donor-derived) EBV-CTL

5 CR, 1 died (no response to CTL secondary to tumor mutation resistant to CTL)

Gustafsson et al68

6

T-cell-depleted HSCT or unmanipulated HSCT with ATG/ OKT3 conditioning (mismatched or matched unrelated donor or matched related donor)

No—Treatment based on Allogeneic (donor-derived) ↑ EBV DNA levels EBV-CTL

5 patients had ↓ EBV DNA levels; 1 patient subsequently died of PTLD (CTL showed poor specificity for EBV targets on cytotoxicity assay)

Lucas et al70

1

T-cell-depleted HSCT

Yes

Allogeneic (donor-derived) EBV-CTL

Patient attained CR

Imashsuki71

1

Mismatched HSCT with ATG

Yes

Allogeneic (donor-derived) EBV-CTL

Patient failed to respond

Slobod et al72

40

T-cell-depleted HSCT (mismatch related donor or matched unrelated donor)

No—prophylaxis study

Allogeneic (donor-derived) EBV-CTL

No patients developed PTLD

O’Reilly et al38

17 (includes 3 patients after solid-organ transplantation)

T-cell-depleted HSCT (mismatch related donor or matched unrelated donor)

Yes

Allogeneic (donor-derived) EBV-CTL

11 CR, 2 disease stabilization

Haque et al73

2

Allogeneic HSCT (unmanipulated graft)

Yes

Closely matched allogeneic EBV specific CTL

2 CR

Leen et al74

11

Allogeneic HSCT (unmanipulated and T-cell- depleted grafts)

Yes (n  1) Remaining patients had normal or high virus load

Multivirus specific polyclonal CD4
and CD8
T cells specific for CMVpp65, EBV and adenovirus

Patient with PTLD had CR with CTL alone All cleared EBV with CTL alone

EBV  Epstein-Barr virus; HSCT  hematopoietic stem cell transplantation; CR  complete response; ATG  antithymocyte globulin.

for the development of PTLD with incidence dependent of the type of organ transplanted and the degree of immunosuppression. Because reducing immunosuppression can often control LPD, administration of EBV-specific CTLs might be an alternate means of restoring the immune response. In this clinical scenario, the organ donor is not HLA-matched and often is not available, and the tumor often arises from recipient cells. Therefore, most studies in solid-organ recipients have used autologous CTLs generated from peripheral blood of transplant recipients.77-79 The results of these studies are summarized in Table 59-3. These studies have shown that the approach is safe and that EBV-specific immunity can be transiently restored. However, the persistence of CTLs is less than that observed after HSCT transplantation, perhaps because of the continuing immunosuppression that these patients receive to prevent rejection. An alternative approach is to use closely matched allogeneic EBV-specific CTLs. A Phase II clinical trial has evaluated this

strategy in HSCT and solid-organ recipients who had failed to respond to conventional PTLD therapy. Response rates were 64% and 52% at 5 weeks and 6 months, respectively.73 Patients with closer HLA matching donors showed better responses at 6 months. EBV-Associated Type II Latency Tumors EBV-associated nasopharyngeal carcinoma, Hodgkin disease, and NHL that develop in the immune competent host show Type II latency where viral gene expression is limited to the immunosubdominant latent membrane protein (LMP)1 and LMP2, EBNA1, and EBERs,85 which are weak targets for CTL activity. Nevertheless, these subdominant EBV antigens are potential targets for immunotherapy approaches. Phase I dose-escalation studies have evaluated the use of autologous EBV-specific CTL for patients with EBV-positive Hodgkin disease or nasopharyngeal carcinoma.7,86 The Hodgkin disease study involved

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Section V

Table 59-3. Clinical Trials on Use of EBV-Cytotoxic T Cells as Prophylaxis or Treatment for Posttransplant Lymphoproliferative Disease (PTLD) after Solid-Organ Transplantation Study

No. of Patients

Types of Solid-Organ Transplants Included

Pathologic Evidence of PTLD

Cytotoxic T-cell (CTL) Lines

Results

Comoli et al78

7

Heart, liver, kidney

Prophylaxis

Autologous EBV specific CTL

No patient developed PTLD

Haque et al80

3

Liver, kidney

Prophylaxis

Autologous EBV specific CTL

No patient developed PTLD

77

1

Lung

Yes

Autologous EBV specific CTL

Significant regression

Sherritt et al81

1

Heart

Yes

Autologous EBV specific CTL

CR

82

5

Kidney

Yes

Autologous EBV specific CTL

CR (used as adjuvant after chemotherapy rituximab)

Sun et al83

2

Kidney, liver

Yes

Closely matched allogeneic EBV specific CTL

2 CR

Haque et al84

7

Liver, small bowel, kidney

Yes

Closely matched allogeneic EBV specific CTL

3 CR; 1 PR; 3 did not respond

Savoldo et al79

12

Liver, heart

Prophylaxis (n  10) and treatment

Autologous EBV CTL

No patient developed PTLD after CTL; 1 attained CR; 1 PR (12 months)

Haque et al73

31

Heart, liver, kidney, lung, small bowel

Yes

Closely matched allogeneic EBV specific CTL

At 6 months after CTL: 12 CR, 3 PR, and 16 persisting/ progressive PTLD

Khanna et al

Comoli et al

EBV  Epstein-Barr virus; CR  complete response; PR  partial response.

14 treated patients, of whom seven received gene-marked CTLs. The infused CTLs expanded in vivo, persisted up to 12 months and trafficked to tumor sites.7 EBV-CTLs were well tolerated, and had some antitumor activity with five patients (two treated with active disease) remaining in complete remission up to 40 months, one having a partial response, and five having stable disease. Lucas and colleagues also saw activity using EBV-CTL from partially HLA-matched normal donors.87 Five of six patients with Hodgkin disease had reductions in measurable disease and the maximal duration of response was 22 months.87 EBV-CTLs have been administered to 10 patients with EBVpositive nasopharyngeal carcinoma who were in relapse or who had high-risk disease.86 The four patients with high risk disease remain in remission after treatment and two of the six patients in relapse entered complete remission and remained disease free 24 to 42 months after CTL infusion.86 Comoli et al88 also treated 10 patients with relapsed disease and saw responses in six—two with partial responses and four with stable disease. Although these results using EBV-specific CTL in Type II latency tumors have shown some activity (Table 59-4), the response rate is less impressive than that seen in EBV-LPD after HSCT. Moreover, apart from the patients with relatively low tumor burden who have demonstrated durable responses, the majority of the antitumor responses have been transient, and no patient with aggressive, bulky relapsed disease has been cured by CTL therapy alone. This may be due to a lack of specificity of the EBV-specific CTLs for the immunosubdominant LMP1 and LMP2 antigens present on the Type II latency tumor (Fig 59-1). A follow-up study has shown that LMP2-CTLs could be generated from normal donors using dendritic cells genetically modified with a recombinant adenovirus encoding

926

LMP2 (Ad5LMP2). for the initial stimulation, followed by stimulation with LCL that had been genetically modified to overexpress LMP2a or LMP1 and LMP2 by transduction with an Ad5f35LMP2a vector or a Ad5f35LMP1-I-LMP2 vector, respectively.91 LMP2-specific CTL lines using this methodology were administered to 16 patients with EBV
ve Hodgkin disease or EBV-positive B-cell or T/NK-cell NHL.89 The LMP2specific CTLs expanded and persisted in vivo without adverse effects. Nine of 10 patients treated in remission of high-risk disease remain in remission, and four of six patients with active relapsed disease had a complete response.89 These studies are being extended using autologous T cells enriched for both LMP1 and LMP2 for the treatment of EBV-positive lymphoma and nasopharyngeal carcinoma.

CMV-Specific Cytotoxic T Cells Inability to control CMV reactivation following allogeneic HSCT is caused not only by low antigen-specific T-cell numbers but also by impairment of the T-cell function in vivo.92 CMV prophylaxis and treatment with adoptively transferred, donor-derived CMV-specific CTLs has been successfully used with CMVspecific CD8
clones or polyclonal CTL lines (Table 59-5). In the first study from Riddell and colleagues, CMV-specific T-cell clones were derived from sibling donors after stimulation with autologous fibroblasts pulsed with CMV.2,99 There were no adverse effects and CMV-specific immune responses were reconstituted, although long-term persistence of infused CTLs was seen only in recipients who developed recovery of CD4
CMV-specific T-cell responses. Other groups have also reported successful reconstitution of immunity to CMV by administering CMV-specific CTLs generated by stimulation of PBMCs with

Chapter 59: Adoptive Immunotherapy

Table 59-4. Clinical Trials on Use of EBV-Cytotoxic T Cells as Treatment for Type II Latency EBV-Positive Tumors Study

Number of Patients

Type of Disease

Cytotoxic T-Cell (CTL) Lines and Dose

Results

Sun et al83

4

EBV-positive HD and NHL (including 1 HIV associated and 2 after solid-organ transplant)

Allogeneic (2 partially HLA-matched and 2 from HLA matched siblings) EBV-CTL 5  106/kg

1 CR, 1 CRU, 1 PR and 1 NR

Lucas et al87

6

Relapsed EBV-positive HD

Allogeneic (partially HLA matched) EBV-CTL. 1.5  107/kg  pretreatment with fludarabine

1 CR, 4 PR, 1 NR (although ?effect of fludarabine)

Bollard et al7

14

Relapsed EBV-positive HD—either as adjuvant treatment after HSCT or for treatment of active disease

Autologous EBV-CTL 4  107/m2  1.2  108/m2

5 CR (includes 2 with active disease),1 PR, 5 SD, 3 NR

Bollard et al89

16

Relapsed EBV-positive HD or NHL—either as adjuvant treatment after HSCT/chemo or for treatment of active disease

Autologous LMP2-CTL 4  107/m2  3  108/m2

13 CR (includes 4 with active disease),1 PR, 2 NR

Comoli et al90

1

NPC—active disease

HLA matched (sibling) EBV-CTL

SD (transient)

88

Comoli et al

10

NPC—all with active disease

Autologous EBV-CTL

2 PR (3-5 months) and 4 SD (4-15 months)

Straathof et al86

10

NPC—either as adjuvant treatment after HSCT/ chemo or for treatment of active disease

Autologous EBV-CTL 4  107/m2  3  108/m2

6 CR (includes 2 with active disease) 1 PR, 1 SD, 2 NR

EBV  Epstein-Barr virus; HD  Hodgkin disease; NHL  non-Hodgkin disease; HIV  human immunodeficiency virus; CR  complete response; CRU  complete response unconfirmed; PR  partial response; NR  no response; SD  stable disease; HSCT  hematopoietic stem cell transplantation; NPC  nasopharyngeal carcinoma.

dendritic cells pulsed with CMV antigens100 or the HLA-A2restricted peptide NLV derived from the CMV-pp65 protein.101 CMV-specific CTLs have also been used therapeutically with encouraging results.93 These methodologies for generating CMV-specific CTLs all require prolonged ex-vivo activation and expansion. To overcome this problem a recent study used tetramer selection of CMV peptide-specific T cells directly from peripheral blood and relied on expansion in vivo after adoptive transfer. Infused CD8
T cells expanded by several logs after infusion, and cleared CMV infection in eight of nine cases.97

Adenovirus-Specific T Cells Another alternative to expand virus-specific T cells is to stimulate T cells with the infectious antigen of interest and then “capture” the responding cell that is producing effector cytokines such as interferon-γ. This strategy has been used to isolate adenovirusspecific CTLs for clinical use. Cells were incubated with adenovirus lysate in vitro to produce CD4
and CD8
T cells that were highly specific for adenovirus and exhibited markedly reduced alloreactivity. Feuchtinger et al102 infused adenovirus-specific CTLs generated using this approach into nine pediatric patients with systemic adenovirus infection following allogeneic HSCT. All patients tolerated the infusions, and they exhibited a dosedependent and sustained in-vivo expansion of adenovirus-specific CTLs associated with clearance or decrease in viral load in five of six evaluable patients.102 Only one patient developed acute exacerbation of chronic GVHD. The efficacy was independent of T-cell dose, implying in-vivo expansion in the presence of active viremia from even low numbers of transferred cells.

Trivirus-Specific CTLs Because it would be preferable to target more than one virus, methods have been developed to generate multivirus-specific CTLs for adoptive immunotherapy. The initial focus was on CMV, EBV, and adenovirus as common viral reactivations seen in the posttransplant period. Trivirus-specific T cells were reactivated by using mononuclear cells transduced with a recombinant adenoviral vector encoding the CMV antigen pp65 for the first stimulation followed by subsequent stimulations with EBV-LCLs transduced with the same vector. The resulting CTLs had specificity for all three viruses and were administered to 10 HSCT recipients.74 After adoptive transfer there was recovery of immunity to CMV and EBV in all patients, but adenovirus-specific T cells were detected only in recipients who had evidence of adenovirus infection preinfusion. Reactivations or infections with all three viruses were cleared. This approach is being extended to additional pathogens. Fungal-Specific T Cells Although profound neutropenia following HSCT remains an important risk factor for opportunistic invasive fungal infections, most cases arise after neutrophil recovery in the context of potent immunosuppressive therapy for GVHD prevention or treatment. Perruccio et al recently detailed the results of the first use of Aspergillus-specific adoptive therapy following HSCT.103 Ten patients with Aspergillus pneumonia and positive galactomannan antigenemia were treated with Aspergillus- and CMV-specific donor-derived T-cell clones. In all, galactomannan antigenemia decreased to normal within 6 weeks, with nine patients clearing the Aspergillus infection.103

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Table 59-5. Published Reports on the use of CMV-Cytotoxic T Cells as Prophylaxis or Treatment for CMV Infection after Marrow Transplantation Study

No. of Patients

Type of Transplant

Walter et al2

14

MRD marrow transplantation No (unmanipulated graft)

Einsele et al93

8 (7 evaluable)

MRD, MMRD and MUD (4 patients received CD34 selected stem cells)

CMV colitis (n  1) CMV interstitial pneumonitis (n  1)

Peggs et al94,95

16

Allogeneic MRD or MUD

No — administered when Polyclonal CD8
and CD4
CMV DNA detected lines generated using CMV-lysate-pulsed antigen-presenting cells

Rauser et al96

8

Allogeneic HSCT (unmanipulated graft)

No — administered when Polyclonal CD8
and CD4
6/7 cleared virus with CTL CMV DNA detected 1 patient did not respond lines generated using CMV lysate and CMV-peptide-pulsed antigen-presenting cells

Cobbold et al97

9

Allogeneic HSCT (unmanipulated graft)

No — administered when HLA-peptide tetramer selected CMV DNA detected CD8
T cells specific for pp65 and IE1

6 cleared virus with CTL alone 3 required antiviral therapy as well as CTL

10

Allogeneic HSCT (unmanipulated and T-cell-depleted grafts)

No

Mulitvirus specific polyclonal CD4
and CD8
T cells specific for CMVpp65, EBV, and adenovirus

Functional CMV immunity detected in all None required antiviral agents All cleared virus with CTL alone

7

Allogeneic HSCT (unmanipulated graft)

No

Direct selection of IFNγ secreting T cells after incubation with recombinant pp65

Functional immunity detected in all 5 required antiviral agents but for shorter time than controls

Leen et al74

Mackinnon et al98

Clinical Evidence of CMV Disease

Cytotoxic T Lymphocyte (CTL) lines

Results

CD8
CMV-specific CTL clones

Functional immunity detected in all; no patients developed CMV viremia or disease

Polyclonal CD4  CD8
CMV-specific CTL

CMV DNA undetectable in 6/7 patients after CTL. ↑ functional immunity in 5 patients 2 nonresponders had increased immune suppression Expansion of CMV-specific immune response 8 cleared virus with CTL alone 8 required antiviral agents

MRD  matched related donor; CMV  cytomegalovirus; MMRD  mismatched related donor; MUD  matched unrelated donor; HSCT  hematopoietic stem cell transplantation; IFN-  interferon gamma.

Tumor-Specific T Cells Clinical immunotherapy targeting nonviral antigens is challenging especially when considering adoptive immunotherapy for malignant disease. Many of these tumor antigens are self antigens, and as a result of self-tolerance mechanisms, CTLs against these antigens are of low frequency, have low T-cell receptor avidity, or are anergic. In the allogeneic setting, preclinical studies have shown that it is possible to generate leukemia-specific CTLs using dendritic cells as antigen-presenting cells.104 A report has detailed a patient with relapse CML who attained complete remission following infusion of leukemia-specific CTLs generated from the original allogeneic stem cell donor.105 In the autologous setting, dendritic cells pulsed with defined tumor peptide epitopes have been used as antigen-presenting cells. With this approach, CD8
CTLs specific for the melanocyte differentiation antigens, Melan-A and gp100 peptides, could be generated from the PBMCs of HLA-A2-positive patients.106,107 In 10 patients with metastatic melanoma who received autologous CTLs in conjunction with low-dose IL-2, three had mixed

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responses and five had stable disease. The HLA-A*0201 melanoma line pulsed with Melan-A peptide analogue could also be used successfully as an antigen-presenting cell, obviating the need to generate dendritic cells from individual patients. In this study,108 10 patients with metastatic melanoma were infused with Melan-A specific CTLs followed by exogenous IL-2 and interferon-α. One achieved complete remission, three had partial or mixed responses, and three had stable disease. Limitations of this approach are that CTLs generated from peptide-pulsed antigen-presenting cells are predominantly, if not exclusively, CD8
and that targeting a single peptide epitope increases the potential for tumor escape. An alternative strategy, that circumvents some of the concerns regarding peptide pulsing, is to generate tumor-infiltrating lymphocytes (TILs), which can be screened for recognition of melanoma cells using an interferon-γ secretion assay, and then expanded with OKT3. Such TIL lines usually contain both CD4
and CD8
T cells and can be shown to have specificity for MART-1 or gp100 epitopes.109,110 Clinical trials using TIL and high-dose IL-2 in nonlymphocyte-depleted patients resulted

Chapter 59: Adoptive Immunotherapy

in objective responses in roughly a third of patients with most responses being transient.111

Genetically Modified T Cells T Cells Transduced with αβ-T-Cell Receptor An alternative strategy to target weakly immunogenic tumor antigen is to alter the antigen specificity of T cells by gene modification. T cells can be transduced with genes encoding a different antigen receptor: either physiologic αβ-T-cell receptor (TCR) heterodimers or artificial chimeric T-cell receptors.112,113 Several αβ-TCR heterodimers specific for tumor antigens have been cloned, either from autologous CTL culture114,115 or from an HLA-A*0201 transgenic mouse model whereby highavidity murine TCRs can be cloned.116,117 These include the TCR recognizing MART-1 and gp100 melanoma/melanocyte differentiation antigens, the NY-ESO-1 CTA, and a p53 epitope expressed on approximately 50% of cancers of common epithelial origin.118 Although some inefficiency from heterologous pairing between endogenous and transduced TCR chains is expected, primary T cells transduced with cloned α and β TCR chains do mediate anti-tumor activity in vitro. 114,116-118 To investigate the anti-tumor ability of genetically engineered T cells in vivo, one group administered peripheral blood lymphocytes transduced with the genes encoding the alpha and beta chains of the anti-MART-1 TCR. After transduction, approximately 30% of the CD8
T cells expressed the αβTCR heterodimer specific for MART-1. The genetically modified T cells were administered to 15 patients with metastatic melanoma and persistence of the T cells was seen in all patients for 2 months after infusion. Two of the 15 patients had antitumor responses and in both patients the T cells were still detected in the peripheral blood at 1 year after infusion. This study suggested that T cells expressing αβ-TCR heterodimers specific for tumor antigens may have therapeutic potential in vivo.118 However, the main drawback of cloned TCR is HLArestriction which limits its use to a subset of patients with particular HLA type. T Cells Transduced with Chimeric Antigen Another strategy for the generation of tumor-specific T cells is the transduction of T cells with chimeric surface proteins that transmit TCR signals in response to target cells. Such proteins are composed of an extracellular domain (ectodomain) usually derived from immunoglobulin variable chains, which recognizes and binds target antigen. This domain is attached via a spacer to an intracytoplasmic signaling domain (endodomain) usually the cytoplasmic segment of TCR-ς chain, which transmits an activation signal to the T cell. Genetic engineering of CTLs for redirected CD30-, kappa-, CD20-, and CD19-specific target cell recognition has been achieved and these approaches are in the process of being translated to the clinic.9,119-121 However, chimeric receptor signaling produces only limited activation of the T cells, and clinical trials in patients with solid tumors have shown that T cells expressing transgenic antigen-specific chimeric

receptors have limited therapeutic activity, in part because engagement of the chimeric receptor alone is insufficient to sustain T-cell growth and activation.122 One means of solving this problem is to transduce antigen-specific T cells rather than nonspecifically activated cells and take advantage of the costimulation provided to the native TCR by antigen. This approach has been evaluated in vitro using CTLs specific for EBV, CMV, and influenza.123-125 An alternate strategy is to provide additional costimulatory signals, and the signaling domain of the TCR has been combined with specific endodomains derived from different costimulatory molecules including CD28, 4-1BB, or OX40.126-128

T Cells Transduced with Suicide Genes As discussed above, the infusion of an unmanipulated T-cell product will contain alloreactive cells able to induce GVHD. One means of reducing this risk is to transduce the donor T cells with a suicide gene that can be activated if the recipient develops GVHD. The most commonly used suicide gene is herpes simplex virus thymidine kinase (HSV-TK) that renders transduced T cells sensitive to the cytotoxic effects of ganciclovir. HSV-TK-transduced T cells have been evaluated in several clinical trials129-131 that have shown that GVHD can be effectively controlled by ganciclovir-induced elimination of the transduced T cells. However, the thymidine kinase suicide gene is virus derived and potentially immunogenic132; therefore, new nonimmunogenic suicide genes based on inducible Fas and caspase 9 have been developed.133-135 In these constructs the Fas and caspase 9 molecules have been fused to a human FK506 binding protein to allow conditional dimerization. In both in-vitro and in-vivo studies, T cells stably transduced with a retroviral vector coding for the dimerizable gene can be selectively eliminated after the exposure to a chemical inducer of dimerization.133,135

NK Cells NK cells are effectors from the innate immune system that also mediate antiviral and antitumor immunity.136 Unlike T cells, NK cells do not undergo clonotypic gene rearrangement in order to express antigen receptors. Through a complex array of receptors they are able to determine non-self from self. There are several classes of receptors, including killer immunoglobulin-like receptors. NK-cell receptors may be activating or inhibitory so that NK-cell activity is regulated by quantitative differences in cumulative inhibitory and activating signals. The presence or absence of the respective ligands on recipient cells determines if NK cells will be primed to kill the targets.137 In the clinical setting of mismatched HSCT, donor vs recipient NK-cell alloreactivity has been associated with better outcome, particularly in patients with acute myeloid leukemia who undergo transplantation while in remission.138 Several immunotherapy approaches using NK cells have been tested in the clinic and recent studies have shown that haploidentical NK cells infused after lymphocyte depleting chemotherapy can have antitumor effects.139

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Section V

Improving Cellular Immunotherapy Approaches

shortly now that in-vivo safety studies in a murine model have been completed.153

Improving Ex-Vivo Generation One of the limitations of adoptive immunotherapy is the time taken to generate sufficient numbers of antigen-presenting cells to generate antigen-specific CTLs. Several groups have evaluated if artificial antigen-presenting cells can serve as a substitute. A number of approaches have been used including grafting ligands for the TCR and costimulatory surface molecules onto mouse fibroblasts or beads.62,140,141 A second limitation is suboptimal persistence of infused cells and several investigators have attempted to identify the optimal phenotype of infused T cells. It is likely that a product containing both CD4 and CD 8 cells and both effector and memory cells will be required.142-144 It is therefore important to correlate in vivo function with the source of antigen-presenting cells and T-cell culture conditions.

