Postgraduate Haematology, Sixth Edition

Postgraduate Haematology

Companion website This book has a companion website: www.wiley.com/go/hoffbrand/postgraduate with: • Figures and tables from the book for downloading • Interactive multiple-choice questions prepared by the authors

Postgraduate Haematology E D IT E D BY

A Victor Hoffbrand

MA, DM, FRCP, FRCPath, FRCP (Edin),

DSc, FMedSci Emeritus Professor of Haematology at University College London and Honorary Consultant Haematologist at the Royal Free Hospital, London, UK

Daniel Catovsky

MD, DSc (Med), FRCPath, FRCP, FMedSci

Emeritus Professor of Haematology, Institute of Cancer Research, Sutton, Surrey, UK

Edward GD Tuddenham

MD, FRCP, FRCPath, FMedSci

Professor of Haemophilia at University College London and Director of the Haemophilia Centre at Royal Free Hospital, London, UK

Anthony R Green

PhD, FRCP, FRCPath, FMedSci

Professor of Haemato-oncology, Department of Haematology and Cambridge Institute for Medical Research, University of Cambridge; Honorary Consultant at Addenbrooke’s Hospital, Cambridge, UK

Sixth edition

A John Wiley & Sons, Ltd., Publication

This edition first published 2011 © 2005 by Blackwell Publishing Ltd Blackwell Publishing was acquired by John Wiley & Sons in February 2007. Blackwell’s publishing program has been merged with Wiley’s global Scientific, Technical and Medical business to form Wiley-Blackwell. Registered office: John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial offices: 9600 Garsington Road, Oxford, OX4 2DQ, UK The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK 111 River Street, Hoboken, NJ 07030–5774, USA For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/wiley-blackwell First published as Tutorials in Postgraduate Haematology © William Heinemann Ltd 1972 Reprinted 1975 Second edition 1981 published © Butterworth Ltd Reprinted 1983, 1986 Third edition 1989 published © Butterworth Ltd Reprinted 1992 Fourth edition 1999 published © Butterworth-Heinmann Ltd Revised and reprinted 2001 by Arnold Fifth edition 2005 Reprinted 2005 The right of the author to be identified as the author of this work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. 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, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. The contents of this work are intended to further general scientific research, understanding, and discussion only and are not intended and should not be relied upon as recommending or promoting a specific method, diagnosis, or treatment by physicians for any particular patient. The publisher and the author make no representations or warranties with respect to the accuracy or completeness of the contents of this work and specifically disclaim all warranties, including without limitation any implied warranties of fitness for a particular purpose. In view of ongoing research, equipment modifications, changes in governmental regulations, and the constant flow of information relating to the use of medicines, equipment, and devices, the reader is urged to review and evaluate the information provided in the package insert or instructions for each medicine, equipment, or device for, among other things, any changes in the instructions or indication of usage and for added warnings and precautions. Readers should consult with a specialist where appropriate. The fact that an organization or Website is referred to in this work as a citation and/or a potential source of further information does not mean that the author or the publisher endorses the information the organization or Website may provide or recommendations it may make. Further, readers should be aware that Internet Websites listed in this work may have changed or disappeared between when this work was written and when it is read. No warranty may be created or extended by any promotional statements for this work. Neither the publisher nor the author shall be liable for any damages arising herefrom. Library of Congress Cataloging-in-Publication Data Postgraduate haematology / edited by A. Victor Hoffbrand ... [et al.]. – 6th ed. p. ; cm. Includes bibliographical references and index. ISBN 978-1-4051-9180-7 1. Blood–Diseases. 2. Hematology. I. Hoffbrand, A. V. [DNLM: 1. Blood. 2. Hematologic Diseases. WH 100 P857 2011] RC633.P67 2011 616.1′5–dc22 2009046376 A catalogue record for this book is available from the British Library. This book is published in the following electronic formats: ePDF 9781444323177; Wiley Online Library 9781444323160 Set in 9.5/12pt Minion by Toppan Best-set Premedia Limited 1 2011

Contents

Contributors, vii Preface to the sixth edition, xi Preface to the first edition, xii 1 Stem cells and haemopoiesis, 1