Lymphocyte Depletion to Improve Expansion of T Cells In Vivo As discussed above, outside the setting of HSCT, poor lymphocyte survival or function has been a frequent limitation in immunotherapy studies. Several groups have evaluated whether administration of lymphocyte-depleting chemotherapy such as fludarabine or monoclonal antibodies may induce an environment that would promote the homeostatic expansion of infused T cells.109,145 This strategy may also have a second benefit by depleting Treg cells. Clinical studies have already shown expansion of T cells specific for antigens expressed on solid tumors after lymphocyte depletion.146

Genetic Modification of T Cells to Overcome Tumor Evasion Mechanisms Tumor cells evade a transferred T-cell response by several mechanisms such as downregulation of MHC and costimulatory molecules and secretion of inhibitory cytokines by the malignant cell population.147 One cytokine, which has devastating effects on CTL proliferation and function, is transforming growth factor-beta (TGFβ), which is secreted by a wide variety of tumors as a powerful mechanism by which the tumor cells can escape the immune response.148,149 The importance of this mechanism is illustrated by the observation that transgenic mice genetically engineered so their T cells are insensitive to TGF signaling are able to eradicate tumors.150 Moreover, adoptive transfer of T cells transduced with a dominant-negative TGFβ Type II receptor (DNR) resulted in eradication of tumor cells.151 Human in-vitro studies have shown that that DNR-transduced CTL were resistant to the antiproliferative effects of recombinant TGFβ and long-term expression of this construct had no deleterious effects on the function, phenotype, or growth characteristics of the transduced-CTL lines.152 CTL expressing the DNR may, therefore, have a selective advantage in vivo in patients with TGFβ-secreting tumors. A study to test this hypothesis will open

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Regulatory Considerations for Adoptive Immunotherapy In the United States, human cells, tissues, cellular and tissuebased products (HCT/Ps) that have been more than minimally manipulated (cultured ex vivo, transduced or activated ex vivo) are referred to as Type 351 products and must be prepared under good manufacturing practice (GMP) regulations to ensure their safety, purity, and potency. In cellular therapy, guidance from the Food and Drug Administration show GMP as a continuum, so that as an approach moves through Phase I and II clinical trials the application of GMP will become more rigorous, until in a Phase III study manufacture would be a fully validated process. A Phase III product would also need validated release criteria, which are test specifications designed to ensure that the product is sterile and pure, and has the desired functionality. In the United States new good tissue practice (GTP) regulations came into effect in May 2005 to cover HCT/Ps that were minimally manipulated (eg, were not cultured ex vivo, genetically modified, activated ex vivo, etc), and that were intended for autologous use, or use in a first- or second-degree blood relative. Cellular products that fall into this classification are referred to as Type 361 products and require manufacturing of the product under GTP regulations. In general, these cover personnel, procedures, facilities, environmental control and monitoring, equipment, supplies and reagents, recovery, processing and process controls, process changes, process validation, labeling controls, storage receipt, predistribution shipment and distribution, records, tracking, and complaints. Implementation of the components of either GMP or GTP is a time-consuming and labor-intensive process that requires the development, implementation, and maintenance of numerous components. Professional societies, such as the Foundation for the Accreditation of Cellular Therapy and AABB have developed an accreditation process that takes into account GTP regulations and provides a framework around which compliance can be built. Both organizations have developed standards that are harmonized with regulatory requirements, so that accreditation by either organization assists with regulatory compliance. The challenge of bringing adoptive cellular therapy protocols from the laboratory bench to the bedside has also been recognized by funding agencies, and the NIH has designated specialized centers for product assistance to provide consulting, manufacturing, and regulatory support to other research centers.154

Conclusions T-cell therapies have produced definitive benefits in the treatment of relapsed leukemia and lymphoma after transplantation

Chapter 59: Adoptive Immunotherapy

and EBV-associated malignancy. However, clinical studies have also identified limitations of such therapies including inadequate persistence or expansion. With increased knowledge of the optimal methods for generation of T-cell products, identification of additional antigen targets, and optimization of gene therapy approaches to enhance the function of adoptively transferred, adoptive immunotherapy strategies may find broader indications in the therapy of cancer and infections.

Disclaimer The authors have disclosed no conflicts of interest.

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T cells. Blood 2002;100:3690-7. 93. Einsele H, Roosnek E, Rufer N, et al. Infusion of cytomegalovirus (CMV)-specific T cells for the treatment of CMV infection not responding to antiviral chemotherapy. Blood 2002;99:3916-22. 94. Peggs K, Verfuerth S, MacKinnon S. Induction of cytomegalovirus (CMV)-specific T-cell responses using dendritic cells pulsed with CMV antigen: A novel culture system free of live CMV virions. Blood 2001;97:994-1000. 95. Peggs KS, Verfuerth S, Pizzey A, et al. Adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virus-specific T-cell lines. Lancet 2003;362: 1375-7. 96. Rauser G, Einsele H, Sinzger C, et al. Rapid generation of combined CMV-specific CD4
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T-cell lines for adoptive transfer into recipients of allogeneic stem cell transplants. Blood 2004;103:3565-72. 97. Cobbold M, Khan N, Pourgheysari B, et al. Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA-peptide tetramers. J Exp Med 2005;202:379-86. 98. Mackinnon S, Thomson K, Verfuerth S, et al. Adoptive cellular therapy for cytomegalovirus infection following allogeneic stem cell transplantation using virus-specific T cells. Blood Cells Mol Dis 2008;40:63-7. 99. Riddell SR, Watanabe KS, Goodrich JM, et al. Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones. Science 1992;257:238-41. 100. Peggs KS, Verfuerth S, Pizzey A, et al. Adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation with virus-specific T-cell lines. Lancet 2003;362: 1375-7. 101. Micklethwaite K, Hansen A, Foster A, et al. Ex vivo expansion and prophylactic infusion of CMV-pp65 peptide-specific cytotoxic T-lymphocytes following allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2007;13:707-14. 102. Feuchtinger T, Matthes-Martin S, Richard C, et al. Safe adoptive transfer of virus-specific T-cell immunity for the treatment of systemic adenovirus infection after allogeneic stem cell transplantation. Br J Haematol 2006;134:64-76.

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103. Perruccio K, Tosti A, Burchielli E, et al. Transferring functional immune responses to pathogens after haploidentical hematopoietic transplantation. Blood 2005;106:4397-406. 104. Montagna D, Maccario R, Locatelli F, et al. Ex vivo priming for long-term maintenance of antileukemia human cytotoxic T cells suggests a general procedure for adoptive immunotherapy. Blood 2001;98:3359-66. 105. Falkenburg JH, Wafelman AR, Joosten P, et al. Complete remission of accelerated phase chronic myeloid leukemia by treatment with leukemia-reactive cytotoxic T lymphocytes. Blood 1999;94:1201-8. 106. Meidenbauer N, Marienhagen J, Laumer M, et al. Survival and tumor localization of adoptively transferred Melan-A-specific T cells in melanoma patients. J Immunol 2003;170:2161-9. 107. Yee C, Thompson JA, Byrd D, et al. Adoptive T cell therapy using antigen-specific CD8
T cell clones for the treatment of patients with metastatic melanoma: In vivo persistence, migration, and antitumor effect of transferred T cells. Proc Natl Acad Sci U S A 2002;99:16168-73. 108. Vignard V, Lemercier B, Lim A, et al. Adoptive transfer of tumorreactive Melan-A-specific CTL clones in melanoma patients is followed by increased frequencies of additional Melan-A-specific T cells. J Immunol 2005;175:4797-805. 109. Dudley ME, Rosenberg SA. Adoptive-cell-transfer therapy for the treatment of patients with cancer. Nat Rev Cancer 2003;3:666-75. 110. Dudley ME, Wunderlich JR, Yang JC, et al. A phase I study of nonmyeloablative chemotherapy and adoptive transfer of autologous tumor antigen-specific T lymphocytes in patients with metastatic melanoma. J Immunother 2002;25:243-51. 111. Rosenberg SA, Yannelli JR, Yang JC, et al. Treatment of patients with metastatic melanoma with autologous tumor-infiltrating lymphocytes and interleukin 2. J Natl Cancer Inst 1994;86:1159-66. 112. Dotti G, Heslop HE. Current status of genetic modification of T cells for cancer treatment. Cytotherapy 2005;7:262-72. 113. Sadelain M, Riviere I, Brentjens R. Targeting tumours with genetically enhanced T lymphocytes. Nat Rev Cancer 2003;3:35-45. 114. Clay TM, Custer MC, Sachs J, et al. Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes confers anti-tumor reactivity. J Immunol 1999;163:507-13. 115. Zhao Y, Zheng Z, Robbins PF, et al. Primary human lymphocytes transduced with NY-ESO-1 antigen-specific TCR genes recognize and kill diverse human tumor cell lines. J Immunol 2005;174:4415-23. 116. Cohen CJ, Zheng Z, Bray R, et al. Recognition of fresh human tumor by human peripheral blood lymphocytes transduced with a bicistronic retroviral vector encoding a murine anti-p53 TCR. J Immunol 2005;175:5799-808. 117. Stanislawski T, Voss RH, Lotz C, et al. Circumventing tolerance to a human MDM2-derived tumor antigen by TCR gene transfer. Nat Immunol 2001;2:962-70. 118. Morgan RA, Dudley ME, Wunderlich JR, et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 2006;314:126-9. 119. Jensen M, Cooper L, Wu A, et al. Engineered CD20-specific primary human cytotoxic T lymphocytes for targeting B-cell malignancy. Cytotherapy 2003;5:131-8. 120. Savoldo B, Rooney CM, Di Stasi A, et al. Epstein Barr virus-specific cytotoxic T lymphocytes expressing the anti-CD30{zeta} artificial chimeric T-cell receptor for immunotherapy of Hodgkin’s disease. Blood 2007;110:2620-30.

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121. Vera J, Savoldo B, Vigouroux S, et al. T lymphocytes redirected against the kappa light chain of human immunoglobulin efficiently kill mature B lymphocyte-derived malignant cells. Blood 2006;108:3890-7. 122. Kershaw MH, Westwood JA, Parker LL, et al. A phase I study on adoptive immunotherapy using gene-modified T cells for ovarian cancer. Clin Cancer Res 2006;12:6106-15. 123. Rossig C, Bollard CM, Nuchtern JG, et al. Epstein-Barr virus-specific human T lymphocytes expressing antitumor chimeric T-cell receptors: potential for improved immunotherapy. Blood 2002;99:2009-16. 124. Heemskerk MH, Hoogeboom M, Hagedoorn R, et al. Reprogramming of virus-specific T cells into leukemia-reactive T cells using T cell receptor gene transfer. J Exp Med 2004;199:885-94. 125. Cooper LJ, Al Kadhimi Z, Serrano LM, et al. Enhanced antilymphoma efficacy of CD19-redirected influenza MP1-specific CTLs by cotransfer of T cells modified to present influenza MP1. Blood 2005;105:1622-31. 126. Maher J, Brentjens RJ, Gunset G, et al. Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta/ CD28 receptor. Nat Biotechnol 2002;20:70-5. 127. Pule MA, Straathof KC, Dotti G, et al. A chimeric T cell antigen receptor that augments cytokine release and supports clonal expansion of primary human T cells. Mol Ther 2005;12:933-41. 128. Imai C, Mihara K, Andreansky M, et al. Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia. Leukemia 2004;18:676-84. 129. Bonini C, Ferrari G, Verzeletti S, et al. HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft versus leukemia. Science 1997;276:1719-24. 130. Tiberghien P. Use of suicide gene-expressing donor T-cells to control alloreactivity after haematopoietic stem cell transplantation. J Intern Med 2001;249:369-77. 131. Ciceri F, Bonini C, Marktel S, et al. Antitumor effects of HSV-TK-engineered donor lymphocytes after allogeneic stem-cell transplantation. Blood 2007;109:4698-707. 132. Riddell SR, Elliot M, Lewinsohn DA, et al. T-cell mediated rejection of gene-modified HIV-specific cytotoxic T lymphocytes in HIVinfected patients. Nat Med 1996;2:216-23. 133. Berger C, Blau CA, Huang ML, et al. Pharmacologically regulated Fas-mediated death of adoptively transferred T cells in a nonhuman primate model. Blood 2004;103:1261-9. 134. Marktel S, Magnani Z, Ciceri F, et al. Immunologic potential of donor lymphocytes expressing a suicide gene for early immune reconstitution after hematopoietic T-cell-depleted stem cell transplantation. Blood 2003;101:1290-8. 135. Straathof KC, Pule MA, Yotnda P, et al. An inducible caspase 9 safety switch for T-cell therapy. Blood 2005;105:4247-54. 136. Freud AG, Caligiuri MA. Human natural killer cell development. Immunol Rev 2006;214:56-72. 137. Ruggeri L, Aversa F, Martelli MF, Velardi A. Allogeneic hematopoietic transplantation and natural killer cell recognition of missing self. Immunol Rev 2006;214:202-18. 138. Ruggeri L, Mancusi A, Capanni M, et al. Donor natural killer cell allorecognition of missing self in haploidentical hematopoietic transplantation for acute myeloid leukemia: Challenging its predictive value. Blood 2007;110:433-40. 139. Miller JS, Soignier Y, Panoskaltsis-Mortari A, et al. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 2005;105:3051-7.

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140. Oelke M, Maus MV, Didiano D, et al. Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells. Nat Med 2003;9:619-25. 141. Latouche JB, Sadelain M. Induction of human cytotoxic T lymphocytes by artificial antigen- presenting cells. Nat Biotechnol 2000;18:405-9. 142. Sallusto F, Geginat J, Lanzavecchia A. Central memory and effector memory T cell subsets: Function, generation, and maintenance. Annu Rev Immunol 2004;22:745-63. 143. June CH. Principles of adoptive T cell cancer therapy. J Clin Invest 2007;117:1204-12. 144. Riddell SR. Engineering antitumor immunity by T-cell adoptive immunotherapy. Hematology Am Soc Hematol Educ Program 2007;250-6. 145. Klebanoff CA, Khong HT, Antony PA, et al. Sinks, suppressors and antigen presenters: How lymphodepletion enhances T cell-mediated tumor immunotherapy. Trends Immunol 2005;26:111-17. 146. Dudley ME, Wunderlich JR, Robbins PF, et al. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science 2002;298:850-4. 147. Smyth MJ, Dunn GP, Schreiber RD. Cancer immunosurveillance and immunoediting: The roles of immunity in suppressing tumor development and shaping tumor immunogenicity. Adv Immunol 2006;90:1-50.

148. Chemnitz JM, Eggle D, Driesen J, et al. RNA fingerprints provide direct evidence for the inhibitory role of TGFbeta and PD-1 on CD4
T cells in Hodgkin’s lymphoma. Blood 2007;110:3226-33. 149. Zou W. Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat Rev Cancer 2005;5:263-74. 150. Gorelik L, Flavell RA. Immune-mediated eradication of tumors through the blockade of transforming growth factor-beta signaling in T cells. Nat Med 2001;7:1118-22. 151. Zhang Q, Yang X, Pins M, et al. Adoptive transfer of tumorreactive transforming growth factor-beta-insensitive CD8
T cells: Eradication of autologous mouse prostate cancer. Cancer Res 2005;65:1761-9. 152. Bollard CM, Rossig C, Calonge MJ, et al. Adapting a transforming growth factor beta-related tumor protection strategy to enhance antitumor immunity. Blood 2002;99:3179-87. 153. Lacuesta K, Buza E, Hauser H, et al. Assessing the safety of cytotoxic T lymphocytes transduced with a dominant negative transforming growth factor-beta receptor. J Immunother 2006;29:250-60. 154. McCullough J, Wagner J, Gee A, et al. Production assistance for cellular therapies. Transfusion 2004;44:1793-5.

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60

Gene Therapy in Transfusion Medicine Emanuela M. Bruscia1 & Diane S. Krause2 1 2

Associate Research Scientist, Department of Pediatrics, Yale University, New Haven, Connecticut, USA Professor, Laboratory Medicine and Pathology, Yale University School of Medicine, New Haven, Connecticut, USA

Gene therapy, the use of DNA that encodes or corrects a gene for medical therapy, is achieved by transfer of genetic material to a patient’s cells. Gene therapy can be achieved by repairing the defect of the endogenous gene (called targeting a gene) or by introducing additional genetic material into the genome. The specific manner by which gene therapy is performed varies significantly based on the disease to be treated, the gene to be inserted, the target cells of insertion, the vector used to transmit the gene, and the route of administration of the gene. This chapter discusses these variables as well as the current successes and challenges in the field of gene therapy. Some of the many potential diseases that may be treated using gene therapy are listed in Table 60-1. It is hoped that the target diseases for gene therapy will eventually include most serious diseases for which the molecular etiology is well understood. Gene therapy may prove useful for gene replacement for congenital enzyme deficiencies or gene abnormalities. For treatment of cancer, gene therapy may be used to induce selective cancer cell death. Alternatively, cancer treatment could be achieved by using gene therapy to replace a missing tumor suppressor gene, to insert a chemotherapy drug-resistance gene selectively into normal cells, or to enhance the immunogenicity of cancer cells. It is also hoped that gene therapy will be developed to treat infectious diseases such as AIDS.

Gene Therapy and Transfusion Medicine Transfusion medicine is the science of administering cells or other biologic materials (eg, serum, isolated clotting factors) to treat patients. The growing field of gene therapy has affected transfusion medicine on several levels. First, gene therapy is introducing new biologicals for transfusion in the clinic setting. Gene therapy falls within the purview of transfusion medicine Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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Table 60-1. Diseases that May Be Treated Using Gene Therapy Disease Type

Examples of Diseases

Gene deficiency/mutation

Hemophilia Cystic fibrosis Thalassemia Severe combined immunodeficiency Gaucher’s disease Sickle cell anemia

Cancer

Lack/mutation of tumor suppressor gene

Autoimmune diseases

Rheumatoid arthritis

Infections

AIDS

because it often requires the infusion of either gene-modified cells or direct administration of vectors that contain the therapeutic gene. As with blood components, the safety and efficiency of gene therapy agents must be optimized. Therefore, as gene therapy strategies are found to be effective in preclinical studies, quality control and infection control measures need to be developed for the clinical laboratory. Second, hematopoietic cells are excellent targets for gene therapy trials. They are easily accessed and may be incubated ex vivo with the vectors containing the genetic repair material. Autologous marrow or peripheral blood stem cell (PBSC) transplantation already provides a relatively safe standard of care for some malignancies, and the circulation of these cells suggests that hematopoietic cells may be used as gene delivery systems even for nonhematologic diseases. Ideally, one needs only to infect the “stem” cells in order to get large-scale amplification and long-term expression of a transgene. Third, gene therapy strategies are under development to treat several diseases that directly affect transfusion medicine, including hemoglobinopathies and clotting factor disorders. For example, hemophilia A, in which patients lack expression of functional clotting Factor VIII, is likely to be one of the first diseases to be treated successfully with gene therapy. This is because

Chapter 60: Gene Therapy in Transfusion Medicine

functional Factor VIII protein product can be produced by any cells in the body, not necessarily the normal site of production in the liver, as long as the protein is secreted into the circulation. Also, the levels of circulating Factor VIII that are necessary for therapeutic efficacy need not be highly regulated as long as there is at least 5% of the normal physiologic level present.

Vector Design The many aspects that go into designing an ideal gene therapy vector are based largely on the specific disease to be treated, the target cells, and the therapeutic strategy. Three major considerations are which gene to insert, which vector to use, and how to administer the vector.

What Gene Should Be Inserted and Where Should It Be Inserted into the Genome? Based on the disease to be treated, the gene that will be inserted can confer a variety of attributes. The gene can provide a functional form of a missing or defective gene, it can be a gene designed to give the cell in which it is expressed a survival advantage, or it can act as a suicide gene designed to kill the cell in which it is expressed. See Table 60-2 for examples of each of these gene insertion strategies. For inherited diseases in which there is a nonfunctional gene product, such as cystic fibrosis, gene therapy approaches are being taken to add a functional gene. Similarly, for inherited diseases in which a gene is missing, such as thalassemia, the gene for the fully functional protein product is inserted. For cancer, the genes to be inserted can take one of several different forms. For example, one could insert a suicide gene selectively into the cancer cells. An example of one such “suicide” gene is HSV-TK (herpes simplex virus thymidine kinase), which can phosphorylate ganciclovir, which is then incorporated into the DNA, preventing its replication and thereby killing the cell. Alternatively, one could replace a missing or mutated p53 gene

with the wild-type gene, which could restore normal apoptotic (cell death) machinery to the cell. In addition to determining which gene to administer, gene regulatory elements must be inserted in order to achieve appropriate gene expression levels. The process by which a gene is transcribed into messenger RNA (mRNA) and then translated into protein is controlled at multiple levels. For some therapeutic genes, the level of expression does not need to be highly regulated (eg, circulating level of clotting Factor VIII in hemophilia or adenosine deaminase levels). In contrast, other therapeutic genes, if expressed at either too high or too low a concentration, could be deleterious (eg, β-globin). If diabetes is going to be treatable by gene therapy, then insulin must be produced in response to elevated circulating glucose levels and reduced when the glucose level falls below normal levels. Every cell type in the body, although containing the same genomic DNA, presents a different pattern of gene expression. This process is controlled at multiple levels, the most common of which is transcriptional control by gene promoters, enhancers, and silencers in the specific context of a cell’s nucleus. Promoters that are used only in specific cell types (eg, the insulin promoter in pancreatic cells) are referred to as cell-type-specific or tissue-specific promoters. Research has been focused on designing the regulatory domains of the transgenes (the promoters) so that the gene is either regulated as it would normally be in the cell with a tissue specific promoter, or regulated by an inducible promoter. Another level of control for gene expression is to design gene therapy vectors that affect only specific target cells, so that the transgene is not expressed inappropriately in the body, and is not expressed by cells in which it would be deleterious. In the last few years, control of where a gene therapy vector may integrate in the genome has grown more critical. For example, in the case of recent clinical trials in X-linked immunodeficiency, discussed in further detail below, the therapeutic retroviruses integrated into a region of the genome, where they led to activation of proto-oncogenes that caused the patients to have leukemia. In addition the genome architecture adjacent to the proviral

Table 60-2. Genes Targeted in Gene Therapy to Date Gene Type

Disease

Gene

Target Cell

Replacement of missing or defective gene

Thalassemia Sickle cell anemia Severe combined immunodeficiency Cystic fibrosis Hemophilia

β-globin β-globin Adenosine deaminase Cystic fibrosis conductance regulator Clotting Factor VIII or IX

Hematopoietic stem cell Hematopoietic stem cell T lymphocyte Respiratory epithelial cells Hepatocyte

Survival advantage

Chemotherapy for multiple forms of cancer

Multiple drug resistance

Hematopoietic stem cell

Suicide gene

Graft-vs-host disease caused by allogeneic donor T lymphocytes

Thymidine kinase gene (kills cell when exposed to ganciclovir) Cytosine deaminase (kills cell when exposed to 5 FU)

Hematopoietic stem cell Hematopoietic stem cell

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Section V

integration site can lead to silencing of transgene expression. The use of “insulating” genetic material at the extremity of the therapeutic cassette may prevent the ability of the inserted genetic material to perturb the activity of surrounding genes of the area of integration. Each of these concerns theoretically can be overcome if efficient gene targeting can be attained.

Table 60-3. Common Viruses Used in Gene Therapy Research Family/Subfamily and Characteristics Retroviruses (single-stranded RNA, with maximal insert size of approx. 8 kb) Oncoviruses

How Should the Gene Therapy Be Administered? Gene therapy vectors can be applied to cells ex vivo and then returned to the patient. Alternatively, viral vectors that are replication incompetent (incapable of replication) and nonviral vectors can be directly administered to the patient for infection of target cells in vivo. So far, most animal and human clinical trials have used ex-vivo infection of patients’ cells with retroviruses. This allows for selection of virally infected cells before use in order to guarantee vector insertion.1 The ex-vivo approach minimizes infection of unintended cellular targets, and because the cells are washed free of unincorporated viral vector before infusion, this approach significantly decreases the potential risks of exposing the patient to large amounts of viral vector. This approach has been used for hematologic disease in which the target cells can be harvested from patients and returned by intravenous transplantation. On the other hand, direct administration of vectors to the patient overcomes the difficulties of maintaining fully functional target cells ex vivo. However, because there are risks associated with transgene expression in unintended host cells, it is critical that the vector administered in vivo have target specificity or have little toxicity if expressed by cell types other than the intended target tissue. This in-vivo infection has been used mostly in clinical trials to treat patients with cystic fibrosis. Adenoviral vectors containing a normal version of the cystic fibrosis conductance regulator gene that is mutated in cystic fibrosis are inhaled and subsequently enter respiratory epithelial cells. Viral vectors have also been developed that can be administered either by intramuscular or by direct injection into the liver for the treatment of hemophilia.

Specific Virus

Murine leukemia virus Spleen necrosis virus Rous sarcoma virus Avian leukocytosis virus

Lentiviruses

Human immunodeficiency virus, type 1 Human immunodeficiency virus, type 2

Spumaviruses

Foamy virus

Adenoviruses (double-stranded DNA, with maximal insert size of approx. 8 kb for first generation; up to 37 kb for new generation)

Adenovirus, type 5

Adeno-associated virus (single-stranded DNA with maximal insert size of approx. 5 kb)

Adeno-associated virus, type 2

of specific gene elements for insertion into specific vectors that will target appropriate levels of gene expression to the appropriate tissues. Of course, no viral or nonviral vector yet achieves all of these goals; different vectors have different advantages and disadvantages. Many research laboratories are developing strategies to optimize current vectors to meet the criteria for an ideal vector. Provided below is an overview of the successes and remaining challenges in three of the most well established viral vector systems for clinical trials, retroviruses, adeno-associated viruses, and adenoviruses. Table 60-3 summarizes the salient features of these viral vectors. Nonviral techniques that are under development for gene therapy are discussed below as well.

What Vector Should Be Used? The ideal vector should have no toxicity to the patient, should not cause an immune response, should be able to hold a large amount of genetic information (large gene-carrying capacity), should have target specificity or the ability to program target specificity, should infect target cells with a high efficiency, should have controllable gene expression (persistent versus inducible), and should have a controlled integration site that prevents nonspecific effects. The choice of which vector to use may also be based on the level and longevity of expression desired. Depending on the application, some genes need to be expressed over the long term in the individual (eg, replacement of a missing globin gene in thalassemia) and, therefore, must be incorporated into the genomic DNA of a cell population that will endure (the hematopoietic stem cell), and others may require only transient expression (eg, induction of tumor cell death) for which adenoviral vectors can be useful. Each application requires the design

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Viral Vectors Retroviruses Retroviruses, the first gene therapy vectors to be used, generally have an enveloped single-stranded RNA genome and are approximately 100 nm in diameter. For applications using retroviruses, the therapeutic gene is inserted between the two long terminal repeats (LTRs) at each end of the retroviral genome and replaces three key normal viral genes (gag, pol, and env). Without these genes, the virus is no longer able to proliferate. The LTRs are critical for incorporating the therapeutic genes into the genomic DNA of the target cells and may also be required for expression of the inserted genes. The maximal insert size for the expressed gene(s) is approximately 8 kb, which can be quite limiting because the coding region for many genes is longer than 8 kb, and often more than one gene (eg, a therapeutic gene and

Chapter 60: Gene Therapy in Transfusion Medicine

a selectable marker) may need to be included in the retroviral vector. There are multiple types of retroviruses used in gene therapy research, including gamma retrovirus or oncoretroviruses, lentiviruses, and spumaviruses. To date, most retroviral vectors assessed for clinical trials have been based on murine leukemia viruses (MLVs). As described below, lentiviral vectors based on RNA sequences from human immunodeficiency virus (HIV) are better than oncoretroviruses at infecting nondividing cells. Although the therapeutic vector, in this case, is based on the HIV genome, these vectors do not contain the genes in the virus that can cause AIDS. The ability to transduce nondividing cells is associated with the viral genes (eg, gag matrix protein MA, the regulatory protein Vpr, and viral integrase) that efficiently shuttle the virus into the nuclei of target cells. Lentivirus can transduce primate2 and human3 hematopoietic stem cells (HSCs) more efficiently than the oncoretroviruses because the majority of HSCs are in G1 or G0 phase of the cell cycle. Recently, the human foamy virus (HFV) has been used as an alternative gene therapy vector to murine leukemia viruses and HIV-1 vectors. HFV is nonpathogenic, has the unique property of completing reverse transcription before entering its target cells, and infects multiple cell types including hematopoietic cells.4 Although progress has been made in the last few years, the full cell biology of this virus is not yet well defined. Studies showing infection of nondividing cells5,6 including unstimulated HSCs suggest that this vector system should be pursued more thoroughly. The unique capability of this virus to transduce metabolically inactive cells appears to be related to the fact that the foamy virus particles contain active reverse transcriptase and, as mentioned, the transcription of double-strand DNA is completed before the entry of the particles in the cell. Therefore, these viruses do not rely on the limited host nucleotide supply for their replication.7

Optimization of Retroviral Vectors Optimization of retroviral vectors should combine several features, which are described below. Virus Inactivation For safety reasons, all gene therapy vectors must be incapable of replication in the human host. By removal of the gag, pol, and env genes, replication-incompetent retroviruses (RIR) are produced. Because the genes removed are necessary for packaging the RNA genome into retroviral particles, special “packaging” cells are required to produce retrovirus for use in gene therapy. These packaging cells express the gag, pol, and env proteins because the three genes have been introduced into their genomes. When an RIR vector containing the LTRs and the packaging signal is introduced into such packaging cell lines, its RNA is packaged into infectious particles by the gag, pol, and env proteins. The DNA encoding viral gag, pol, and env is not contained within the viral particles produced, so that even though these retroviruses are capable of infecting target cells, they are not capable of replication. However, if some of the gag, pol, and

env sequences within the packaging cell genome share regions of DNA overlap with the retroviral coding region, there is a risk of recombination between the two, which could lead to the production of replication-competent retroviruses. MLV has the advantages of having the lowest overlap between viral elements and the genes inserted into packaging cells. In contrast, lentiviruses have a large portion of overlap in the N-terminus of the gag gene, and HFV requires the greatest overlap between coding sequence and RIR for generating potentially therapeutic viruses. Of note, the oncoretroviruses used for recent clinical studies were produced several years ago, and still contain large portions of the gag andpol genes, which increase the risk of making replicationcompetent viruses. Before any retroviruses can be used in clinical trials, however, each batch must be tested extensively in order to ensure that there are no replication-competent viruses present. Ongoing studies are focused on reducing the sequence overlaps without decreasing the titer of the viruses produced. A step forward in the design of viral vectors was to create selfinactivating vectors by deleting the enhancer promoter sequences from one of the two viral LTRs containing the retroviral promoter and enhancer elements. Given the retroviral cycle, mutation of the transcriptional element at the 3⬘ end of the virus does not affect the ability of the vector to infect cells and incorporate into the genomic DNA. However, after entry into the target cells, the 5⬘ LTR, which is derived from the 3⬘ LTR, also becomes mutated, and the LTR is incapable of promoting transcription. This approach greatly reduces the risk of creating replicationcompetent virus. The downside is that now the vector no longer has its own promoter, so it needs to be designed with another promoter that will drive transcription of the desired transgene once inside the target cells. Efficiency Some of the best target cells for in-vitro retrovirus infection are hematopoietic cells. Highly proliferative hematopoietic progenitors, measured by in-vitro colony-forming assays, have been infected successfully by retroviruses. The biggest obstacle to using retroviruses to infect hematopoietic stem cells is that oncoretrovirus do not infect nondividing cells, and hematopoietic stem cells cycle very slowly and are usually in G0 phase of the cell cycle. The efficiency of infection of HSCs has been increased in recent years by addition of cytokines to the ex-vivo culture medium before and during retroviral infection in order to increase the number of HSCs in the cell cycle. However, such cytokines may also cause HSCs to differentiate so that they lose their long-term repopulating “stem cell” capacity. Retroviral infection and modification of mouse HSCs has been demonstrated using mouse marrow transplantation,8 and efficiencies obtained have been very high with 80% to 100% of mouse stem and progenitor cells being infected with retrovirus. Infection of human HSCs is less efficient than murine cells. In contrast to oncoretroviruses, lentiviruses and foamy viruses are able to efficiently infect quiescent cells and, therefore, appear to be more promising vectors for HSC-based gene therapy. Lentiviral vectors have a high efficiency of infection (up to 80%) in human

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CD34⫹ cells from cord blood, cells in peripheral blood that have undergone granulocyte colony-stimulating factor (G-CSF) mobilization, and marrow cells.9 Similarly, HFV is found to infect more than 80% of human CD34⫹ hematopoietic progenitors from cord blood.6 Clinical trials using lentiviral vectors have been initiated for inherited diseases and HIV. Experimental models for human HSCs, take advantage of the ability of human cells to engraft the marrow of immunocompromised animal hosts. These xenogeneic in-vivo models have been quite useful as preclinical systems. One such model uses sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. The human cells that maintain multilineage long-term engraftment in these mice are referred to as SCID-repopulating cells (SRC).10 In a related immunodeficient mouse system, human CD34⫹ marrow cells were infected with an efficiency of approximately 10% by retroviral vectors, and these cells survived in the immunocompromised mouse hosts for at least 8 months.11 Specificity and Efficiency The different degree of retroviral infection between mouse and human HSCs is caused by multiple factors that must be optimized in order to obtain high levels of infection. Much research has focused on two major differences between infection of mouse and human cells, 1) the percentage of stem cells in mitosis and 2) the surface expression of appropriate retroviral receptors. As alluded to above, in order for viral vectors to be expressed in the target cells, they must be able to enter the nucleus. For all viruses, except lentiviral vectors, this requires nuclear membrane breakdown, which occurs when a cell undergoes mitosis. Thus, quiescent, nondividing cells are more difficult to infect with gene therapy vectors than are actively dividing cells. In mice, approximately 8% of HSCs enter the cell cycle per day in vivo, and about 75% are quiescent in G0 phase at any time. It is estimated that human HSCs cycle less often than murine cells, and therefore a higher percentage of human HSCs are quiescent than are murine cells.12,13 The efficiency of retroviral infection is also highly dependent upon whether the retrovirus can bind to and enter the cell. Different retroviruses have different receptors. (A list of viral vectors and their receptors is given in Table 60-4.) For the retrovirus used most often in mouse cells, the receptor on the cell is CAT-1, an amino acid transporter.13 In contrast, the retroviruses that have been used to infect human cells, called amphotropic retroviruses, bind to Pit-2, which is a phosphate transporter.14 It is estimated that the CAT-1 retroviral receptor is expressed on a higher percentage of mouse HSCs than is Pit-2 on human HSCs.15,16 Preselection of a subpopulation of hematopoietic cells that expresses high levels of the retroviral receptor and stimulation of receptor expression are two strategies that have been attempted to increase retroviral infection of human HSCs.17,18 Higher levels of infection of human cells have also been achieved using “pseudotyped” retroviruses in which the envelope-encoding gene env, which encodes the ligand on the virus coat that binds to the target cells, has been replaced with a gene that is known to bind

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Table 60-4. Viral Vectors and Their Receptors Virus Retrovirus ● Ecotropic murine leukemia virus ●

Amphotropic murine leukemia virus

●

Human immunodeficiency virus

Adenovirus

Adeno-associated virus Parvovirus B19

●

●

AAV2

Receptor or Coreceptor

CAT-1 (cationic amino acid transporter) Pit-2 (sodium-dependent phosphate transporter) CD4, CXCR4 CAR (coxsackie virus and adenovirus receptor) αvβ5 or αvβ3 integrins Red-cell-specific protein Heparin sulfate proteoglycans Fibroblast growth factor receptor 1 αVβ5

well to HSCs.19,20 One protein that has been studied extensively is the gibbon ape leukemia virus (GaLV) envelope protein, which binds to a receptor called Pit-1.21 Retroviruses pseudotyped with GaLV infect more than twice as many hematopoietic progenitors than the standard amphotropic retrovirus.22,23 Gammaretrovirus and lentivirus are much easier to pseudotype than foamy virus.24 After effective pseudotyping approaches are developed, this should allow for construction of vectors that are targeted to specific cells. An additional level of specificity is to express the transgene on a tissue-specific promoter. For hematopoietic diseases, limiting expression of the therapeutic gene to differentiated cells rather than more primitive stem and progenitor cells could reduce the risk of genotoxicity of the vector. For example, vectors have been designed to treat erythroid disorders in which the β-globin gene is driven by a promoter that is active only in red cell precursors; preclinical studies using mouse models have shown promising therapeutic results.25 A similar vector has also given promising results for a vector encoding human Factor IX.26 Lineage-specific expression has also been achieved in T cells and B cells.27,28 Specific Chromosomal Targeting All retrovirus vectors integrate randomly into genomic DNA. Based on the integration site of the viral DNA, expression of endogenous genes can be modified (genotoxicity). If this gene were a pro-oncogene, the activation induced by viral insertion could cause cancer. As discussed below, this is what occurred in a recent clinical trial in which four patients developed leukemia. The components that influence retroviral integration sites in the genome are not known. Ongoing studies are revealing general patterns of integration that characterize different vectors. For example, oncoretroviruses such as MLV integrate preferentially within a few kilobases upstream of transcriptional start sites in

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DNA regions containing CpG islands (regions rich in cytodine residues next to and 5⬘ to guanine residues). Lentiviruses also tend to insert near actively transcribed genes, but the integrations are not necessarily clustered near transcription start sites. Foamy viruses tend to integrate outside of genes, with both clusters and gaps.29,30

Risks The major risks associated with viral vector-based gene therapy are the generation of replication-competent viruses and integrations that lead to activation of oncogenes or silencing of tumor suppressor genes. Replication-Competent Viruses Replication competent retroviruses develop either by recombination of the constituent parts of the vector system with endogenous viral sequence in the viral producer cell lines or by activation of endogenous proviral sequences. The significant risks of retroviral vectors for gene therapy were elucidated when primate studies were initially performed before 1992. CD34-selected marrow cells of the monkeys were infected with replication competent viruses from packaging cell lines. There were, however, some replication competent viruses that formed by rearrangement that had not been detected by the existing assays for replication-competent viruses. Upon infusion, three of eight monkeys subsequently developed lymphomas containing the active rearranged retrovirus.31 As mentioned, investigators are optimizing the safety by creating self-inactivating (SIN) vectors, and minimizing the regions of homology between the vectors and the producer cells. Currently, all clinical trials require very stringent testing to guarantee that the retroviral vector to be used in humans is entirely replication incompetent. Genotoxicity (insertional mutagenesis) Genotoxicity is caused by the insertion of the provirus in proximity to a tumor suppressor gene or pro-oncogene. The presence of a strong promoter of viral origin near a proto-oncogene can lead to transcriptional activation of the gene and ultimately the development of neoplasia. Unfortunately, this has occurred in a clinical trial for X-linked immunodeficiency.32-35 More details regarding this trial are provided below in the Clinical Trials section. The best approach for minimizing oncogene activation may be the use of vectors such as lentiviruses and foamy viruses, which tend to integrate into areas of the genomes that are not oncogenic. However, additional evaluation of the possible risks associated with these vectors is required before they are ready for clinical trials. No strategies are available currently for directing the integration of the transgenes in a safe, specific spot in the genome. Preliminary data suggest that an alternate approach, the addition of insulator sequences at each end of the transgene, can reduce the genotoxicity of retroviral insertion.36 These cis-acting insulator sequences are designed to prevent the genome information contained inside the vector from interfering with the adjacent

endogenous neighboring genes. The best characterized chromatin insulator to date is derived from the chicken β-globin locus control region.37,38 Developing vectors with tissue-specific promoters also addresses genotoxicity because these promoters allow improved spatial and temporal control of transgene expression.

Challenges The most important challenges for translating viral gene therapy into a compelling alternative treatment for hematologic diseases are 1) to create vectors with no adverse effects, 2) to ensure long-term therapeutic effects, and 3) to standardize protocols for highly controlled, efficient, high quality vector production. Prevention of Gene Silencing Even if the insertion site causes no genotoxicity, expression of the therapeutic transgene can get silenced because of epigenetic modulation of the chromatin (eg, histone methylation) presumably because the host cell recognizes that the inserted DNA sequences are being expressed inappropriately. The elimination of a known silencing element within the U3 domain of the LTR, the use of a robust promoter (eg, PGK), the introduction of an intron sequence, and the incorporation of other transcriptional regulatory elements (eg, WPRE enhancers) are all strategies that have been evaluated to ensure long-term expression of the transgene. For example, SIN vectors, which lack functional domains of the LTR region, are less prone to silencing. These strategies are undergoing evaluation, and it is not yet clear that they will be useful for clinical studies.39 Vector Production and HSC Modification Additional problems with long-term retroviral infection of HSCs currently being addressed include establishing techniques for concentrating retrovirus onto the cells,40,41 improving retroviral survival in vivo,42,43 and optimizing infection efficiency into specific target cells.20

Adeno-Associated Virus Adeno-associated virus (AAV) is a nonpathogenic, replicationdefective, 4.68 kb, single-stranded DNA virus that requires external factors for infection. The name AAV was given because helper virus, usually adenovirus, is necessary for infection and replication. Advantages to AAV vectors over retroviruses include the ability to infect nondividing cells and insertion as DNA rather than RNA. This is a potential advantage over retroviral vectors because some genes, most notably β-globin, require the presence of introns for optimal expression, and these introns are likely to be spliced out when the retroviral RNA is reverse transcribed into DNA. The inverted terminal repeats (ITRs) that are located at each end of the AAV genome are sufficient to package the single-stranded DNA into viral particles. When a gene therapy vector is made from AAV, the therapeutic gene can be inserted in

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place of nearly the entire normal viral protein-coding domain (4.5 kb) as long as the flanking ITRs remain intact. These vectors are packaged into viruses by supplying the required rep and cap AAV proteins separately. This is usually achieved by inserting both a vector plasmid encoding the therapeutic gene and a packaging plasmid encoding the rep and cap genes into cells also infected with adenovirus. Replication-incompetent AAV vectors are produced by lysing the cells to release viruses, and destroying the “helper” adenovirus with heat. In the absence of helper virus, AAV does not replicate and remains integrated in the genomic DNA of the producer cell. AAV is not pathogenic (approximately 80% of humans are seropositive for AAV type 2), making it suitable for gene therapy. AAV can infect nondividing cells and remain latent for prolonged periods in an episomal state. That is, AAV persists in cells without inserting into the genomic DNA (thus avoiding insertional mutagenesis) and does not trigger a robust immune response. Unlike the retroviruses, different naturally-occurring AAV variants, with more than 100 different capsid proteins, confer target cell specificity for AAV infection.44 In addition, because of its genetic simplicity, AAV virus sequences can be mutated to increase the specificity of gene delivery; such modified AAVs are called recombinant AAVs. With repeated infections, AAV incorporates stably into the endogenous genome with a frequency of 1%, but most AAV remains in the episomal state. When AAV does integrate, it integrates randomly in the cells.45 When more of the initial AAV genome is maintained, although the integration frequency is still quite low, the integration is targeted to a specific area of the genome (19q13.3), which is transcriptionally active. The targeted integration of the AAV is a unique characteristic among all known eukaryotic viruses, and, because the integration site appears to be nononcogenic, investigators are working on methods to maintain the targeted integration while making the AAV replication incompetent. AAV vectors efficiently infect CD34⫹ human marrow progenitor cells. From 80% to 90% of CD34⫹ cells in suspension cultures and 50% to 95% of myeloid colonies differentiating in vitro from transduced CD34⫹ cells demonstrate AAV-induced transgene expression. A high percentage of transduction (up to 23%) has also been reported for CD34⫹ human cord blood cells.46 In murine transplantation models with AAV-infected hematopoietic cells, no cytotoxic response to AAV occurs; mice transplanted with AAV-transduced marrow have long-term multilineage reconstitution,46 and the transgene is still detectable after 6 months. In secondary mouse recipients (another test for whether self-renewing stem cells have been infected), a small but significant percentage (1%) of marrow cells were still transduced. A study in nonhuman primates demonstrated that 15 months after transplantation of AAV-modified autologous marrow cells, approximately 1 in 10 cells in the circulation was still modified. Modified cells were also detected in the marrow, and in specific subpopulations including granulocytes, B cells, and T cells.47 Wang et al48 developed capsid-modified, helper-dependent AAV vectors carrying the β-globin locus control region (LCR) to drive green fluorescent protein (GFP) expression. GFP expression was

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detected in an erythroleukemia cell line. In primary CD34⫹ human cells, more than 40% expressed GFP at high levels. AAV has been used in Phase I and II clinical trials for various diseases including Parkinson’s, α1-antitrypsin deficiency, cystic fibrosis, and hemophilia B; however, no clinical trials using AAV-infected hematopoietic cells have been reported. Although this virus has been considered the safest vector in gene therapy (explaining the abundant ongoing clinical trials that are in place using this vector), the use of AAV in a clinical trial was recently associated with a severe adverse event.49 In this clinical trial for the treatment of rheumatoid arthritis, a tumor necrosis factor, alpha (TNF-α) inhibitor protein was delivered by an AAV vector that shuttled the gene into the joints. The trial, beginning in the fall of 2005, enrolled 127 patients without any serious adverse effects, but one patient during the second administration of the vector in the summer of 2007 developed a severe reaction to the vector that led to death in few days. This event is now, appropriately, under intense scrutiny. In addition, other investigators have shown that after several administrations of AAV in a mouse model of the lysosomal storage disease mucopolisaccharidosis VII, the vector integrated in a specific area of the genome on chromosome 12, which led to dramatic overexpression of the provirus and development of hepatocellular carcinoma.50 Another major problem with using AAV vectors is the highly variable efficiency of infection reported by different laboratories. One possible explanation for this variability is that different laboratories have different amounts of remaining intact adenovirus, which is coinfected with the AAV. This is problematic because adenoviral vectors are replication competent. Improved techniques to effectively remove contaminating helper virus are currently being developed. Also, it has been difficult to obtain high titers of pure AAV. A “hybrid” between viral AAV vectors and liposomes (phospholipid-bound vesicles) has been proposed for gene therapy in which the AAV ITRs are used to flank the gene of interest in order to facilitate its insertion into the host genomic DNA, but rather than a viral particle, the DNA is packaged simply in a liposome carrier. Future studies in the AAV field are focused on 1) increasing the tropism of AAV for specific cells and 2) evaluating the “target” integration of the vector and the mechanism and proteins involved in this unique propriety of AAV, which could be therapeutically very important. In addition, deeper evaluation of the risks associated with the use of AAV is under way.

Adenoviral Vectors Adenoviruses are double-stranded DNA viruses that are approximately 36 kb in size. Four early genes are expressed at the beginning of the viral cycle, and are required for DNA replication and viral propagation. The late genes, which encode structural proteins, are transcribed after DNA replication. Unlike retroviruses, adenoviral vectors are quite stable in vitro, can be produced and concentrated to high titers, and can infect quiescent, postmitotic cells. After the adenovirus enters a cell, its

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genome is released and maintained as an episome (circular DNA separate from the nuclear genomic DNA) in the cell. Because the adenoviral DNA does not incorporate stably into the genomic DNA of the cell, adenoviral gene expression is transient. The duration of expression of a gene on an adenoviral vector varies significantly based on the longevity and cell cycle status of the cell that is infected. Adenoviruses infect many different cell types, and there are more than 50 different types of adenoviruses that are associated with disease in humans. The diseases caused by adenoviruses are transient and usually are not serious unless the patient is immunocompromised. The cell membrane receptor for adenovirus fiber protein is called CAR for coxsackievirus and adenovirus receptor. The normal cellular function of CAR is not yet known. Similar to other types of viruses, interaction with a second membrane coreceptor is necessary for internalization of bound adenovirus. For adenoviruses, this second interaction is between the penton base of the virus and integrins on the cell surface including αvβ3 and αvβ5. Adenoviral vectors have been tested extensively in models to treat cystic fibrosis, and have been used for modifying HSCs as well. The most widely used adenoviral vector is based on the Ad5 virus, belonging to adenovirus subgroup C. Human CD34⫹ hematopoietic stem/progenitor cells do not express CAR or the αv integrins at levels high enough for efficient Ad5 vector-mediated gene transfer. Therefore, when hematopoietic cells are exposed to adenovirus, there is only low-level transgene expression.51 In contrast, human monocytes have relatively high levels of αv integrin and CAR.52 Strategies to increase gene transfer efficiency of Ad5-based vectors include prolonged incubation of CD34⫹ cells with high concentrations of vectors, formation of virus-polycation complexes before infection, and cytokine stimulation of CD34⫹ cells. However, none of these approaches has led to significant improvement of transduction. More recent approaches have been focused on enhancing recognition of hematopoietic cells by adenoviruses by genetic or chemical modifications of the fiber/knob domain of the virus.53 For example, investigators have replaced the entire fiber gene or the knob part of the Ad5 vector genome with the corresponding gene from Ad35, which does not require CAR expression on the target cell.54,55 These modified adenovirus vectors show efficient (30-70%) gene transfer with little evident of cellular toxicity into hematopoietic cells, including CD34⫹ cells and primitive progenitor cells.56 The infected populations were viable, and retained their ability to form colonies in short-term culture assays54 as well as their ability to confer multilineage repopulation in vivo after transplantation into immunodeficient mice.53,56 The transient expression of this vector could have some potential applications for hematologic diseases such as transient overexpression of immune-response molecules for immunotherapies against leukemias or for transient expression of “stem cell genes” for expansion of HSCs in vitro without permanently affecting the genome of the cells and their progeny. Ongoing challenges with adenoviral vectors include the immune response mounted by the host, the transient nature of

transgene expression due to lack of genomic insertion, and the inherent low affinity for hematopoietic cells. In clinical trials using adenoviral vectors to treat cystic fibrosis, problems arose because of an inflammatory response to the vectors. In addition, in cystic fibrosis there is a relatively low level of integrin expression on the airway epithelium.57 Inflammation against adenovirus was also to blame in September 2000, when an 18-year-old patient with ornithine transcarbamylase deficiency, an inherited liver disease that causes life-threatening levels of ammonia to build up in the blood, became the first known death caused by gene therapy. This event demanded the attention of the public as well as that of regulatory agencies and committees that oversee and approve human gene therapy protocols. The patient, who had managed his disease with oral medication and a special diet, developed acute hepatitis after receiving an adenoviral gene therapy vector via the portal vein. Since that time, the Food and Drug Administration (FDA) has investigated several laboratories across the country and has found one failure to comply with National Institutes of Health (NIH) and FDA procedures. These findings raise the ethical quandary of whether to stop or slow the progress of gene therapy by tightening all regulations or to move ahead because patients with rare and untreatable diseases and their families want the trials to continue.

Nonviral Gene Therapy Nonviral vectors for gene therapy are also undergoing intensive investigation.58 Unlike viral vectors, nonviral vectors do not need to overcome the extensive immune mechanisms that destroy some viral vectors in vivo, and they do not have the risk of there being replication-competent forms of the vector. However, the application of nonviral gene therapy to humans has been severely limited by the poor efficiency of gene delivery to the cells. Several nonviral strategies under development are described briefly below.

Episomal Plasmid DNA The most common nonviral gene therapy approach is based on the transfer of plasmid DNA containing the correct cDNA sequence and regulatory elements of the gene of interest. The main challenges with this technology have been 1) the efficiency of gene delivery inside the cells, 2) appropriate shuttling of the vector from the cytoplasm to the nucleus where the plasmid is transcribed, and 3) the transient expression induced. Although plasmid DNA is not thought to be immunogenic, methylation of plasmid DNA may contribute silencing of transgene expression, which occurs when this strategy is tested in vivo. The DNA methylation issue can be overcome at least in part by making the therapeutic plasmids in cells that are incapable of methylating the DNA. However, the delivery of the plasmid DNA to the nuclei of the target cells is still a major concern. Approaches for increasing the passage of nonviral vectors into the cell, and into its nucleus include addition of 1) ligands for receptor-mediated endocytosis,

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2) polypeptide sequences that assist DNA compaction, 3) endosomal disruption sequences, and 4) nuclear-import signals. Among the technologies being developed for plasmid delivery are physical methods such as DNA microinjection, electroporation and gun injection, or chemical methods in which the plasmid DNA is combined with liposomal formulations or basic proteins and polymers such as polyethylenimine, to assist the plasmid cell entry by binding and/or enveloping the DNA through a charge interaction. Because of the transient expression of the transgene, the use of plasmid DNA for hematologic diseases has not been extensively studied.

Integrated Exogenous DNA Exogenous DNA, in circular as well as linear form, can spontaneously integrate into genomic DNA. Although this is a very rare event, there are some enzymes that have the capability to catalyze such reaction. This is the case for enzymes such as transposase, which are involved in the insertion of transposon elements into the genome, and site-specific integrases, which are found in bacteriophages and are able to first recognize unique nucleic acid sequences and then to mediate integration at that site. On the basis of this knowledge, investigators have developed nonviral vectors in which plasmid DNA is integrated into the genome of a target cell with the help of these enzymes. Two of these systems— the Sleeping Beauty vector (transposase-based method) and the phiC31 vector (integrase-based method)—are described below.

Sleeping Beauty Vector Transposable elements were not available for genome manipulations in vertebrates until recently, when an active element was resurrected from transposon fossils found in fish genomes.59 This element, called Sleeping Beauty transposon, shows efficient transposition in cells of a wide range of vertebrates, including humans. The integration of a corrected copy of the gene of interest is achieved by transfecting the target cell with two plasmids: one containing the therapeutic gene flanked by the transposable element and the other providing expression of the transposase. The Sleeping Beauty transposon carrying the therapeutic gene is “cut” out of the plasmid vector by the transposase and then “pasted” directly into the chromosome. The efficiency of this process is quite high (up to 10%) and the tropism is for both dividing and nondividing cells. The vector does not appear to be toxic or to be associated with a dramatic immune response after repeated administration in vivo. The integration of the vector in the genome is random (transcribed and nontranscribed regions), with a general preference for adenine and thymine (AT)-rich DNA, microsatellite repeats, and a small bias toward genes and their upstream regulatory sequence.60 The next challenge for this kind of technology will be to confer “specificity” for the integration site. Such targeted transposon integration at specific DNA sites is being tested by coupling a site-specific DNA-binding domain to the transposase.61 This system has been used for gene transfer into human CD34⫹ cord blood cells. When the vector containing GFP and the transposase was delivered to the cells via electroporation,

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transgene integration reached a frequency of 6% and was stable for over 4 weeks. However, the gene transfer of more primitive cells (CD34⫹/CD38⫺) was less efficient because these cells were particularly sensitive to damage induced by the electroporation. Thus, although this vector may have an interesting potential application in gene therapy for hematologic diseases, the delivery of vector into primitive cells without damage is a significant challenge.62 A Sleeping Beauty vector with erythroid-specific expression of β-globin is under investigation.63

Recombinase The phiC31 integrase is a site-specific recombinase originally isolated from a Streptomyces bacteriophage. The phiC31 integrase catalyzes precise, unidirectional recombination between its 30 to 40-bp attP and attB recognition sites in the genomic DNA. In mammalian cells, the enzyme also mediates integration of plasmids bearing attB into genomic DNA at sequences that have partial sequence identity with attP, termed pseudo attP sites. The integrase approach involves delivering the gene of interest as a plasmid also containing the integrase recognition site attB (40 bp). The integrase pairs attB with the endogenous pseudo attP sites and catalyzes genomic integration. The system has been used successfully in gene therapy experiments in mouse liver and muscle, human skin, rat retina, and rabbit joints with an efficiency of 10% and with just a single copy integrated. The great advantages of this vector in gene therapy is that pseudo attP sites are rare in mammalian cells.64 A bioinformatics analysis revealed that phiC31 integrase-mediated integration into human pseudo attP sites was mostly (56%) distributed among 19 pseudo attP sequences. The integration sites were not close to cancer genes in the genome, suggesting that cancer risk may be minimal. However, a serious concern is that aberrant events were described, including large deletions and chromosomal rearrangements.65 To date, published data on using phiC31 for hematologic diseases have focused on optimization of phiC31 integrasemediated integration into human T-cell lines.66 The phiC31 integration sites in these cells were frequently located in intergenic regions on chromosomes 13 and 18 that may be preferred in hematopoietic cells. PhiC31 was used effectively to restore functional γc protein in a mutant cell line, supporting the possible application of the phiC31 integrase-mediated genomic integration strategy as a gene therapy approach for treating SCID-X1.

Gene Targeting Gene targeting strategies attempt to directly correct the endogenous DNA sequence rather than introducing the therapeutic DNA by random insertion into the genome. In addition to allowing for potentially permanent correction of a gene disorder, such gene targeting avoids the risks associated with harmful mutations caused by the chance integration of the therapeutic DNA, prevents delivery of extra copies of the transgene, and allows for control of gene expression by the endogenous regulatory mechanisms. The technology is based on promoting homologous

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recombination, which mediates the exchange between the endogenous (dysfunctional) target sequence and the corrected donor DNA sequence. Investigators have used several different targeting approaches including chimeric RNA-DNA oligonucleotides, short single-stranded oligonucleotides, triplex DNA, and small DNA fragments, in order to modify specific bases of various reporter genes, as well as different gene mutations associated with human disorders. These vectors, which vary in length and chemistry, in most cases are designed to be homologous to the target locus except for the nucleotides to be replaced.67,68 Although each of these approaches has induced targeted homologous recombination, the frequency to date is too low for clinical applications.

Zinc Finger Nucleases Novel strategies are being developed in order to better target the site of homologous recombination. Based on the observation that double-strand breaks in the genome enhance gene targeting by homologous recombination, bifunctional zincfinger (ZF) proteins are being designed to induce double-strand DNA breaks within specific DNA sequences. The two functional domains of the zinc-finger nucleases (ZFNs) are ZFs customized to recognize specific DNA sequences, and a nuclease domain. The amino acid composition of the ZF domains of proteins determines the specific DNA sequences to which they bind. ZFNs specifically designed to recognize different DNA sequences that flank a specific genomic locus, are fused in frame with the DNA cleavage domain from FokI nuclease. After two ZFNs dimerize, one that binds to a DNA sequence upstream and one that binds a different DNA sequence downstream of the target DNA, the nuclease will induce a double-strand break in that specific location. If donor DNA is present, homologous recombination will occur with a frequency up to 10%.69,70 Although this technology is far away from translation to the clinic, ZFNs are able to stably and specifically modify primary human CD4⫹ T cells at the endogenous IL2Rγ locus with a frequency up to 6.6%.71 Ongoing studies are focused on using this strategy for gene correction of sickle cell anemia, X-linked SCID, and Wiskott-Aldrich syndrome. Recently, investigators have combined technology from lentiviruses and zinc finger proteins using the advantages of lentiviruses, their ability to enter the nucleus, with the site specific DNA nicks created by zinc finger proteins. They have inserted the cDNA for a zinc finger gene into a defective lentiviral vector that cannot incorporate into the DNA. Template (corrected) DNA plus the integrase gene are also introduced in order to achieve targeted gene correction.

Clinical Protocols Basic Design of Clinical Trial Stages In designing a clinical trial, different goals are defined for Phases I through IV. In Phase I trials, researchers test the treatment in a small group of people (usually 20 to 80) for the first time to

evaluate its safety, determine a safe dosage range, and identify side effects. Phase I studies are thus primarily concerned with safety and secondarily with the feasibility of the work proposed. In Phase II clinical trials, more people (100 to 300) are recruited in order to evaluate whether the therapy is effective and to further evaluate its safety. Most gene therapy protocols are currently in the Phase I or Phase II stages. In Phase III studies, larger groups of people (1000 to 3000) are treated in order to confirm that the therapy is effective, to monitor side effects, and to compare it with existing treatments. Factors involved in safety include production of sterile replication-incompetent vectors, prevention of adverse reactions, and minimization of patient immune responses against vector. Phase IV studies are postmarket trials that provide additional confirmation of the efficacy and safety of the regimen. Regulatory considerations for all clinical trials are assessed by multiple reviewers with different levels of oversight, including an institutional scientific review committee, institutional human investigation committee, institutional biosafety committee, the NIH recombinant advisory committee, and the FDA.

Clinical Gene Therapy Successes and Failures Several clinical trials of gene therapy for hematologic diseases have been completed. In all of these trials oncoretroviruses were used and a functional LTR promoter drove expression of the transgene. Overall, approximately 20 patients have been treated, and 80% are healthy and have recovered from their genetic defect.

Clinical Trials for X-Linked Severe Combined Immunodeficiency The first success in gene therapy was achieved by a French group who treated 10 children with X-linked SCID, in which there is a complete lack of functional T and natural killer (NK) lymphocytes in affected boys because of mutation of the gene encoding the shared subunit of the receptors for interleukins 2, 4, 7, 9, and 15.33,72,73 A murine retrovirus vector was used to introduce the cDNA for the missing receptor ex vivo into autologous CD34⫹ marrow cells, and the cells were returned to the patients. Because of the survival advantages of the corrected HSCs over the endogenous defective cells, preconditioning was not necessary to obtain long-term engraftment in these patients. Circulating T and NK cells that contained the therapeutic transgene were detected in nine of 10 patients and the functional activity of the T and NK cells were the same as in normal unaffected children for at least the first 2.5 years following gene transfer. However, four of the 10 patients developed leukemia caused by insertional mutagenesis of the proviral vector near the proto-oncogene LMO2.32 Unfortunately, one child died of leukemia; the other three patients are responding well to antileukemic therapy.74 At the same time, a very similar clinical trial was performed by a British group in which four children75 and two adults76 affected by X-linked SCID, were treated with autologous CD34⫹ marrow HSCs that were transduced with a gammaretrovirus (almost

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identical to the vector used in the other clinical trials) encoding the corrected sequence for the receptor. In this study, the four children had restoration of functional immunity whereas the treatment was not effective in the older patients. To date, at least one patient treated in the British trial has developed leukemia. No reason is yet known for the different incidence of leukemia in the French and British trials; more time has elapsed since the French trial was performed so the comparison is not yet final.

Clinical Trials for Adenosine Deaminase Severe Combined Immunodeficiency Retroviral vectors containing the missing adenosine deaminase (ADA) gene were used to transfect autologous cord blood cells, which were then returned to three newborns who had been diagnosed with ADA deficiency in utero. Using in-vitro retroviral infection protocols that achieved levels of infection of 5% to 40% of hematopoietic stem and progenitor cells, the patients had low percentages (0.01 to 0.001%) of detectable circulating blood cells containing vector sequences after 1 year.77 In these human studies, no replication-competent retrovirus has been detected. After 5 years, the authors reported that there were still some circulating cells that contain the transgene. Because these children are being treated with an oral form of pegylated-ADA (the standard of care for the disease), the potential therapeutic effect of the inserted transgene cannot be assessed.78 Aiuti et al79 have performed more recent clinical trials for ADA deficiency using an improved protocol for gene transfer into HSCs. In this clinical trial, the patients were not treated with pegylated-ADA and, to provide an initial engraftment advantage for the modified HSCs, patients received a low intensity, nonmyeloablative conditioning regimen using busulfan. Unlike X-SCID, the corrected cells in ADA deficiency do not have a strong survival advantage. Autologous CD34⫹ marrow cells were transduced with the retroviral vector ex vivo, and infused into the patients.79 After 47 months, all of the patients were healthy and cured from the genetic immunodeficiency associated with ADA deficiency.80 Similar success was also recently obtained in another ADA-SCID patient treated with autologous CD34⫹ cells transduced with ADA-expressing gammaretrovirus. As in the trials conducted by Aiuti et al, the patient received a nonmyeloablative conditioning regimen. However, in this case, the patient was undergoing pegylated-ADA treatment, which was stopped just before transplantation.81 In all of these clinical trials the vector used and the technical procedures for preparing the HSCs were very similar. A recent analysis of the different viral insertion sites in the genomes of patients from the three clinical trials82 showed that the proviral insertion was similar in all three trials, and was not random but clustered near promoters of genes in GpC islands. No significant differences were found in the viral insertion sites between the patients who did or did not develop leukemia in the French trial.74

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Clinical Trial for Granulomatous Disease To date, two patients affected by chronic granulomatous disease (CGD), characterized by neutrophil dysfunction with impaired oxidative antimicrobial activity, have been treated with gene therapy. As in the ADA-SCID trials, autologous HSCs were transduced with a gammaretrovirus encoding a functional copy of the transgene called gp91(phox), and the patients were treated with nonmyeloablative conditioning before infusion of the genetically modified cells. The patients had a robust amelioration of symptoms including clearance of infection associated with engraftment of corrected cells.83-85 A careful evaluation of insertion sites and clonality analysis, revealed that although the initial marrow reconstitution was polyclonal, with time there was selection for cells having the provirus inserted in the promoter of genes involved in survival and cells division. Both patients showed insertions in the MDS-Evil locus, which is known to be associated with leukemia. One patient lost the transgene expressing cells and died of infection and, at last report, the other has remained well, without progression to leukemia.86 First Clinical Trials Using Lentivirus Vectors Lentiviruses are extremely promising vectors for gene therapy. The first Phase I clinical trial using this vector has recently been performed for the treatment of five patients with chronic human immunodeficiency virus (HIV). In this case, the vector is not used for delivering a corrected copy of a gene but, rather, for transcribing an antisense RNA sequence against the HIV envelope with the goal of impairing viral infection. Autologous CD4⫹ cells were expanded ex vivo, transduced with the lentivirus vector, and infused into the patients. The treated patients showed an increased cellular response to HIV and improved the memory response of T cells. In one of the patients, the lentivirus-based gene therapy led to a robust antiviral response. At 21 to 36 months after treatment, there was no evidence of genotoxicity from insertional mutagenesis.87

Designing a Clinical Vector Laboratory Gene therapy holds much promise for the treatment of malignant and nonmalignant diseases. But, just as there is the potential for benefit, there is also the potential to cause harm. Safety measures are being put into place at all levels from the basic science laboratories and centers where the work is being tested to the establishment of federal regulations designed to safeguard the patients, the people working with the transgenes, and the general public. The clinical stem cell laboratory, where gene therapy vectors are applied to the cells ex vivo, must be compliant with very stringent regulations designed to maintain safety and sterility. These complex and thorough regulations need to be followed in order to be within the limits of current good manufacturing practice (cGMP) regulations. By definition, cGMP regulations are “methods to be used in, and the facilities or controls to be used for, the manufacture, processing, packing, or holding

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of a drug to assure that such drug meets [safety] requirements … and meets the quality and purity characteristics that it purports or is represented to possess.”88 The cGMP requirements are put into place to ensure that all procedures are performed in as safe and controlled a manner as possible. All of the general principles of cGMP are outlined by the FDA in the Code of Federal Regulations (CFR) in 21 CFR 210 and 211, 21 CFR 600s, and 21 CFR 820. Some of the key elements of cGMP encompass raw materials, maintenance of equipment, complete standard operating protocols, validation, record-keeping, and personnel training. Several professional organizations including the Foundation for the Accreditation of Cell Therapy and AABB have voluntarily established comprehensive standard-setting and accreditation programs that encompass all phases of cell collection, processing, and transplantation. Additional standards specifically for gene therapy are under constant development and improvement by the United States Pharmacopoeia (USP), the USP Biotechnology and Gene Therapy Subcommittee, and its Advisory Panel on Gene and Cell Therapies.88

Summary Gene therapy continues to offer much promise for the treatment of genetic and acquired diseases. Gene therapy has been used to treat patients with X-SCID, ADA-SCID, CGD, and HIV, and applications to other diseases are currently being tested in clinical trials. Transfusion medicine laboratories are currently involved in clinical gene therapy trials and, in the future, when these therapeutic modalities are approved, will oversee what may be “routine” administration of gene therapy vectors and vector-infected cells. This oversight requires extensive knowledge of cGMP as well as risk prevention/infection control in gene therapy.

Disclaimer The authors have disclosed no conflicts of interest.

References 1. Phillips K, Gentry T, McCowage G, et al. Cell-surface markers for assessing gene transfer into human hematopoietic cells. Nat Med 1996;2:1154-6. 2. Hanawa H, Hematti P, Keyvanfar K, et al. Efficient gene transfer into rhesus repopulating hematopoietic stem cells using a simian immunodeficiency virus-based lentiviral vector system. Blood 2004;103:4062-9. 3. Case SS, Price MA, Jordan CT, et al. Stable transduction of quiescent CD34(⫹)CD38(⫺) human hematopoietic cells by HIV-1-based lentiviral vectors. Proc Natl Acad Sci U S A 1999;96:2988-93. 4. Hirata RK, Miller AD, Andrews RG, Russell DW. Transduction of hematopoietic cells by foamy virus vectors. Blood 1996;88:3654-61.

5. Mergia A, Chari S, Kolson DL, et al. The efficiency of simian foamy virus vector type-1 (SFV-1) in nondividing cells and in human PBLs. Virology 2001;280:243-52. 6. Vassilopoulos G, Trobridge G, Josephson NC, Russell DW. Gene transfer into murine hematopoietic stem cells with helper-free foamy virus vectors. Blood 2001;98:604-9. 7. Leurs C, Jansen M, Pollok KE, et al. Comparison of three retroviral vector systems for transduction of nonobese diabetic/severe combined immunodeficiency mice repopulating human CD34⫹ cord blood cells. Hum Gene Ther 2003;14:509-19. 8. Williams DA. Expression of introduced genetic sequences in hematopoietic cells following retroviral-mediated gene transfer. Hum Gene Ther 1990;1:229-39. 9. Amsellem S, Ravet E, Fichelson S, et al. Maximal lentivirusmediated gene transfer and sustained transgene expression in human hematopoietic primitive cells and their progeny. Mol Ther 2002;6:673-7. 10. Larochelle A, Vormoor J, Hanenberg H, et al. Identification of primitive human hematopoietic cells capable of repopulating NOD/ SCID mouse bone marrow: Implications for gene therapy. Nat Med 1996;2:1329-37. 11. Dao MA, Hannum CH, Kohn DB, Nolta JA. FLT3 ligand preserves the ability of human CD34⫹ progenitors to sustain long-term hematopoiesis in immune-deficient mice after ex vivo retroviralmediated transduction. Blood 1997;89:446-56. 12. Cheshier SH, Morrison SJ, Liao X, Weissman IL. In vivo proliferation and cell cycle kinetics of long-term self-renewing hematopoietic stem cells. Proc Natl Acad Sci U S A 1999;96:3120-5. 13. Gothot A, van der Loo JC, Clapp DW, Srour EF. Cell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD34(⫹) cells in non-obese diabetic/severe combined immune-deficient mice. Blood 1998;92:2641-9. 14. Kavanaugh MP, Miller DG, Zhang W, et al. Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters. Proc Natl Acad Sci U S A 1994;91:7071-5. 15. Crooks GM, Kohn DB. Growth factors increase amphotropic retrovirus binding to human CD34⫹ bone marrow progenitor cells. Blood 1993;82:3290-7. 16. Orlic D, Girard LJ, Anderson SM, et al. Transduction efficiency of cell lines and hematopoietic stem cells correlates with retrovirus receptor mRNA levels. Stem Cells 1997;15 (Suppl 1):23-28; discussion 28-29. 17. Orlic D, Girard LJ, Jordan CT, et al. The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction. Proc Natl Acad Sci U S A 1996;93:11097-102. 18. Scott-Taylor TH, Gallardo HF, Gansbacher B, Sadelain M. Adenovirus facilitated infection of human cells with ecotropic retrovirus. Gene Ther 1998;5:621-9. 19. Miller N, Vile R. Targeted vectors for gene therapy. FASEB J 1995;9:190-9. 20. Kasahara N, Dozy AM, Kan YW. Tissue-specific targeting of retroviral vectors through ligand-receptor interactions. Science 1994;266:1373-6. 21. Kavanaugh MP, Kabat D. Identification and characterization of a widely expressed phosphate transporter/retrovirus receptor family. Kidney Int 1996;49:959-63.

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22. Dybing J, Lynch CM, Hara P, et al. GaLV pseudotyped vectors and cationic lipids transduce human CD34⫹ cells. Hum Gene Ther 1997;8:1685-94. 23. von Kalle C, Kiem HP, Goehle S, et al. Increased gene transfer into human hematopoietic progenitor cells by extended in vitro exposure to a pseudotyped retroviral vector. Blood 1994;84:2890-7. 24. Mergia A, Heinkelein M. Foamy virus vectors. Curr Top Microbiol Immunol 2003;277:131-59. 25. Puthenveetil G, Scholes J, Carbonell D, et al. Successful correction of the human beta-thalassemia major phenotype using a lentiviral vector. Blood 2004;104:3445-53. 26. Chang AH, Stephan MT, Sadelain M. Stem cell-derived erythroid cells mediate long-term systemic protein delivery. Nat Biotechnol 2006;24:1017-21. 27. Kowolik CM, Hu J, Yee JK. Locus control region of the human CD2 gene in a lentivirus vector confers position-independent transgene expression. J Virol 2001;75:4641-8. 28. Marodon G, Mouly E, Blair EJ, et al. Specific transgene expression in human and mouse CD4⫹ cells using lentiviral vectors with regulatory sequences from the CD4 gene. Blood 2003;101:3416-23. 29. Mitchell RS, Beitzel BF, Schroder AR, et al. Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences. PLoS Biol 2004;2:E234. 30. Trobridge GD, Miller DG, Jacobs MA, et al. Foamy virus vector integration sites in normal human cells. Proc Natl Acad Sci USA 2006;103:1498-503. 31. Donahue RE, Kessler SW, Bodine D, et al. Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer. J Exp Med 1992;176:1125-35. 32. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, et al. LMO2associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003;302:415-19. 33. Hacein-Bey-Abina S, von Kalle C, Schmidt M, et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003;348:255-6. 34. Check E. Gene-therapy trials to restart following cancer risk review. Nature 2005;434:127. 35. Check E. Gene therapy put on hold as third child develops cancer. Nature 2005;433:561. 36. Emery DW, Tubb J, Nishino Y, et al. Selection with a regulated cell growth switch increases the likelihood of expression for a linked gamma-globin gene. Blood Cells Mol Dis 2005;34:235-47. 37. Recillas-Targa F, Pikaart MJ, Burgess-Beusse B, et al. Positioneffect protection and enhancer blocking by the chicken beta-globin insulator are separable activities. Proc Natl Acad Sci USA 2002;99: 6883-8. 38. Burgess-Beusse B, Farrell C, Gaszner M, et al. The insulation of genes from external enhancers and silencing chromatin. Proc Natl Acad Sci U S A 2002;99(Suppl 4):16433-7. 39. Frederickson RM. Nucleic acid medicines move towards the clinic. Mol Ther 2005;12:775-6. 40. Chuck AS, Palsson BO. Consistent and high rates of gene transfer can be obtained using flow-through transduction over a wide range of retroviral titers. Hum Gene Ther 1996;7:743-50. 41. Chuck AS, Clarke MF, Palsson BO. Retroviral infection is limited by Brownian motion. Hum Gene Ther 1996;7:1527-34. 42. Takeuchi Y, Porter CD, Strahan KM, et al. Sensitization of cells and retroviruses to human serum by (alpha 1-3) galactosyltransferase. Nature 1996;379:85-8.

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43. Rother RP, Fodor WL, Springhorn JP, et al. A novel mechanism of retrovirus inactivation in human serum mediated by antialpha-galactosyl natural antibody. J Exp Med 1995;182:1345-55. 44. Gao G, Alvira MR, Somanathan S, et al. Adeno-associated viruses undergo substantial evolution in primates during natural infections. Proc Natl Acad Sci U S A 2003;100:6081-6. 45. Russell DW, Hirata RK. Human gene targeting by viral vectors. Nat Genet 1998;18:325-30. 46. Nathwani AC, Hanawa H, Vandergriff J, et al. Efficient gene transfer into human cord blood CD34⫹ cells and the CD34⫹CD38⫺ subset using highly purified recombinant adeno-associated viral vector preparations that are free of helper virus and wild-type AAV. Gene Ther 2000;7:183-95. 47. Schimmenti S, Boesen J, Claassen EA, et al. Long-term genetic modification of rhesus monkey hematopoietic cells following transplantation of adenoassociated virus vector-transduced CD34⫹ cells. Hum Gene Ther 1998;9:2727-34. 48. Wang H, Shayakhmetov DM, Leege T, et al. A capsid-modified helper-dependent adenovirus vector containing the betaglobin locus control region displays a nonrandom integration pattern and allows stable, erythroid-specific gene expression. J Virol 2005;79:10999-1013. 49. Kaiser J. Gene therapy. Questions remain on cause of death in arthritis trial. Science 2007;317:1665. 50. Donsante A, Miller DG, Li Y, et al. AAV vector integration sites in mouse hepatocellular carcinoma. Science 2007;317:477. 51. Chen L, Pulsipher M, Chen D, et al. Selective transgene expression for detection and elimination of contaminating carcinoma cells in hematopoietic stem cell sources. J Clin Invest 1996;98:2539-48. 52. Huang S, Kamata T, Takada Y, et al. Adenovirus interaction with distinct integrins mediates separate events in cell entry and gene delivery to hematopoietic cells. J Virol 1996;70:4502-8. 53. Jaras M, Brun AC, Karlsson S, Fan X. Adenoviral vectors for transient gene expression in human primitive hematopoietic cells: Applications and prospects. Exp Hematol 2007;35:343-9. 54. Yotnda P, Onishi H, Heslop HE, et al. Efficient infection of primitive hematopoietic stem cells by modified adenovirus. Gene Ther 2001;8:930-7. 55. Knaan-Shanzer S, Van Der Velde I, Havenga MJ, et al. Highly efficient targeted transduction of undifferentiated human hematopoietic cells by adenoviral vectors displaying fiber knobs of subgroup B. Hum Gene Ther 2001;12:1989-2005. 56. Nilsson M, Karlsson S, Fan X. Functionally distinct subpopulations of cord blood CD34⫹ cells are transduced by adenoviral vectors with serotype 5 or 35 tropism. Mol Ther 2004;9:377-88. 57. Goldman MJ, Wilson JM. Expression of alpha v beta 5 integrin is necessary for efficient adenovirus-mediated gene transfer in the human airway. J Virol 1995;69:5951-8. 58. Glover DJ, Lipps HJ, Jans DA. Towards safe, non-viral therapeutic gene expression in humans. Nat Rev Genet 2005;6:299-310. 59. Koga A, Suzuki M, Inagaki H, et al. Transposable element in fish (letter). Nature 1996;383:30. 60. Ivics Z, Izsvak Z. Transposons for gene therapy! Curr Gene Ther 2006;6:593-607. 61. Yant SR, Huang Y, Akache B, Kay MA. Site-directed transposon integration in human cells. Nucleic Acids Res 2007;35:e50. 62. Hollis RP, Nightingale SJ, Wang X, et al. Stable gene transfer to human CD34(⫹) hematopoietic cells using the Sleeping Beauty transposon. Exp Hematol 2006;34:1333-43.

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63. Zhu J, Kren BT, Park CW, et al. Erythroid-specific expression of beta-globin by the Sleeping Beauty transposon for sickle cell disease. Biochemistry 2007;46:6844-58. 64. Calos MP. The phiC31 integrase system for gene therapy. Curr Gene Ther 2006;6:633-45. 65. Chalberg TW, Portlock JL, Olivares EC, et al. Integration specificity of phage phiC31 integrase in the human genome. J Mol Biol 2006;357:28-48. 66. Ishikawa Y, Tanaka N, Murakami K, et al. Phage phiC31 integrasemediated genomic integration of the common cytokine receptor gamma chain in human T-cell lines. J Gene Med 2006;8:646-53. 67. Gruenert DC, Bruscia E, Novelli G, et al. Sequence-specific modification of genomic DNA by small DNA fragments. J Clin Invest 2003;112:637-41. 68. Parekh-Olmedo H, Ferrara L, Brachman E, Kmiec EB. Gene therapy progress and prospects: targeted gene repair. Gene Ther 2005;12:639-46. 69. Bibikova M, Beumer K, Trautman JK, Carroll D. Enhancing gene targeting with designed zinc finger nucleases. Science 2003;300:764. 70. Porteus MH, Baltimore D. Chimeric nucleases stimulate gene targeting in human cells. Science 2003;300:763. 71. Urnov FD, Miller JC, Lee YL, et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 2005;435:646-51. 72. Cavazzana-Calvo M, Hacein-Bey-Abina S, Fischer A. Gene therapy of X-linked severe combined immunodeficiency. Curr Opin Allergy Clin Immunol 2002;2:507-9. 73. Hacein-Bey-Abina S, Fischer A, Cavazzana-Calvo M. Gene therapy of X-linked severe combined immunodeficiency. Int J Hematol 2002;76:295-8. 74. Deichmann A, Hacein-Bey-Abina S, Schmidt M, et al. Vector integration is nonrandom and clustered and influences the fate of lymphopoiesis in SCID-X1 gene therapy. J Clin Invest 2007;117:2225-32. 75. Gaspar HB, Parsley KL, Howe S, et al. Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector. Lancet 2004;364:2181-7.

76. Thrasher AJ, Hacein-Bey-Abina S, Gaspar HB, et al. Failure of SCID-X1 gene therapy in older patients. Blood 2005;105:4255-7. 77. Kohn DB. The current status of gene therapy using hematopoietic stem cells. Curr Opin Pediatr 1995;7:56-63. 78. Gaspar HB, Thrasher AJ. Gene therapy for severe combined immunodeficiencies. Expert Opin Biol Ther 2005;5:1175-82. 79. Aiuti A, Slavin S, Aker M, et al. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 2002;296:2410-13. 80. Aiuti A, Cassani B, Andolfi G, et al. Multilineage hematopoietic reconstitution without clonal selection in ADA-SCID patients treated with stem cell gene therapy. J Clin Invest 2007;117:2233-40. 81. Gaspar HB, Bjorkegren E, Parsley K, et al. Successful reconstitution of immunity in ADA-SCID by stem cell gene therapy following cessation of PEG-ADA and use of mild preconditioning. Mol Ther 2006;14:505-13. 82. Bushman FD. Retroviral integration and human gene therapy. J Clin Invest 2007;117:2083-6. 83. Ott MG, Seger R, Stein S, et al. Advances in the treatment of chronic granulomatous disease by gene therapy. Curr Gene Ther 2007;7:155-61. 84. Stein S, Siler U, Ott MG, et al. Gene therapy for chronic granulomatous disease. Curr Opin Mol Ther 2006;8:415-22. 85. Ott MG, Schmidt M, Schwarzwaelder K, et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med 2006;12:401-9. 86. Dunbar CE. The yin and yang of stem cell gene therapy: insights into hematopoiesis, leukemogenesis, and gene therapy safety. Hematology Am Soc Hematol Educ Program 2007; 460-5. 87 Levine BL, Humeau LM, Boyer J, et al. Gene transfer in humans using a conditionally replicating lentiviral vector. Proc Natl Acad Sci U S A 2006;103:17372-7. 88. States P. Cell and gene therapy products. Cytotherapy 2000;2:123-71.

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Tissue Engineering and Regenerative Medicine Clay Quint1 & Laura Niklason2 1 2

Fellow, Biomedical Engineering, Yale University School of Medicine, New Haven, Connecticut, USA Associate Professor, Anesthesiology and Biomedical Engineering, Yale University School of Medicine, New Haven, Connecticut, USA

The fields of tissue engineering and regenerative medicine have evolved into a unified discipline with an interdisciplinary approach being a large part of the merger. The first use of the term tissue engineering in the public domain was at the proceedings of the Granlibakken workshop in 19851: “Tissue engineering is the application of principles and methods of engineering and life sciences toward fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and the development of biological substitutes to restore, maintain, or improve tissue function.” This was a broad definition that focused on the incorporation of living cells into acellular, related structures as a source for tissue replacements. In addition to providing a definition of tissue engineering, the basic components necessary to create a tissue-engineered product were identified. These components included a cell source, a method to induce specific tissue growth, and a biomaterial to act as a scaffold.2 The principles of tissue engineering have been translated to clinical applications with the Food and Drug Administration (FDA) approval of products in the areas of skin substitutes, bone formation, and cartilage. Tissue engineering has the potential to revolutionize medical care by replacing diseased or damaged tissue without the use of conventional organ or tissue transplantation.

Overview of Tissue Engineering A cell source or method that can be used for all cell lineages and has the required characteristics to make it safe, reproducible, and without any immunologic rejection has not been developed. The options for a cell source are primary autologous, allogeneic, xenogeneic, or stem cells. Cells can be cultured with low rates of bacterial contamination through the use of antibiotics

and sterile technique. However, concerns about xenogeneic contamination remain if feeder cells or sera from other species are used in the cell culture conditions. An alternative to primary tissue is to induce progenitor cells to differentiate into a specific cell type. Difficulties associated with the usage of primary tissue often include the need for an operative procedure, inability to obtain sufficient normal cells, inability of cells to proliferate, and the presence of cells with the same genetic predisposition for development of a disease. Another complication of primary cell sources is the need to efficiently isolate a specific cell lineage with low contamination rates from other cell types. Cell sorting methods, such as magnetic or fluorescent cell sorting, provide a reliable method if the cell type has specific membrane proteins with known antibodies.

Stem Cell Sources Stem cells are an attractive cell source for tissue engineering applications. Adult stem cells or human embryonic stem cells each offer their own set of advantages and challenges. Adult stem cells can typically be harvested by less invasive procedures from marrow, fat, or skin. Because these cells are autologous, no immunosuppressive medications are needed when the tissue is implanted in the donor. Purification difficulties and a reduced differentiation potential of adult stem cells as compared to embryonic cells, however, prevent use of these cells for all applications. Adult stem cells do not involve human embryos or any of the ethical controversies related to the collection of embryonic cells. The potential use of embryonic stem cells has created a national debate over ethical issues, because an embryo must be destroyed to harvest the cells. In the United States a limitation currently exists on public funding but not on private funding for human embryonic stem cell research.

Scaffolds Rossi’s Principles of Transfusion Medicine, 4th edn. Edited by T. L. Simon, E. L. Snyder, B. G. Solheim, C. P. Stowell, R. G. Strauss and M. Petrides. © 2009 AABB published by Blackwell Publishing, ISBN: 978-1-4051-7588-3.

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A scaffold should enable the creation of a three-dimensional formation of tissue in vitro and in vivo. Properties for the ideal tissue-engineered scaffold are the support of cell attachment, ability for the cells to proliferate and differentiate, allowance for

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diffusion of nutrients and waste, and ability to simulate mechanical properties found in vivo.3 Biomaterials can be categorized as synthetic polymers, natural materials, or inorganic matter; they can also be classified as being biodegradable or permanent.4 The preference for tissue-engineered scaffolds has been on biodegradable natural materials and polymers. Natural materials include collagen, gelatin, fibrinogen, dextran, glycosaminoglycans, and chitin. Examples of biodegradable synthetic polymers are polylactic acid (PLA), polyglycolic acid (PGA), and polycaprolactone (PCL). The properties of natural materials are their similarity to the native biologic environment, minimal toxicity, and degradation by natural enzymes.4 The limitations of natural materials include batch-to-batch variability from different animal sources, and potential immunologic reactions if materials are of xenogeneic origin. The most commonly used FDA-approved biodegradable synthetic polymers are polyglycolic acid and polylactic acid. These synthetic polymers are degraded by hydrolysis. Copolymers of PGA and PLA can be designed in specific ratios to match the degradation process for the individual tissue requirements.4 Scaffold fabrication techniques provide a variety of methods to form a structure, alter the mechanical properties, and change the degradation rates. Fibers can be woven or knitted to form structures with controlled pore sizes.5 The molecules selfassemble under the appropriate conditions. Natural materials, such as components in the extracellular matrix, often are capable of forming scaffolds by self-assembly.5 Solvent casting and particulate leaching techniques involve dissolving a polymer in a solvent, and then casting into a mold filled with a porogen (eg, sodium chloride or gelatin). Gas foaming is a process wherein a polymer is prepared by compression molding, or where the gas, for instance CO2, is dissolved into the polymer melt. The structure is exposed to high pressure CO2; then, as the pressure is reduced, pores remain when the CO2 evacuates.3 Electrospinning is a method in which a polymer forms nanofibers as an electrified jet is deposited on a metallic collector.3

Skin Tissue Engineering The field of skin tissue engineering has developed in response to the clinical need for skin substitutes. The clinical applications that skin substitutes have focused on are burn injuries and chronic wounds. The estimated incidence of deaths from fire and burns in the United States is 4500 each year, and there are approximately 45,000 hospitalizations per year to treat burn injuries.6 Chronic wounds that require skin substitutes are caused by arterial occlusive disease, venous disease, and pressure ulcers. These wounds affect more than 2 million people in the United States.7 Although the need for skin substitutes in this group is less acute than in the burn group, the morbidity as a result of these chronic wounds is significant. For patients with nonhealing lower leg ulcers, such wounds can eventually lead to an amputation.

Structure and Function The structure of skin consists of the surface epidermis and the deeper layer of connective tissue, the dermis. The epidermis is mostly keratinocytes, which are responsible for the barrier function and strength of the epidermis. The connective tissue layer of the dermis contains fibroblasts, few adipose cells, blood vessels, mast cells, nerve endings, hair follicles, and glands. The main component of the connective tissue matrix is collagen, with other matrix molecules such as elastin and reticulin. The skin functions to provide a barrier between the body and the external environment, and to provide sensation, temperature regulation, and vitamin D production. The most important function of the skin as related to skin substitutes is for the epidermis to restore the skin barrier. The origins of the field of tissue engineering derived from the establishment of skin substitutes. The early leaders in this field in the 1980s were Burke and Yannas at the Massachusetts General Hospital and Massachusetts Institute of Technology.8 They developed a bilayer artificial skin substitute with a temporary silastic epidermis and collagen dermis that could support the growth of dermal fibroblasts from the wound bed.8 Other investigators developed similar methods using allogeneic keratinocytes with collagen gel or on PGA mesh.9 The first skin substitutes were approved by the FDA in the late 1990s for applications of venous leg ulcers and for temporary covering of partial-thickness burns.

Design Goals for Skin Substitutes An intact epidermal layer provides a barrier function to prevent fluid losses and to inhibit infection. An ideal skin substitute will permanently heal into the wound bed to replace the dermal and epidermal layers in a timely manner, to form a desirable cosmetic result, to have minimal risk of infection or immunologic rejection, and to have a low cost. The standard material for covering large burn wounds is an autologous splitthickness skin graft. Autologous skin grafts function to permanently heal into the wound bed, contain an epidermal and dermal component, and have no risk of immunorejection. Skin substitutes are a viable alternative to autografts because they are readily available, do not require donor site harvest, and allow for better healing than dressing changes alone. The skin substitutes provide a temporary coverage over the wound; eventually, they get rejected or require a secondary thin epidermal autograft. Because most of the skin substitutes do not heal into the wound, they promote healing by stimulating cytokines and growth factors in the wound bed to recruit and direct host cells to repair the wound site.10 Early alternatives to the standard of autologous skin grafts were allogeneic skin grafts and porcine xenogeneic skin grafts. Allogeneic skin grafts have been the preferred material for temporary coverage as a skin substitute over xenografts. The beneficial properties of the allogeneic skin graft are that it prevents desiccation of the wound, prevents fluid losses, has a dermal component, and stimulates repair of the wound bed for eventual

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placement of an autograft.11 Porcine skin grafts are temporary skin grafts that have similar properties to allogeneic skin grafts, with the addition of being more readily available and cheaper. Because allogeneic and xenogeneic grafts do not incorporate into the wound, these grafts carry the potential risk of infection and eventual immunorejection.11

Acellular Skin Substitutes The group of acellular skin substitutes includes Biobrane, AlloDerm, Integra, and Transcyte. Biobrane (UDL Laboratories, Rockford, IL) is a synthetic bilaminate skin substitute that was introduced in 1979. The outer layer is silicon rubber, while a secondary layer consists of a nylon mesh that is covalently bound to Type I porcine collagen.4 The outer layer silicon pore size allows for drainage of exudate and permeability to topical antibiotics, but the small pore size acts as a barrier to bacterial invasion.4 Additional properties of Biobrane are adherence to clean wounds, stimulation of ingrowth of cells from the wound bed, ease of application, and a shelf life of at least 3 years. Biobrane may be used for superficial partial-thickness burns, over a meshed autograft, and in donor sites. Biobrane has demonstrated a reduced time to re-epithelialization when compared to traditional therapy of topical antibiotics and dressing changes for burn injuries. AlloDerm (LifeCell, Branchburg, NJ) is a dermal skin substitute that removes epithelial cells from harvested human skin to leave an acellular dermal matrix. AlloDerm is approved for treatment of partial to full-thickness burns and chronic wounds. AlloDerm has similar properties to allogeneic grafts, except the immunologic response is reduced because of the processing of the tissue.12 The clinical use of AlloDerm for partial-thickness or full-thickness burns is similar to that of the dermal component, but an ultra-thin autograft is needed over the AlloDerm. The advantage of the ultra-thin autograft is that it reduces the morbidity or loss of donor site grafts, due in particular to scar formation. It has been shown that an acellular allograft with a thin autograft produces equivalent healing rates at 14 days in comparison to split-thickness skin grafts.13 Further case studies have demonstrated high success rates of AlloDerm with a splitthickness autograft.14 Integra (Integra Life Sciences, Plainsboro, NJ) is a bilayer skin substitute that was approved in 1996 for use in partialthickness and full-thickness burns. The dermal component is a biodegradable collagen-chondroiten-6-sulfate matrix with an epidermal layer that is a thin silicone elastomer. The thin silicone layer acts as a barrier to bacteria and controls moisture loss. The dermal component encourages the adherence and growth of the wound bed. After 2 to 3 weeks when a neodermis has formed, the thin silicone layer is removed and then covered with a thin autograft. TransCyte (Advanced Tissue Sciences, La Jolla, CA) is also a bilayer skin substitute that was approved in 1997 for temporary coverage for partial-thickness or full-thickness burns with autografting. The outer epidermal component is a silicone thin

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film with an inner dermal layer consisting of a nylon mesh coated with porcine dermal collagen with ingrowth of neonatal human fibroblasts. The material is cryopreserved, which destroys the metabolic activity of the fibroblast cells, but the tissue growth factors and matrix remain intact. The growth factors and complex matrix components may create an environment more favorable for the adherence and migration of fibroblasts. TransCyte has demonstrated faster healing times and reduced scar formation in comparison to topical antibiotics in partial-thickness burns.15

Allogeneic Skin Substitutes The allogeneic skin substitutes that are FDA-approved include Apligraf, Dermagraft, and Orcel. Apligraf (Organogenesis, Canton, MA) is a bilayer structure with Type I collagen gel and allogeneic fibroblasts that form a dermal equivalent, onto which allogeneic keratinocytes are seeded to provide an epidermal layer. Dermagraft (Dermagraft, Westport, CT) is a singlelayer skin substitute using allogeneic fibroblasts cultured onto a bioresorbable glycolic acid scaffold. Orcel (Forticell Bioscience, New York, NY) is a bilayer material with fibroblasts cultured in a porous sponge as the dermal layer with keratinocytes seeded on the nonporous side of the matrix. Although the fresh form of Orcel is available, a cryopreserved formulation is in clinical trials. The mechanism of wound healing for these skin substitutes is not clearly understood, but they seem to stimulate epithelialization by the fibroblasts’ release of growth factors and extracellular matrix to provide recruitment of endogenous cells.16 The function of the allogeneic fibroblasts likely contributes a temporary response to the wound healing, because at 4 weeks after treatment, only two of 10 patients had Apligraf DNA in the wound site.17 Apligraf, Dermagraft, and Orcel have all shown improved rate of wound closure when used for chronic ulcers in comparison to conservative treatment with dressing changes.18,19 The primary application for the allogeneic skin substitutes is for the treatment of chronic ulcers or acute wounds, with limited use for partial-thickness or full-thickness burn injuries. Currently available skin substitutes are listed in Table 61-1. The current skin substitutes constituted the first step in using tissue-engineered products for clinical applications. These skin substitutes have demonstrated some of the potential successes and challenges that remain to create an ideal tissue-engineered equivalent. The limitations of the existing materials are related to the allogeneic cells that seem to function by recruiting host cells for wound healing instead of primarily contributing to wound healing. Cultured epidermal autograft techniques are not suitable for widespread application because only the epidermis can be created, without a true bilayer structure. The results of the cultured epidermal autografts have been disappointing because they are fragile, require a dermal layer, and require weeks to grow in culture. The future of skin substitutes may incorporate adult mesenchymal stem cells (MSCs) and gene therapy strategies for optimization. The design of a tissue-engineered skin graft could contain

Chapter 61: Tissue Engineering and Regenerative Medicine

Table 61-1. Clinical Skin Substitutes Reference

Skin Substitute

Application

Result (time to healing)

Kumar22/Barrett23

Silver sulfadiazine

Pediatric partial-thickness burn

11.2-16.1 days

Kumar22/Barrett23 Still24

Biobrane

Pediatric partial-thickness burn donor site

9.5-9.7 days 18.4 days

Callcut14

AlloDerm over split-thickness skin graft

Deep partial or full-thickness burn

26/27 graft take

Apligraf

Chronic ulcers

Heal % at 12 weeks: Apligraf, 56% Dressing, 38%

Blight25/Still26/Clugson27

Culture epithelial autograft

Full-thickness burn

Heal %: 15-80%

Purdue28/Martson18

Dermagraft

Full-thickness burn—temporary coverage; diabetic foot ulcers

Heimbach29

Integra

Full-thickness or deep partial-thickness burn

95% take Heal % at 12 weeks: Dermagraft, 30% Dressing, 18% Take Integra: 76% Take epidermal graft: 88%

Still24

Orcel

Donor site; venous ulcer

13.2 days Heal % at 12 weeks: Orcel, 59% Dressing, 36%

Kumar22/Noordenbos15

TransCyte

Pediatric partial-thickness burn; partial-thickness burn

7.5 days 11 vs 18 days

Veves

19

autogenous cells in a bilayer structure with a dermal and epidermal component. The use of marrow MSCs cultured with autogenous dermal fibroblasts may enhance epidermal regeneration.20 Gene therapy can be used to upregulate the delivery of growth factors or cytokines to expedite the regenerative process. In animal models, gene delivery with epidermal growth factor has been shown to dramatically accelerate healing.21 However, for wound healing the upregulation of growth factors should be transient, because continued gene expression might result in adverse effects. The advancement of skin substitutes and a better understanding of the biology of wound healing will likely lead to the development of rationally based designs to specifically treat the pathology of the wound.

Blood Vessel Tissue Engineering Cardiovascular disease is the leading cause of mortality in the United States.30 More than a million arterial bypass procedures performed annually require autologous vessel or synthetic grafts to replace coronary or peripheral arteries.31 More than 500,000 coronary bypass artery graft procedures are performed annually in the United States, and peripheral disease results in more than 100,000 amputations each year.30,32 The therapeutic modalities to treat cardiovascular disease are medical treatments, catheter-based interventional procedures, and surgical procedures. The medical treatments focus on disease

modification by treating conditions that lead to cardiovascular disease such as management of hypertension, hyperlipidemia, and diabetes, and cessation of smoking. The interventional procedures such as angioplasty, stents, or thrombolytic therapy offer a less invasive treatment than does surgery for patients with occlusive vessel disease. The surgical options for vascular disease are bypass surgery, endarterectomy, or open thrombectomy. The currently available conduits for bypass procedures include autologous artery or vein, prosthetic material, and cadaveric vein. Autologous tissue provides the best outcome; however, autologous tissue in many patients may not be available or may not be suitable. Prosthetic materials, such as polytetrafluoroethylene (PTFE) and Dacron, perform well for large-diameter arteries in peripheral vascular disease. In contrast, no prosthetic material is well-suited for small-diameter (less than 6 mm) applications.33Another growing area of need for bypass procedures is hemodialysis—more than 250,000 patients require chronic hemodialysis access and frequent graft replacement.34 Tissue engineering technology has the potential to generate engineered arterial grafts for multiple applications in cardiovascular surgical therapy.

Structure and Function A blood vessel is composed of the intima, media, and the adventitia.35 The intima contains the endothelium, laminia propria, and the internal elastic lamina. The media contains the smooth

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muscles arranged concentrically, elastin and collagen fibers, and the external elastic lamina. The adventitia contains connective tissue, fibroblasts, vasa vasorum, and nerve cells. Arterial blood vessels are classified as elastic arteries, muscular arteries, and arterioles. The large elastic arteries have higher elastin content and fewer smooth muscle cells relative to diameter than other vessel types. Muscular arteries contain a greater number of smooth muscle cells in proportion to their diameter, and the adventitia is composed of a thicker layer of connective tissue in comparison to the elastic arteries. The arterioles consist of a thin layer of smooth muscle and range from 40 microns to 9 microns in diameter.35

Design Goals for a Tissue-Engineered Vessel The primary design goal of a tissue-engineered blood vessel is to create a graft that will maintain patency in small-diameter applications, eg, 2 to 6 mm. The desirable features of the tissue-engineered vessel are a patency rate comparable to autologous vein, similar mechanical properties to a native artery or vein, minimal immunologic or infective risk, a reasonable time to produce the grafts, and cost effectiveness. The reported 5-year patency rates using autologous vein grafts for below-the-knee applications are 50% to 76%.33 However, the patency rates for below-the-knee bypass with PTFE have a 2-year patency rate of 30% and only 18% after 5 years.36,37 In cardiac bypass, the 10-year patency rate of autologous vein is 50%.38 The current blood vessel prosthetics, Dacron and PTFE, both cause platelet and fibrin deposition that leads to thrombosis. Tissue-engineered grafts probably need some amount of lead time to culture, which could limit their clinical utility for some patients. New technology using tissue-engineered grafts will likely be more expensive than the current prosthetic materials. However, because there are no effective, synthetic, small-diameter grafts, the increase in cost for a tissue-engineered graft could be justified when considering reduced cost of chronic wound management, multiple surgical procedures with ineffective material, and reduction in patient morbidity.

Cell Source The predominant two cell types that form a vessel are smooth muscle cells and endothelial cells. The contribution of the live smooth muscle cells may not be critical for the function of the tissue-engineered vascular graft. Many tissue-engineered models take advantage of the process of decellularization to reduce the immunologic reaction.39 The decellularized scaffold is able to maintain the tissue architecture and the extracellular matrix components to encourage repopulation of the graft with host cells.40,41 The function of the endothelial cell layer on the lumen is to maintain an antithrombotic environment. Studies have shown that without an endothelial coating for a tissueengineered small vascular graft, there is a significantly higher rate of thrombosis.41,42 The options for obtaining endothelial cells

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from a recipient patient are to use primary vascular endothelial cells, or to isolate progenitor endothelial cells from the marrow or the peripheral blood. Primary endothelial cells from endogenous veins have the benefit of established isolation techniques that reliably produce endothelial cell cultures. The limitations to using a primary endothelial cell source are that it requires an operation to harvest a vein, derived endothelium is venous rather than arterial, and that they will have been exposed to potential disease processes. The most common source for endothelial progenitor cells is from the peripheral blood, although there are also reports of obtaining progenitor cells from marrow. The endothelial progenitor cell can be defined as a cell that has high proliferative capacity, is capable of clonal expansion, and can differentiate into endothelial cells. The first description of endothelial progenitor cells was by Asahara and colleagues in 1997.43 They demonstrated that the isolated cells from the peripheral blood mononuclear fraction that were sorted for CD34, expressed cell surface markers of endothelial cells such as vascular endothelial growth factor receptor-2 (VEGFR-2) and were positive for CD31. In addition, these progenitor cells exhibited functional properties of endothelium, by production of nitric oxide in response to acetylcholine and to VEGF.

Scaffold There are several approaches to developing a scaffold for tissue-engineered vessels. One approach is to use cells to form an extracellular matrix without a synthetic scaffold, while the other approach is to use synthetic polymers or natural materials to form the cell culture scaffold. In the first approach, L’Heureux and colleagues cultured sheets of fibroblasts or smooth muscle cells and wrapped the sheets around a mandrel (scaffold) in several layers to form a tubular structure.44 The burst pressure, compliance, and the wall thickness were all similar to native arteries.44 The biomimetic system developed by Niklason and colleagues for small-diameter vessels calls for placing aortic smooth muscle cells on a PGA scaffold under pulsatile flow for 8 weeks.42 The PGA scaffold degrades, and the resulting vessel is composed primarily of smooth muscle cells and collagen. The tissue-engineered vessel does exhibit contractile properties, although at 20% that of native vessels. The mechanical properties demonstrate burst pressure and compliance similar to native vessels.42 Other approaches use a collagen gel that is coated with a microporous polyurethane film.45 The collagen gel is compressed to remove water, and then wrapped with the polyurethane film. The compliance of such constructs in the higher strain range is similar to native arteries.45 Bruch et al created a tissue-engineered graft by mixing fibrinogen and thrombin in equal volumes into a mold with specific length and diameter.46 The only mechanical characterization was burst strength, which was significantly less than native arteries.46 A combination method evaluated in animal models has been to decellularize a native vessel and then to seed this with endothelial cells.41 There is evidence to suggest that decellularization invokes minimal immunologic

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reaction, in comparison to cryopreserved vessels, which lead to moderate immunologic reaction.47 The tissue-engineered graft developed by Shin’oka et al was designed for low-pressure venous systems, using a copolymer lactide and ε-caprolactone 1:1 scaffold.48 The graft is seeded with marrow cells at the time of operation and then sprayed with fibrin sealant, after which the graft is implanted.

Bioreactor Design The two most successful bioreactor designs for tissue-engineered vessels are to wrap sheets of fibroblasts around a mandrel or to use a flow bioreactor system to culture a vessel from smooth muscle cells. The first method involves obtaining a skin biopsy to culture fibroblasts.44 The fibroblasts are grown in sheets, and the first sheet is wrapped around a mandrel to form a tubular structure. The inner sheet is dehydrated to form an acellular inner membrane. Additional sheets of living cells are then placed around the inner membrane, and the tissue remains in culture to mature. The last step of this process involves obtaining endothelial cells harvested from saphenous vein to coat the luminal surface of the graft. The entire process takes approximately 28 weeks to form a vessel for implantation. The second technique is to obtain smooth muscle from a vessel biopsy, and to seed the smooth muscle on a biodegradable PGA scaffold.42 The scaffold with the cells remains static for 1 week to allow for attachment, and then the construct is exposed to pulsatile flow for 7 to 8 weeks. The luminal side of the graft can be seeded with endothelial cells. This process takes around 10 weeks total to create a vessel for implantation.

Preclinical Data Table 61-2 lists some of the important animal models that have combined a cell source with a scaffold to produce a functional vascular graft. L’Heureux recently reported using the fibroblast

sheet scaffold and primary endothelial cells in a nude rat model and an immunosuppressed primate model.49 The patency rates in both of these studies are high, and there were no graft mechanical failures. In Argentina this has been approved for a clinical study for arteriovenous graft in kidney failure patients and in England for coronary bypass graft.50 These studies are ongoing and actively enrolling patients. The tissue-engineered smooth muscle grafts seeded with primary endothelial cells by Niklason (unpublished observations) also had high patency rates in a porcine carotid model and no incidence of mechanical failure. Niklason’s group has demonstrated the mechanical stability of the tissue-engineered grafts after a decellularization process. The option of decellularization would allow for increased availability with the advantage of being able to create an artery and then to store the construct until needed. Other in-vivo studies have used a decellularized native vessel, and then coated the acellular construct with endothelial cells derived from peripheral blood endothelial progenitor cells.41 The patency rate could be attributed to the activity of the endothelial cells because they were fluorescently labeled and remained on the graft at the time of harvest. The Matsuda group that used a collagen gel with a microporous polyurethane film combined their unique scaffold with endothelial cells that were derived from endothelial progenitor cells.48,51 They also had very high patency rates in their carotid canine implant model, and the endothelial cells derived from progenitor cells provided a nonthrombotic environment. An interesting blood vessel graft for the application of large vein bypass for infants has achieved clinical success in Japan. Shin’oka has used a polylactide/caprolactone-reinforced PGA mesh coated with marrow mononuclear cells.48 In the case study, they performed a cavopulmonary bypass in 23 patients and a sheet graft in 19 patients. The median follow-up was 16.7 months with a 100% patency rate and without any major complications.

Table 61-2. Selected Animal Models that Have Produced a Functional Vascular Graft Reference

Model

Cell Source

Scaffold

Outcome

Human tube graft cavopulmonary connection (n ⫽ 23) Sheet graft patch (n ⫽ 19)

Endothelial cells—Marrow

Polylactide /caprolactone- reinforced PGA mesh

Follow-up: median 16.7 months

L’Heureux49

Canine aortic graft Rat aortic graft (n ⫽ 27) Primate aortic graft (n ⫽ 3)

Scaffold—fibroblast

Niklason42

Porcine saphenous artery graft (n ⫽ 4) Endothelial—primary SMC—porcine aortic

Tissue-engineered SMCs over PGA mesh 100% patent at 4 weeks

Cho52

Canine carotid (n ⫽ 6)

Endothelial—marrow SMC—marrow

Decellularized canine carotid

100% patent for 8 weeks

Endothelial—peripheral blood

Decellularized porcine iliac vessel

100% patent at 15 or 130 days

Endothelial—Peripheral Blood

Collagen mesh reinforced with polyurethane film

11/12 patent for up to 3 months

48

Shin’oka

Bischoff and Mayer41 Sheep (n ⫽ 7) Matsuda51

Canine (n ⫽ 12)

Patency: 100% No complications Fibroblast sheet

Rat: patency 85% at 90-225 days Primate: all patent at 6-8 weeks

Endothelial cell—primary cells

PGA ⫽ polyglycolic acid; SMC ⫽ smooth muscle cell.

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Future of Vascular Tissue Engineering The field of vascular tissue engineering has demonstrated proofof-principle methods with various scaffolds and cell sources that are now ready to emerge in the clinical arena. The animal studies provide insight into the potential of the application, but further human trials are needed to determine efficacy once the safety of these devices has been verified in animals. Tissue-engineered vessels appear to be the next generation of small-diameter bypass grafts to address the growing need for alternative materials for small-caliber arterial replacement. If the tissue-engineered grafts perform close to autologous veins in terms of mechanical characteristics and nonthrombotic surface, the morbidity and mortality of cardiovascular disease could be dramatically reduced.

Bone Tissue Engineering Bone tissue regeneration is a potential clinical treatment to replace loss of skeletal function for orthopedic and oral-maxillary applications. The most common needs for bone replacement materials are for trauma, tumor, infection, and inborn skeletal disorders. The current bone replacement materials are derived from autologous bone, allogeneic bone, demineralized bone matrices, and synthetic biomaterials. The first choice for bone repair has been the autologous bone graft.53,54 The advantages of autologous tissue are a lack of an immune reaction and the provision of bone cells for osteogenesis. In addition to the direct osteogenic effects, the autologous bone graft can stimulate recruitment of other mesenchymal cells through stimulation by bone morphogenic proteins.54 However, the use of autologous bone grafting is limited by the morbidity associated with the donor site. The usual sites to harvest bone grafts are the iliac crest and the fibula. After the procedure, up to 20% of patients have significant pain at the donor site.55 In a large review of iliac crest donor sites, major complications included vascular injuries, deep infections, neurologic injuries, and deep hematoma, with a combined incidence of 6%.56 An alternative to autologous bone grafting is allogeneic bone grafts. The mechanical properties of processed allogeneic bone tissue are similar to autologous bone, although the ability to induce bone healing is reduced.53 For smaller defects, such as bone chips in a distal radial fracture or for use in a cervical fusion, allogeneic bone seems to be comparable to autologous bone.57,58 Allogeneic bone groups had a longer time to fusion in cervical fusion, but this did not affect the fusion rate or collapse rate.58 The acrylate bone cements are a biomaterial that has been introduced for fixation of hip prostheses and to repair craniofacial defects. They provide mechanical support, but do not integrate into the bone and are minimally reabsorbed. This results in long-term failure at the interface between the bone and the prosthetic, which has led to metal prostheses coated with hydroxyapatite that do not use bone cement. The cementless hydryoxyapatite-coated prosthesis is under investigation to improve osseointegration and prevent bone remodeling that

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leads to long-term failure.59 Another new material for craniofacial defects is a hydroxyapatite-based paste. The paste is readily available, can be easily molded into shape, demonstrates osteoconduction, has good mechanical strength, lacks immune response or risk of viral transmission, and has the potential for bone replacement.60

Structure and Function Bones are composed of extracellular bone matrix and bone cells that form the shape of long, short, flat, or irregular bones.35 The bone matrix consists of an organic component of collagen and proteoglycans. The inorganic component of the bone matrix is a calcium phosphate crystal called hydroxyapatite. The bone cells can be classified as osteoblasts, osteoclasts, and osteocytes. Osteoblasts are bone cells that produce collagen, proteoglycans, and form bone. The osteoclasts are the cells responsible for the breakdown of bone by producing an acidic environment that causes decalcification of the bone matrix. The osteocytes are osteoblasts that are surrounded by matrix. The interaction of bone cells with hormones regulates the homeostasis of calcium and phosphate in the blood.

Scaffold The structure of the scaffold for bone regeneration is related to the function of bone. A bone scaffold should provide means of cell migration and proliferation, generate appropriate mechanical strength, integrate into the native bone matrix, stimulate vascularization, slowly degrade with increasing cellular infiltration, and not elicit an immunologic reaction. For example, the different requirements for the strength of long bones and for the flexibility of craniofacial bones may lead to the development of different scaffolds for these applications. The matrices used for bone tissue engineering can be classified as calcium phosphate derivatives or as synthetics. Hydroxyapatite is a calcium phosphate compound that is the primary component of bone. Hydroxyapatite as a scaffold offers a high degree of biocompatibility, the osteoconductive potential of hydroxyapatite, and slow degradation. Hydroxyapatite is a porous material that will allow ingrowth of bone and vascular cells.61 However, hydroxyapatite implants are known to be brittle and lack pliability to conform to irregular forms.61 Hydroxapatite cements have been approved for clinical use for non-stress-bearing cranial defects. The clinical experience with one of the hydroxyapatite bone cements in 100 patients over a minimum of 2 years demonstrated a success rate of 97% and a low rate of infection.62 Hydroxyapatite cements can be used for craniofacial repairs because they integrate into the bone and can be easily shaped to fit unique three-dimensional structures.62 The properties of calcium phosphate or hydroxyapatite grafts can be altered by integrating other biomaterials to form a composite material. The composites that investigators have evaluated include calcium phosphate-chitosan, calcium phosphate-polylactic acid, and hydroxyapatite-collagen scaffolds. The purpose for fabricating composite calcium phosphate is to

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improve the biodegradability and the bioactivity. Pore size and porosity are important factors in bioactivity, affecting cell adhesion, cell growth, the exchange of nutrients and waste products, and vascularization. A calcium phosphate-chitosan composite has shown improved strength characteristics in comparison to calcium phosphate cement alone.63,64 The fast-setting composite material is prepared by mixing a calcium phosphate-mannitol powder with chitosan water to form a paste that can be molded into shape or injected.63 A composite material of collagen and hydroxayapatite has been shown to support bone cell growth and act as a biocompatible material in vivo.65,66 There are different processing techniques to produce collagen-hydroxyapatite composites, such as gels, films, coatings, and hydroxyapatite-collagen matrices. The hydroxyapatite-collagen material creates a favorable environment for cell attachment and growth, and the cells are functional as assessed by the deposition of new bone.66

Vascularization A vascular supply for a tissue-engineered bone graft is necessary for bone growth and survival. The two approaches to vascularize a bone graft are to rely on angiogenesis of the donor, or to form a vascular network within the graft before implantation. The current bone graft substitutes depend on vascularization through angiogenesis from the donor after implantation of the scaffold with bone cells. An explanation for the success of bone tissue engineering in rodents in comparison to larger animal models is that the marrow-seeded grafts in rodents are small enough to allow for diffusion of oxygen and nutrients to allow cell survival before angiogenesis and new vessel formation.67 One method to improve vascularization is to stimulate angiogenesis on the graft. This may be accomplished by adding a combination of growth factors such as VEGF, platelet-derived growth factor-BB, and transforming growth factor-β (TGFβ).68,69 In a rat cranium bone tissue engineering application, the addition of VEGF was shown to improve vascularization and bone mineralization.70 However, it is not clear if the addition of the growth factors will vascularize a bone graft in a short enough time to maintain cell viability in a larger graft, such as that required for patients. The second method would be to vascularize the graft before implantation by heterotopic bone induction or to form a vascular network within the graft in vitro. Warnke and colleagues reported a successful extended mandible reconstruction by growth of a custom bone transplant from the latissimus dorsi muscle of an adult male patient.71 The custom bone transplant was prepared with a titanium mesh that was coated with recombinant bone morphogenic protein 7 (BMP-7) and seeded with marrow aspirate. The bone construct was implanted into the lattisimus dorsi muscle for 7 weeks, then was transplanted as a free bone-muscle flap to repair the mandibular defect. A postoperative computed tomography (CT) scan demonstrated new bone formation, and a nuclear scan showed bone remodeling and mineralization inside the mandibular transplant. In vitro, bone tissue engineering strategies could utilize a co-culture system to

form a vascular network using endothelial cells in a matrix that is later seeded with marrow cells.

Growth Factors Bone morphogenic proteins are osteoinductive factors that can be used to coat scaffolds. BMPs are glycoproteins that belong to the TGF-␤ family and bind to extracellular matrix components. BMPs are the biologically active component in demineralized bone matrix that leads to formation of new bone.72 BMP-2 and BMP-7 are involved in bone formation and osteoblast differentiation. These recombinant proteins have been used for applications such as spinal fusions, internal fixation of fractures, treatment of bone defects, and reconstruction of maxillofacial conditions. BMPs stimulate bone repair through recruitment, proliferation, and differentiation of MSCs.73 Preclinical studies have demonstrated the efficacy of BMPs in healing a variety of bone defects in many different animal models. The bone formation and healing of long bone defects with recombinant BMP2 and collagen scaffolds has been demonstrated in rats, rabbits, and dogs.74-76 Similar results have been shown with BMP-7 and collagen scaffolds in healing long bone defects in multiple animal species.77,78 Two recombinant BMP devices have been approved by the FDA. OP-1 (Stryker Biotech, Hopkinton, MA) has been approved for spinal fusion procedures under the humanitarian device exemption process. The OP-1 device is recombinant BMP-7 and bovine collagen combination that is used to form a paste.79 The other device is the Infuse Bone Graft/LT-CAGE (Medtronic, Minneapolis, MN).79 The indication for the device is for lumbar spinal fusion to treat degenerative disc disease. The device consists of a metallic spinal fusion cage with rh-BMP-2 on a bovine collagen scaffold inside the cage.

Cell Source The options for osteogenic cells in bone engineering are osteoblasts, periosteal cells, and MSCs. Osteoblasts and periosteal cells can be isolated from a biopsy of the calvarium, periosteum, or trabecular bone.80,81 Osteoblasts and periosteal cells are autologous, primary cell sources for bone engineering. Mesenchymal stem cells are utilized for bone regeneration, because the cells have a large proliferative potential, can be differentiated down osteogenic lineage, and can be harvested by a less invasive marrow aspiration procedure. The most common cell source for bone tissue engineering applications is currently MSCs.82 Mesenchymal stem cell differentiation to osteogenic cells was first described by Friedenstein.83 Mesenchymal stem cells have been induced to differentiate into osteoblasts, chondrocytes, adipocytes, and myoblasts.84 Caplan was the first to take advantage of the potential of MSCs by seeding the cells onto a porous calcium phosphate ceramic.85 In a subcutaneous in-vivo rat model, they demonstrated osteogenesis with an early phase resulting from the implanted mesenchymal cells and a late bone growth phase resulting from the host cells. Since these early papers, there have been many studies on the use of MSCs for bone tissue

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engineering applications. The use of MSCs on biomaterial scaffolds has accelerated bone formation and bone integration in vivo. Mesenchymal stem cells improved osteogenic potential directly by differentiating into osteoblasts, and indirectly by releasing bone growth factors that enhance migration and differentiation of endogenous osteogenic cells.86

Preclinical and Clinical Studies The evaluation of new cell-based therapies for bone repair are predominantly in the preclinical stages, with only a few human case reports. There are many studies optimizing scaffolds and evaluating the function of the MSCs. A limited number of groups are studying the challenging task of using a biomaterial in combination with a cell source to repair large osseous defects in animal models. All of the studies in Table 61-3 demonstrate that an MSC-seeded scaffold produces significantly greater bone formation than cell-free grafts, and in most of the studies the MSC-seeded scaffolds resulted in similar bone formation as bone grafts. In addition to the use of calcium phosphate as the scaffold, investigators have also used coral. The rationale for coral as a scaffold is the porous structure and more rapid degradation of coral in comparison to calcium-phosphate-based materials.87 The more rapid degradation time of the coral may reduce the inflammatory response and allow for growth of new bone tissue more quickly.88,89 The number of cells seeded may be an important variable for bone growth in the implanted graft. High cell density at the time of seeding may have a negative impact on the graft by not allowing the cells to spread out on the graft and proliferate. Another consequence of high cell density without adequate vascularization is the formation of a hypoxic environment in the center of the graft.87,89 Case reports have described bone tissue engineering in clinical practice. Vacanti et al reconstructed a distal thumb avulsion with periosteal cells and a coral scaffold.93 At 3 months the patient was able to return to work, and at 28 months the thumb had normal length and strength. In a separate study, three patients with large bone defects in the extremities were treated

with marrow cells and macroporous hydroxyapatite scaffold.94 All three patients recovered limb function. Thus, in the appropriately selected patients, it appears that bone tissue engineering concepts can be applied to repair bone defects in humans.

Conclusion Bone tissue engineering has the potential to provide better treatment options for bone reconstruction in orthopedic, craniofacial, and plastic surgery. Bone tissue engineering may provide a material that will integrate into native bone for a complete functional recovery, without the morbidity associated with autologous donor bone harvest. The integration of marrow MSCs with an optimized scaffold that includes some combination of BMPs will enhance the local environment for bone growth and remodeling by the native tissue. The most efficient technique to vascularize large bone grafts is an important area of research that is needed to improve bone graft survival. The challenges remain to create products that address the wide spectrum of clinical needs, from pliable materials for facial reconstruction to the strength required for long bone reconstruction.

Cartilage Tissue Engineering The motivation for cartilage tissue engineering is to improve treatments for cartilage degeneration that is caused by disease, trauma, or congenital abnormalities. Some examples of cartilage disorders are osteoarthritis, cartilage injuries, herniated disc, costochondritis, and achondroplasia. Osteoarthritis is the most common cause of arthritis and the leading cause of disability that primarily affects middle-aged and elderly population.95 The incidence of osteoarthritis in people older than 75 is greater than 80%, and radiologic evidence in those older than 50 is greater than 50%.96 As the population in the United States continues to age, the demand for treatments that allow patients to maintain an active lifestyle will increase. The surgical options are arthroscopic surgery or joint replacement, although the efficacy of

Table 61-3. Selected Animal Models that Have Produced Bone Formations Reference

Model

Cell Source

Scaffold

Outcome

Schliephake90

Sheep mandible defects 35-mm length

Marrow cells

Heat-treated bovine bone, porous calcium phosphate

Greater bone formation in seeded scaffold at 5 months

Shang91

Sheep cranial bone defect 20-mm diameter

Mesenchymal stem cells (MSCs)

Calcium alginate

At 18 weeks scaffold and MSCs almost healed vs scaffold with open defect

Arinzeh92

Canine femur defect 21-mm length

Allogeneic MSCs

Hydroxyapatite-tricalcium phosphate

No adverse host response; greater amounts of bone vs cell-free implants

Zhu87

Goat femur defect 25-mm length

MSCs

Coral

Cell seeded group at 4 months radiographic union; structural properties similar native bone; cell-free group no bone growth

Hou88

Rabbit cranial defect 15-mm diameter

MSCs

rhBMP-2/coral

At 16 weeks by radiography MSC/rhBMP-2/coral similar to iliac autograft and greater than cell-free rhBMP-2/coral

Viateau89

Sheep metatarsal defect 25-mm length

MSCs

Coral

MSCs and autograft similar bone formation, much greater than cell-free scaffold

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arthroscopy with debridement or joint irrigation of the knee has been recently challenged.97 Joint replacement surgery is indicated when the patient no longer responds to nonpharmacologic and drug treatments.

Structure and Function of Cartilage The structure of cartilage is composed of a dense extracellular matrix with embedded chondrocytes.35 The extracellular matrix includes collagen Type II fibers, elastin fibers, and proteoglycans that give cartilage viscoelastic and mechanical properties. Cartilage is avascular and relies on diffusion for nutrients, oxygenation, and removal of waste. The three types of cartilage are hyaline, elastic, and fibrocartilage. Hyaline cartilage is the most common form of cartilage, and consists primarily of Type II collagen and proteoglycans. Hyaline cartilage forms the skeleton of the embryo, which is later converted to bone as the embryo matures. In adults, hyaline cartilage can be found in the trachea, larynx, nose, bronchi, load-bearing joints, and the ends of the ribs. Elastic cartilage contains collagen and elastin fibers; it is found in the pinna of the ear and forms the epiglottis. Fibrocartilage contains more collagen than hyaline, and is found in areas that require great tensile strength. Fibrocartilage is located between invertebral discs, between the articulations of the hip, and at segments connecting tendons or ligaments to bone. The function of cartilage is to reduce friction of articulating bones and to assist with load bearing.35

Cell-Based Therapies One cell-based treatment for the repair of cartilaginous lesions is autologous chondrocyte implantations. A first procedure requires the harvesting of cartilage from non-load-bearing region of bone. The tissue is cultured and chondrocytes are isolated and expanded. In a second procedure, the lesion is surgically debrided and the chondrocytes are implanted under a periosteal flap. However, the conclusion of a recent systematic review of three trials and nine case reports revealed there was not sufficient evidence that autologous chondrocyte implantation is more effective than alternative therapies for chondral lesions of the knee.98 Two other reviews of available evidence by the National Institute of Clinical Excellence and Blue Cross and Blue Shield Association concluded that autologous chondrocyte implantation is not recommended over alternative treatments.99,100

Cell Source The cell sources for cartilage are harvested primary chondrocytes or MSCs that are induced to differentiate into chondrocytes. Primary chondrocytes can be harvested from articular, septal, auricular, and costal cartilage. Studies have reported different findings on the characteristics of cartilage harvested from various sites in animal models.101-103 Human nasal chondrocytes proliferate faster than articular chondrocytes, possess a higher chondrogenic potential, and survived 6 weeks implantation into nude mice. Hence, nasal cartilage may be a better cell source than articular cartilage for tissue engineering applications.101

Primary chondrocytes can be stimulated to proliferate and differentiate by various factors. Chondrocytes dedifferentiate during in-vitro expansion in monolayer culture, taking on a fibroblast-like morphology and producing macromolecules such as collagen Type I and versican.104 Chondrocytes can be induced to redifferentiate by the addition of growth factors, forming pellets at a high cell density, and by growth in a threedimensional scaffold.105,106 The addition of specific growth factors, such as TGFβ-1 and basic fibroblast growth factor (FGF), to monolayer cultures has been shown to induce dedifferentiation and enhance the proliferative capacity of the cells in animals and in humans.106,107 Many growth factors and combinations of growth factors have been investigated to optimize MSC differentiation into chrondrocytes. Basic FGF (FGF-2) has been identified as the most potent mitogen in the FGF family to promote chondrogenesis and proliferation of MSCs.108 Mesenchymal stem cells induced with FGF-2 in pellets differentiated to chondrocytes in rabbit, dog, and human species.109 The formation of cartilage from MSCs was superior with FGF-2 in comparison to other growth factors, such as insulin-like growth factor, epidermal growth factor, and platelet-derived growth factor.110 BMP-2 is found in the mesenchyme of the developing limb and is known to stimulate cartilage formation in culture. Human MSCs in the presence of BMP-2 in a serum-free pellet culture system resulted in chondrogenic lineage development only. Cells from osteogenic or adipogenic lineage were not stimulated with BMP-2. The addition of BMP-2 to mesenchymal pellet culture system appeared to produce cartilage with higher amounts of proteoglycans in comparison to BMP-4 or BMP-6.111

Scaffold Scaffold materials for cartilage tissue engineering are collagen, hyaluronan, fibrin sealant, and synthetic polymers. Collagen is a good biomaterial for cartilage tissue engineering applications, because it is biocompatible, biodegradable, can be molded into specific forms, and can maintain chondrocyte phenotype visà-vis glycosaminoglycan production.112 In animal models, collagen-based scaffolds with chondrocytes have resulted in better healing in osteochondral injury models than other types of scaffolds. One method to reduce the dedifferentiation of chondrocytes with collagen is to add hyaluronic acid. Hyaluronic acid is a proteoglycan that acts to lubricate joint surfaces and assists cartilage to withstand compressive forces.113 Hyaluronan must be modified by esterification or other means for structural purposes and to optimize solubility, hydration, and degradation.114 A fibrin matrix can also support the growth of chondrocytes, and an in-vivo model demonstrated regeneration of cartilage with articular cartilage defects using a fibrin system.115 The preparation of fibrin needs to result in a stable gel that can support the growth of chondrocytes, otherwise fibrin gels have been known to shrink in size and degrade in a very short time.115 Fibrin gels have been produced that are stable with good mechanical strength, and in-vitro cultures have shown

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chondrocytes proliferate in these gels with the production of glycosaminoglycans and collagen Type II.115 The synthetic polymers PLA and PGA were investigated as cartilage scaffolds early in the study of cartilage tissue engineering. In-vitro studies demonstrated the biocompatibility of PGA with chondrocytes, and early rabbit model studies demonstrated a better healing response of PGA seeded with chondrocytes over PGA scaffold alone.116 PGA-seeded scaffolds have resulted in the greatest production of glycosaminoglycans by chondrocytes at 20 days in comparison to alginate, fibrin, and collagen.117 A concern with the application of PLA/PGA polymers is an inflammatory reaction that develops after hydrolysis of the polymer. However, a laryngotracheal rabbit model with a PLA/PGA copolymer had appropriate clinical function of the implants at the time of harvest at 12 months. The scaffolds were completely resorbed without signs of a chronic inflammatory response.118 Despite the success in animal models, no PLA/PGA-based materials have progressed to clinical trials.

Bioreactor Culture System with Mechanical Stimulation The parameters that have the greatest impact on the production of cartilage in a bioreactor are cell seeding density, mass transfer as modulated by shear stress, and compressive forces.119 The seeding density of chondrocytes must be balanced by maximizing cell number to produce more extracellular matrix, without using too high of a cell number that limits mass transfer. Limited mass transfer can result in non-uniform cell distribution with higher cell numbers at the surface of the scaffold. The methods to apply shear stress and enhance mass transfer are mechanically stirring the bioreactor, direct perfusion of medium through scaffolds, and a rotating-wall bioreactor that imparts low shears and stirring.119 Besides the desirable increased stimulation of extracellular matrix, the stirred mechanical systems also produce a more even distribution of cells over the scaffold. A direct perfusion system is a flow system where a cell suspension or media alone directly passes through the pores of the three-dimensional scaffold. Experiments using a direct perfusion culture system demonstrated an increase in the DNA content and an increase in glycosaminoglycans over static culture conditions.120 However, the tissue-engineered perfused cartilage still has significantly less collagen in comparison to native cartilage. Compressive forces stimulate differentiation of MSCs to chondrocytes and contribute to structural maintenance of articular cartilage in vivo. The two types of forces that in-vitro systems use are hydrostatic force and compressive force. In vivo, hydrostatic pressure is produced on the chondrocytes by the synovial fluid transmitting pressure to the water contained in the cartilage matrix.119 Physiologic levels of hydrostatic pressure are in the range of 7 to 10 MPa, and because cartilage is incompressible in this range, only minimal tissue deformation occurs.121,122 The duration of hydrostatic pressure, total time of application, and magnitude are variables that need to be optimized for cartilage tissue engineering applications. The most common ranges of loads are 0.1 to 15 MPa and the ranges for frequencies are

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0.05 to 1 Hz.119,123,124 Studies have shown that chondrocytes in an agarose scaffold with a strain of 3% to 10% at a frequency of 1 Hz and a culture time of 4 to 6 weeks have an increase in the amount of proteoglycan and collagen in comparison to static cultures.125,126 In another study by Waldman, bovine chondrocytes were seeded onto a calcium polyphosphate scaffold with 5% compression at 400 cycles per day at a frequency of 0.5 Hz.127 The scaffold and cells were stimulated every other day for 1 to 4 weeks, with the addition of either 2% shear or 5% shear. The combined 5% compression and 5% shear increased collagen synthesis by 76% and proteoglycan synthesis by 73% over the static controls. The authors noted that greater responses to uniaxial loading, shear, or compression alone, were observed in prior studies.

Clinical Applications Tissue engineering of articular cartilage could provide an alternative cell-based therapy to autologous chondrocyte implantation. Because autologous chondrocyte implantation has not shown significant improvements in patients with chondral lesions, other therapeutic options are needed for treatment of these lesions.98 The only tissue engineered cartilage graft is Hyalograft C (Fidia Advanced Biopolymers, Abano Terme, Italy), which is not FDAapproved but is available in Europe. Hyalograft C is composed of autologous chondrocytes grown on a three-dimensional hyaluronic-based scaffold called HYAFF 11. The scaffold is an esterified derivative of hyaluronic acid that is biodegradable and biocompatible.128 In a recent multicenter study from Italy, autologous chondrocytes were isolated from a cartilage biopsy harvested arthroscopically from a non-weight-bearing area in the knee. The cells were expanded in vitro and then cultured on the HYAFF 11 scaffold for 14 days. The engineered graft was implanted at the lesion site by a mini-arthrotomy under regional or general anesthesia. In 55 of the 141 patients, a biopsy of the implanted cartilage was taken and evaluated using the International Cartilage Repair Society visual scoring system. At a mean follow-up time of 38 months after the procedure, 71.4% of the patients stated they could do everything or nearly everything that they wanted to do with their joint compared to 4.3% before the surgery. The visual scoring of the repaired tissue at the postsurgery arthroscopy revealed normal or near normal scores in 53 of 55 patients after a mean implantation time of 14.1 months. There were 10 graft failures, which was 5.2% of the study group. The authors stated a limitation of the study was in the design of an observational trial instead of a randomized controlled trial. In a follow-up study to further characterize the nature of the biopsied tissue, biochemical analyses were applied.128 The average follow-up time from grafting to biopsy was 16 months, with a range of 6 to 30 months. Ten of the 23 cases demonstrated regenerated hyaline cartilage, but a larger sample size is needed to make more definite assessments.

Future Challenges The challenge of cartilage tissue engineering remains to develop a therapeutic treatment for cartilage disease or injury. The

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success of Hyalograft C in improving clinical symptoms is encouraging, but further evaluation of the mechanical properties and the long-term graft efficacy needs to be undertaken. A reliable cell source has been obtained by primary cell harvest, and MSCs offer an even better potential. By selecting the appropriate cell density, growth factors, mechanical stimulation, and scaffold, tissue-engineered cartilage can be successfully designed for clinical applications.

Urology Tissue Engineering The urinary system is exposed to a variety of insults that require reconstruction as a treatment. Frequent reasons for reconstruction in the urinary system include congenital abnormalities, cancer, trauma, infection, and iatrogenic injuries. The primary option for bladder and urethral reconstruction involves the use of gastrointestinal tissue. One congenital anomaly that may require the need for bladder reconstruction is exstrophy of the bladder. The prevalence of bladder exstrophy is 3.3 births per 100,000.129 The initial treatment for this bladder defect is early primary closure. However, in some cases the bladder is too small for closure or the bladder does not develop to maintain continence. In these cases, tissue harvested from the small bowel or colon is used to augment the size of the bladder.129 Cancer of the urinary bladder is the fourth most common cause of cancer in men, and the ninth most common in women.129 Invasive bladder cancer is treated by radical cystectomy with urinary diversion. The use of selective bladder preservation or partial cystectomy is inferior to radical cystectomy for invasive cancer, meaning that total bladder replacement could significantly enhance quality of life for these patients.129 Blunt trauma is another cause of urinary tract injury, often associated with pelvic fractures or straddle injuries for the urethra. In the case of large defects that require diversion of the urinary system, use of gastrointestinal tract tissue is the only option. Bowel segments that are mobilized for bladder reconstructions are associated with various complications. Complications include mucus production, chronic bacteruria, stone formation, rupture, electrolyte imbalance, and metabolic acidosis with a range of incidence from 5% to 44%.130,131

Design Goals for Urinary Tissue The objective of urinary tissue engineering is to provide an alternative source of tissue that is more similar to native bladder or urethra. The requirements for bladder tissue engineering are to act as a reservoir for urine, contract until empty, integrate with sites of anastomosis to prevent leakage, have mechanical strength to prevent rupture, and not cause excessive stone formation. The structure of the bladder is a hollow muscular organ that functions as a reservoir to hold urine until time of excretion through the urethra. The bladder is composed of transitional epithelium, lamina propria, muscular layer, and an adventitia. The transitional epithelium forms a layer of four to five cells when the

bladder is empty, and two to three layers when the bladder is distended.35 The viscoelastic properties of the bladder are derived from the smooth muscle, the collagen content of approximately 50%, and elastin content of approximately 2%.129 The bladder has a physiologic compliance that contains neural input to set the volume for micturition.129

Cell Source The most common cell source for bladder tissue engineering is a primary cell source from urothelial cells and visceral smooth muscle cells. Primary urothelial cells are autologous and have been demonstrated to expand the cells from a surface area of 1 cm2 to 4202 cm2 over a period of 8 weeks.132 Phenotype and functional characteristics of harvested human visceral smooth muscle cells from normal, exstrophic, and neuropathic bladders have been evaluated. No differences between the normal and diseased bladder tissue with respect to phenotype and degree of contractility were observed.133 An alternative potential cell source is marrow stem cells or embryonic stem cells that have not been exposed to fibrotic or malignant changes. Because marrow cells can differentiate into smooth muscle, investigators have evaluated if the smooth muscle cells have a similar phenotype and function if placed on a scaffold for bladder applications. One group reported bladder smooth muscle and marrow-derived smooth muscle had similar expression of actin and contractile response to calcium ionophore.134 An in-vivo study in a hemicystectomy dog model with bladder cell-seeded or marrow cellseeded small intestinal submucosa graft had similar outcomes in both grafts. After 10 weeks, the two implants had similar levels of alpha-actin with evidence of smooth muscle regeneration and muscle bundle formation.

Scaffold The main scaffold types for urologic tissue engineering are synthetic polymers and natural materials derived from acellular tissue matrices. It has been demonstrated that the adherence and proliferation of bladder smooth muscle cells appears to be similar across synthetic polymers, eg, poly(lactic-co-glycolic) acid (PLGA) and PCL.135 Hybrid materials, such as a PLGA meshcollagen gel, have also been shown to support the growth of urothelial and smooth muscle cells. The purpose of using a hybrid scaffold is to take advantage of the mechanical strength of the synthetic polymers and the cell adhesion properties of the collagen gel.136 Tissues that are used to create an acellular matrix for bladder scaffolds are small intestine submucosa, native bladder tissue, and bladder submucosa.137,138 Bladder replacement grafts composed of small intestine submucosa have demonstrated regeneration of normal bladder with urothelium, smooth muscle, and serosal layers in canine and rat models.138

Preclinical and Clinical Trials The tissue-engineered bladder has progressed from in-vitro models to a clinical trial. The canine and rabbit models have demonstrated the success in creating an autologous neo-bladder

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in vivo. Table 61-4 lists some of these studies that describe the cell source, scaffold, and the outcome. The Atala group has successfully proliferated the bladder urothelial and smooth muscle cells from a small biopsy, and used a cell-free bladder construct as the scaffold. They demonstrated in a canine 50% cystectomy model that the cell-seeded graft had a 99% increase in the capacity of the bladder.137 Histologic analysis confirmed the presence of nerve fibers in the tissue-engineered bladders. The Atala group has also been applying the same method for urethral replacement.139 In the study by Kropp in 2004, well-expanded bladders were generated with acellular porcine small intestine submucosa (SIS) in a canine cystectomy model using cell-free grafts.138 A later study by Kropp revealed improved results with bladder cellseeded SIS or marrow-derived smooth muscle cell-seeded SIS in comparison to the control group of cell-free scaffolds.134 In a third study of a canine bladder replacement cystectomy of 90%, the cell-seeded and cell-free scaffolds produced similar results of poor bladder regeneration.140 The authors attribute the lack of bladder regeneration to a poor vascular network and increased inflammation produced by the large implants. The viability of the implanted graft depends on vascular ingrowth from the surrounding tissues, and the vascular network may not have been able to develop at a rapid enough rate to support such a large tissue graft. The increased inflammation may have been a result of the technique to create the large defect. In a recent publication, the successful techniques for bladder regeneration in animal models were translated to a human trial.141 The study identified seven patients with a primary diagnosis of myelomeningocele with poorly compliant bladders. The age range of the patients was from 4 to 19 with a mean age of 11 years. The cell source was from a bladder biopsy, and the scaffold was made from collagen or from a composite of collagen and polyglycolic acid. The scaffold for the first four patients was made of decellularized bladder submucosa. The last three

patients used the composite material that was sized for each individual based on age-appropriate bladder size and dimensions obtained from CT scan. The length of time between the bladder biopsy and the tissue-engineered bladder implantation was approximately 7 to 8 weeks. The patients were followed for 22 to 61 months with a median time of 46 months. The postoperative urodynamic studies with the tissue-engineered bladders showed varying degrees of contractility, capacity, and compliance. The volume and the compliance increased postoperatively, with the most significant improvement in the composite tissueengineered scaffold that was implanted with an omental wrap. The results of the urodynamic studies were similar to those of gastrointestinal segment augmentations, and the patients did not develop any of the complications associated with the gastrointestinal segment augmentation. The authors commented that they added the composite material to the study because the PGA improved the structural integrity of the tissue while the collagen continued to support cell growth. The positive preliminary data from this trial led to a Phase II trial to provide more data on the efficacy of the tissue-engineered approach to bladder augmentation.

Future Challenges The use of tissue-engineered bladders in a clinical application demonstrates the exciting opportunities in the field of urothelial tissue engineering. Additional improvements in the existing approach may be to use marrow stem cells as a smooth muscle cell source, providing a vascular network before implantation, stimulation of neuronal integration into the graft, and continued optimization of the scaffold. Although there is evidence to suggest that viable bladder cells can still be harvested from diseased tissue, if bladder smooth muscle cell can be derived from marrow stem cells it may be a more attractive alternative cell source. Another potential clinical application is bladder reconstruction

Table 61-4. Selected Animal Models that Have Produced Autologous Neo-Bladders Reference

Model

Cell Source

Scaffold

Outcome

Canine 50% cystectomy for 2 and 3 months (n ⫽ 10)

Bladder urothelial and smooth muscle

Canine allogeneic bladder submucosa

Cell-seeded with 99% increase in capacity; cell-free only 30% increase

DeFilippo/ Atala139

Rabbit urethral 1-cm segment resection 1, 2, 3, and 6 months (n ⫽ 24)

Bladder urothelial and smooth muscle

Porcine bladder submucosa

Cell-seeded graft normal structure 1 month; cell-free strictures/collapse at all timepoints

Kropp/Zhang138

Canine 40% cystectomy for 10 weeks (n ⫽ 6)

None

Small intestine submucosa

Distal SIS bladders well expanded

Zhang/Kropp134

Canine 40%-50% cystectomy for 10 weeks (n ⫽ 6)

Bladder urothelial and SMC Marrow-derived SMCs

Small intestine submucosa

Both bladder and MSC-seeded grafts with smooth muscle; cell-free with smooth muscle only at edges

Zhang/Kropp140

Canine 90% cystectomy for 1, 5, 9 months (n ⫽ 22)

Bladder urothelial and SMC

Small intestine submucosa

Limited bladder regeneration in cell-seeded and cell-free group

Yoo/Atala

137

SIS ⫽ small intestine submucosa; SMC ⫽ smooth muscle cell; MSC ⫽ marrow stem cell.

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after cancer resection, utilizing harvested cells from an area of bladder without any neoplastic changes. The application of bladder tissue engineering for large cystectomies may require a vascular network before implantation, as suggested by data from the canine model.140 The neuronal integration of the graft is important to restore the natural voiding mechanism. The clinical data demonstrated in myelomeningocele patients that the urodynamics and the dry intervals between straight catheterizations improved postoperatively.141 Optimization of the scaffold for more desirable properties such as improved mechanical characteristics and improved control over degradation in response to tissue remodeling are two key issues. As tissue-engineered products progress from case studies to commercial applications, process methods to reduce the potential of infection and to achieve high-quality standards will be important issues for these new medical devices.

Cardiac Tissue Engineering Cardiovascular disease is the leading cause of mortality in the United States. The number of people in the United States with one or more types of cardiovascular disease is approximately 1 in 3.30 Patients who survive a myocardial infarction experience myocardial damage that can eventually result in congestive heart failure. Other causes of heart failure include hypertension, cardiomyopathy, valvular disease, and cardiac arrhythmias. The number of deaths related to heart failure increased 28% in the period of 1994 to 2004.142 The mortality data for heart failure is comparable to that of cancer. The pathophysiology of heart failure is the inability of the heart to maintain adequate cardiac output because of injury to the cardiac myocytes that causes an increase in wall stress and forward pumping failure.143 The treatment options for severe heart failure range from pharmacologic therapy to heart transplant. The nonpharmacologic treatments include cardiac resynchronization, coronary revascularization, left ventricular assist device implantation, and heart transplantation. The number of people requiring heart transplantation continues to increase, but the number of people receiving heart transplants has been on the decline over the past 10 years.144 The 1-year survival for heart transplants is approximately 87%, and the 5-year survival is approximately 77%.144

Design Goals for Cardiac Tissue The heart is a muscular organ that functions as a continuous pump through rhythmic contraction and relaxation cycles. The three layers of the heart are the epicardium, myocardium, and the endocardium.35 The epicardium is the outer layer of the heart that provides a smooth layer to prevent friction while the heart is beating. The endocardium is the cell layer lining of the interior of the myocardial wall to create an antithrombotic surface and functions to interact with the circulating blood. The primary layer that forms the heart is the myocardium. The myocardium contains specialized cardiac muscle cells that are striated, involuntary

muscle with many branching cells that are joined by unique intercalated disks.35 The intercalated disks are gap junctions that permit electrical communication that can pass impulses to a large area of the heart wall, thereby stimulating contraction. The primary objective of cardiac tissue engineering in the near future will likely be the engineering of patches of myocardium to replace specific areas of diseased heart tissue. The challenge of engineering an entire heart with the ability to contract appropriately with separate chambers, adapt appropriately to physiologic stimuli, and respond to further cardiac disease progression appears too difficult to reach within a reasonable time given current technologies.145 Another application of cardiac tissue engineering would be to use engineered cardiac tissue for reconstruction of pediatric congenital defects. The use of engineered tissue could potentially grow with physiologic development and prevent multiple surgeries for malformations such as single ventricles, atrial septal defects, or ventricular septal defects.145 The alternative cell-based therapies to cardiac tissue engineering are cardiomyocyte transplantation and the injection of marrow-derived cells, or other types of precursor cells, directly into myocardium. Cardiomyocyte transplantation has been studied in animal models by directly injecting fetal or neonatal cardiomyocytes into the injured myocardium. The positive findings of cardiomyocyte transplantation have included beneficial effects on the remodeling process in animal studies.146,147 Despite moderate improvements in contractile performance related to the remodeling process, cardiomyocyte transplantation cannot replace scar tissue and restore synchronously contractile muscle.146,148 The major limitation in translating this technology to human clinical trials would be the availability of cardiomyocytes. The intracoronary injection of marrow mononuclear cells, or other marrowderived cells, in the setting of an acute myocardial infarction in humans, has thus far yielded no improvement in left ventricular function or minimal improvement in recovery.149,150 Further studies, however, are ongoing.

Cell Source Marrow stem cells have been shown in some studies to differentiate into cardiac myocytes.151,152 Primary cardiac cells are probably not a viable cell source in humans, because they are difficult to harvest and do not readily expand. Cardiac cells derived from rat marrow expressed proteins consistent with cardiomyocytes in vitro, and in a rat infarct model the transplanted cells improved the cardiac structure and function of the heart.153 Rodent myocardial infarct animal models with cardiac cells derived from marrow have shown improved cardiac function and better ejection fraction with less remodeling of the left ventricle.152,153 The challenge remains to develop an efficient method to isolate and to differentiate marrow stem cells into cardiac cells, particularly for human patients.

Scaffold The synthetic polymers and natural materials have been used for cardiac tissue engineering scaffolds. A study comparing PGA, gelatin, and polylactide copolymer with PCL using rat aortic

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smooth muscle as the cell source found the PCL material to have better ingrowth of cells in vitro and improved cellular architecture as a cardiac patch compared to the PGA and gelatin.154 The gelatin scaffold was noted to induce an inflammatory reaction, which was attributed to the xenogeneic (bovine) protein in the gelatin. Natural material using alginate-based scaffolds can form a three-dimensional structure with a uniform cell distribution of cardiomyocytes.155 A graft composed of fetal cardiac cells in an alginate scaffold was implanted in a myocardial infarct model in rats and was found to undergo intensive neovascularization and had prolonged survival of the cells in the graft.156 The alginate scaffold was almost completely degraded after 9 weeks, and it was well-invested with differentiated cardiomyocytes.156 Collagen gels have been used to form three-dimensional scaffolds that support cardiomyocyte cells. It has been shown that artificial myocardial tissue can be formed from neonatal rat cardiomyocytes inside a three-dimensional collagen matrix.157 The constructs were prepared by adding a cell solution to preformed, commercially available collagen that was allowed to gel over 4 hours. The collagen matrix was found to support the attachment, proliferation, and synchronous contractions of the neonatal cardiomyocytes in the three-dimensional scaffold. The spontaneous and synchronous contractions were maintained for up to 13 weeks. Zimmermann158 formed engineered heart tissue by mixing a cell solution of neonatal rat cardiac cells with collagen gel and Matrigel (Becton Dickinson, Franklin Lakes, NJ). After a few days, the constructs were placed in a custom-made stretch device to provide mechanical stimulation. The in-vitro tissue demonstrated histologic features of native differentiated myocardium and contractile characteristics of native myocardium. By exposing the cell constructs to stretch stimulation, the force of contraction was greater than twofold in the stretch group compared to the statically grown tissue.159 A novel method to engineer cardiac tissue has been developed by Shimizu. The concept is to use a temperature-responsive polymer called poly(N-iso-propylacrylamide) (PIPA Am) that covalently attaches to tissue culture polystyrene and supports the growth of cells. When the temperature is lowered to 32ºC, the PIPA Am with the cells spontaneously detaches from the underlying polystyrene, with the cell-to-cell connections of the cultured tissues on the upper surface intact.160 This material has been applied to rat neonatal cardiac cells to create sheets that can be layered together to form a three-dimensional myocardial tissue structure. Electrical and morphologic connections between the cell sheets have been demonstrated.160

Bioreactors The objective of bioreactor development in cardiac tissue engineering is to stimulate the growth of a three-dimensional structure that is conditioned by mechanical or electrical stimulation. A bioreactor system with a spinner flask causes turbulent flow that results in improved mixing for nutrient and oxygen delivery in comparison to static cultures.161 Other designs of bioreactor designs have applied stretching or compression to cultured

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cells. The exposure of myocardial cells to cyclic stretch resulted in improved proliferation and more uniform distribution of cardiomyocytes on the scaffold, as well as improved organization and more extracellular matrix.162 The use of electrical stimulation with neonatal rat myocytes has been shown to induce structural differentiation similar to native myocardium, and an increased contractile response in comparison to nonstimulated cultures.163 One important limitation to the growth of cardiac tissue for human applications is the thickness of the tissue. The thickness of human cardiac muscle is about 1 cm, whereas the thickness of cardiac tissues grown in bioreactor systems is only 100 microns.164 Lacking a vascular network, cardiac tissue cannot grow significantly beyond 100 microns because delivery of nutrients and removal of waste is limiting.164 Although bioreactor systems have resulted in great improvements, the next big challenge is to develop a construct and a bioreactor system that provide adequate perfusion to thicker cardiac constructs.

Preclinical Studies The preclinical data for myocardial tissue engineering remain in the early stages, with most investigators using neonatal or fetal rodent cells for evaluation of cardiac tissue engineering patches in animal models. In a study by Krupnick, a cardiac patch 4 mm ⫻ 4 mm ⫻ 2 mm was created with marrow MSCs as a cell source and a scaffold composed of a composite collagen and PLA material reinforced with PTFE.165 The patches were implanted into the left ventricle of a rat nonfunctioning left ventricle model over a period of 4 weeks. The marrow cells integrated into the host and formed differentiated cardiac muscle by the cell morphology and cytoplasmic reactivity of anti-Troponin C. The authors suggested a cardiac patch could be implanted for human use to replace myocardial scar after infarction concurrently with coronary revascularization. However, the authors commented that a limitation of the animal model is that murine species are relatively resistant to arrhythmias. Leor and colleagues engineered cardiac grafts with fetal cardiac cells using three-dimensional alginate scaffolds for the regeneration of infarcted myocardium.156 The porous alginate scaffold was seeded with fetal cardiac cells and incubated for 4 days before implantation. The graft implantation was performed 7 days after myocardial infarct in a rat model and harvested 9 weeks after transplantation. The rats with the grafts demonstrated attenuation of left ventricular dilatation and no change in left ventricular contractility. In contrast, the control group developed significant left ventricular dilatation with progressive deterioration in left ventricular contraction. The cardiac grafts were found to have intensive neovascularization that contributed to the survival of the cells in the graft. The beneficial effects of the graft on cardiac contractility were not likely directly caused by contraction of the implant, because only a small fraction of the implanted cardiac cells survived after 9 weeks. Zimmermann has reported the construction of a engineered heart tissue that can be constructed in vivo and can improve contractile function of an infarcted rat model.166 The engineered

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heart tissue was formed using a suspension of neonatal rat heart cells, liquid collagen, and Matrigel that was formed into circular molds and were subjected to mechanical strain. The myocardial infarct model in the rat was created by ligation of the left anterior descending artery. The engineered heart tissue and control, noncontractile grafts were implanted 14 days after infarct over the area of myocardial scar. The engineered heart tissue showed undelayed electrical coupling comparable to the native myocardium without evidence of arrhythmia at 28 days. The engineered heart tissue prevented further dilation and induced systolic wall thickening of infarcted myocardial segments, superior to control implants.166

Future Challenges The main challenges for cardiac tissue engineering are a reliable autologous cell source, and the vascularization of large tissues. Most of the studies utilize fetal or neonatal cardiac cells as a cell source. However, an autologous cell source from marrow or other cells is critical to advance cardiac tissue engineering for human applications. Clearly, it may be difficult to obtain viable cardiac cells from older patients with diabetes and other diseases because of impaired proliferative and functional capacity.167 Methods to isolate, differentiate, and proliferate marrow-derived or other cells to produce cardiomyocytes need to be developed. After implantation, any cardiac graft will depend on neovascularization to maintain cell survival. Even if cell sheets can be used as layers to create a thicker construct, without a vascular network the cells would likely not survive. The potential applications for cardiac tissue engineering are a concurrent augmentation of scar tissue that occurs at the time of coronary revascularization and the reconstruction of congenital cardiac defects.

Corneal Tissue Engineering A bioengineered cornea has the potential to restore vision when the loss is caused by opacification of the cornea. The cornea is essential to provide the proper anterior refractive surface and to protect the eye against infection and structural damage. The most common causes of opacification of the cornea are injuries or infection that lead to corneal scarring, abnormal shapes of the cornea, and congenital disorders of the cornea.168 Diseases affecting the cornea such as measles, vitamin A deficiency, trachoma, and parasites are common causes of childhood blindness worldwide.168 Corneal transplantation is the only widely accepted treatment for severe corneal diseases. The success rate of traditional allograft corneal transplantation is greater than 90% for patients with corneal disorders such as abnormal corneal shapes, traumatic corneal scars, and degeneration.169

Objectives of Tissue-Engineered Cornea The cornea is composed of an epithelial layer and an endothelial layer. The epithelium is a stratified squamous layer approximately four to six cell layers thick.168 The tight junctions of the

epithelial cells provide a barrier to bacteria and prevent the entry of tears into the intracellular space. The basal layer of the epithelium contains epithelial stem cells that generate new corneal epithelial cells. The corneal endothelium is a hexagonal monolayer that maintains the fluid regulation of the corneal stroma. The endothelial layer maintains a high water content of around 78%, which ensures a cellular architecture that forms a transparent tissue.168 Although the primary treatment for severe corneal disease is corneal allograft transplantation, allograft rejection is the main cause of graft failure in corneal transplantation. The rate of rejection after 5 years has been reported at around 20%.170 Topical corticosteroids are the mainstay treatment for immunologic rejection. Potential risk factors for immunologic rejection are increased stromal vascularization, prior blood transfusions, positive donor-recipient crossmatch due to preexisting lymphocytotoxic antibodies, young recipient age, larger grafts, and the presence of inflammation at the implantation site. Synthetic corneal replacements, or keratoprostheses, provide an additional treatment option to those patients who are not candidates for allograft corneal transplantation. The design of the synthetic corneal prosthesis is a flexible material with an optically clear core that is surrounded by a microporous sponge rim.171 The optical core functions to transmit light and provide refractive power, while the sponge rim is designed to permit fibroblast ingrowth to secure the device into place over the long term. Two FDA-approved keratoprostheses are available for clinical use. The Dolman-Doane keratoprosthesis is double-plated, and is composed of a central core of polymethylmethacrylate (PMMA) that requires corneal donor tissue to form the rim.172 The most favorable outcomes using the Dolman-Doane device are for patients with multiple failed grafts, repeated immunologic rejection of corneal allografts, and neovascularization of a corneal graft.172 The postoperative course must be closely followed for retroprosthetic membrane formation, glaucoma, and retinal detachment. AlphaCor, previously known as Chirila keratoprosthesis, is a one-piece prosthesis consisting of a central transparent optic region and an outer rim that is entirely manufactured from hydrophilic poly(2-hydroxyethyl methacrylate) or PHEMA. The porosity of the PHEMA sponge encourages biointegration with the host tissue to promote fibroblast ingrowth. AlphaCor has been successful in the treatment of corneal opacities that are not suitable for standard allograft corneal transplantation.173 The complications of AlphaCor include corneal melting and the formation of retroprosthetic membranes.

Cell Source The cell source for corneal tissue engineering is corneal limbal epithelial cells or oral epithelial cells. Severe ocular surface diseases such as Stevens-Johnson syndrome, ocular cicatricial pemphigoid, and chemical or thermal burns often destroy the corneal epithelial stem cells.174 In unilateral eye disorders, autologous

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cultured corneal epithelial cells may be harvested from the unaffected eye and cultured as a sheet. Culture systems have been developed to proliferate and expand corneal epithelial cells that are harvested from primary tissue. In the earliest report of autologous cultivated corneal epithelial cells by Pellegrini, a biopsy from the limbus of the uninjured eye was obtained.175 The autologous cultured corneal epithelium was implanted onto the cornea of two patients, and was found to restore the corneal surface with a significant improvement in visual acuity. A similar technique used by other investigators placed the biopsied corneal epithelial cells on denuded amniotic membrane in co-culture with a feeder layer of 3T3 fibroblast cells for 2 to 4 weeks.176 After implantation of the tissue-engineered cornea, the patient had complete epithelialization with a significant improvement in visual acuity that remained stable.177 In patients who have bilateral ocular surface defects, the options are to use allogeneic cultured epithelial cells or epithelial cells from another tissue in the patient. The risk of using allogeneic cultured epithelial cells is an increased incidence of rejection and poor clinical outcome. In a small study by Koizumi, cultured allogeneic epithelial cell transplantation was performed in 13 eyes of 11 patients having total corneal stem cell deficiencies using amniotic membrane as a cell carrier.176 This study resulted in all 13 eyes being free from epithelial defects after 48 hours, visual acuity being improved in all 13 eyes, and 10 of the 13 being restored to good function. Three of the eyes experienced epithelial rejection. To obviate allogeneic cells and immunorejection, epithelial cells from the oral mucosa have also been used for corneal applications.178 A feasibility study was conducted in a rabbit model with an induced ocular surface injury. Oral mucosa biopsies were cultured for 3 weeks on a denuded amniotic membrane carrier. The cultured oral epithelial sheet had four to five layers of stratified cells with a similar micro-architecture to normal corneal epithelium.

Scaffold The scaffolds for corneal tissue engineering function more as carrier substances than as conventional scaffolds. The materials used as the carrier include amniotic membrane and fibrin-based materials. The characteristics that make amniotic membrane appealing for use as a carrier for the growth of epithelial cells are its ready availability, very limited immunogenicity, antimicrobial effect, and the fact that it resembles the basement membrane of the conjunctival epithelium.174 The early reports of use of amniotic membrane for ocular surface reconstruction in rabbit animal models showed that resultant corneas were clear with minimal amounts of neovascularization.179 The effect of the amniotic membrane suggested that it facilitated epithelialization without allowing host fibrovascular ingrowth, which can retard clarity of the membrane.179 These preliminary animal data led to human studies, and amniotic membrane as a carrier substrate has proven to be successful in human corneal implants in multiple studies.177 Fibrin and fibrin-based carriers have also demonstrated success for corneal epithelial systems. Despite the advantageous

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properties of amniotic membrane, the membrane remains permanently beneath the cultured epithelial cells. This may have some long-term effect on biocompatibility or pose a potential risk for infection. In contrast, fibrin and fibrin-based materials are biodegradable and will integrate with the surrounding tissue. Fibrin-fibronectin gel has supported human corneal epithelial stem cell growth and differentiation, with resultant expression of keratin cell markers consistent with corneal epithelium.180 Human studies have demonstrated success in transplanting cultured corneal limbal cells on a fibrin carrier from the healthy eye to the contralateral eye.181

Clinical Applications Corneal tissue engineering has advanced to clinical studies. In patients with unilateral disease, a corneal biopsy from the healthy eye can be used as a cell source to obtain cultured corneal epithelial cells. In the setting of bilateral ocular surface diseases, a replacement cell source comprising autologous oral mucosal epithelial cells demonstrated feasibility in animal models, and this method has been applied to human corneal transplantation.177 Two separate groups using autologous oral mucosal epithelium with different culture methods have performed corneal reconstruction in small case reports. The major difference in culture methods between the two groups is that one group uses a carrier-free epithelial cell sheet that is produced using temperature-responsive culture surfaces, while the other group uses amniotic membrane as a carrier for the epithelial cell sheet.177,182 In the first group, four patients were enrolled in the study with one eye each undergoing transplantation.182 Three of the four patients had undergone allogeneic corneal transplantation that had failed after 1 year despite systemic and local immunosuppression. The oral mucosal cells were harvested and cultured for 2 weeks on temperature-responsive cell-culture surfaces with 3T3 feeder cells.182 The epithelial cell sheets were removed from the cell-culture surface by reducing the temperature and were transplanted directly onto the denuded corneal surface. Complete re-epithelialization of the corneal surface occurred within 1 week in all four treated eyes.182 Over the 14-month postoperative period, the corneal surface remained transparent and there was significant improvement in visual acuity. In the second study, there were a total of six eyes in four patients with Stevens-Johnson syndrome (three eyes) or chemical injuries (three eyes).177 Autologous oral epithelial cells were cultured for 2 to 3 weeks in the presence of 3T3 fibroblasts on a denuded amniotic membrane carrier. The epithelial sheet was transplanted onto the corneal surface and secured in place with sutures. The corneal surface had complete epithelial coverage by 48 hours, and visual acuity significantly improved over the postoperative period.177 The corneal surface remained stable over a mean follow-up time of 13.8 months. In a larger study of ocular surface reconstruction with cultured oral mucosal epithelial cells, mid-term results on a total of 15 eyes over a mean period of 20 months were reported.183 The ocular surface remained stable and transparent in 10 of the 15

Chapter 61: Tissue Engineering and Regenerative Medicine

eyes, and visual acuity significantly improved in 10 of the 15 eyes. There were five eyes with small and persistent epithelial defects. Three of the eyes healed spontaneously, and two required reoperation.183 These studies demonstrate that corneal engineering is a promising treatment for patients with severe ocular disease who fail transplantation, or for patients who are poor candidates for conventional corneal transplantation. The success of autologous oral mucosal epithelial cells for corneal tissue engineering applications obviates the need for immunosuppression and for acquiring corneal stem cells that may not be available when disease is bilateral.

Disclaimer The authors have disclosed no conflicts of interest.

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