Elaine Dzierzak 2 Erythropoiesis, 12

Douglas R Higgs and William G Wood 3 Iron metabolism, iron deficiency and disorders of

haem synthesis, 26 A Victor Hoffbrand, Chaim Hershko and Clara Camaschella 4 Iron overload, 47

Clara Camaschella and A Victor Hoffbrand 5 Megaloblastic anaemia, 61

A Victor Hoffbrand 6 Haemoglobin and the inherited disorders of globin

synthesis, 83 Swee Lay Thein and David Rees 7 Sickle cell disease, 109

Ashutosh Lal and Elliott P Vichinsky 8 Hereditary disorders of the red cell

membrane, 126 Edward C Gordon-Smith and Narla Mohandas 9 Disorders of red cell metabolism, 140

Alberto Zanella and Edward C Gordon-Smith 10 Acquired haemolytic anaemias, 158

Edward C Gordon-Smith and Modupe O Elebute 11 Paroxysmal nocturnal haemoglobinuria, 176

Peter Hillmen 12 Inherited aplastic anaemia/bone marrow failure

syndromes, 186 Inderjeet S Dokal 13 Acquired aplastic anaemia, 206

Judith CW Marsh and Neal S Young

14 Red cell immunohaemotology: introduction, 226

Marcela Contreras and Geoff Daniels 15 Antigens in human blood, 244

Marcela Contreras and Geoff Daniels 16 Clinical blood transfusion, 268

Marcela Contreras, Clare PF Taylor and John A Barbara 17 Phagocytes, 300

John Mascarenhas, Farhad Ravandi and Ronald Hoffman 18 Lysosomal storage disorders, 330

Atul B Mehta and Derralynn A Hughes 19 Normal lymphocytes and non-neoplastic lymphocyte

disorders, 343 Paul AH Moss and Mark T Drayson 20 The spleen, 368

Paul AH Moss 21 The molecular basis of leukaemia and lymphoma, 380

George S Vassiliou and Anthony R Green 22 Laboratory diagnosis of haematological

neoplasms, 395 Barbara J Bain and Torsten Haferlach 23 Acute myeloid leukaemia, 415

Alan K Burnett and Adriano Venditti 24 Adult acute lymphoblastic leukaemia, 433

Nicola Gökbuget and Dieter Hoelzer 25 Childhood acute lymphoblastic leukaemia, 448

Dario Campana and Ching-Hon Pui 26 Supportive care in the management of

leukaemia, 463 Archibald G Prentice and J Peter Donnelly 27 Chronic myeloid leukaemia, 483

John M Goldman and Tariq I Mughal 28 The myelodysplastic syndromes, 503

Timothy JT Chevassut and Ghulam J Mufti v

Contents

29 Chronic lymphocytic leukaemia and other B-cell

disorders, 530 Daniel Catovsky and Emili Montserrat

43 Acquired coagulation disorders, 839

Peter W Collins, Jecko Thachil and Cheng-Hock Toh 44 Thrombotic thrombocytopenic purpura and

30 T-cell lymphoproliferative disorders, 558

haemolytic–uraemic syndrome (congenital and acquired), 860 Pier M Mannucci, Flora Peyvandi and Roberta Palla

Estella Matutes 31 Multiple myeloma, 577

Jesús San-Miguel and Joan Bladé

45 Heritable thrombophilia, 872

Trevor Baglin

32 Amyloidosis, 599

Simon DJ Gibbs and Philip N Hawkins

46 Acquired venous thrombosis, 888

Beverley J Hunt and Michael Greaves

33 The classification of lymphoma: updating the WHO

classification, 614 Elias Campo and Stefano A Pileri

47 Management of venous thromboembolism, 900

Beverly J Hunt

34 Hodgkin lymphoma, 639

48 Congenital platelet disorders, 915

Jonathan Sive and David Linch

Maurizio Margaglione and Paul RJ Ames

35 Non-Hodgkin lymphoma, 655

Kate Cywnarski and Anthony H Goldstone 36 Myeloproliferative neoplasms, 686

Peter J Campbell and Anthony R Green 37 Histocompatibility, 710

Ann-Margaret Little, Steven GE Marsh and J Alejandro Madrigal

49 Primary immune thrombocytopenia, 928

Drew Provan and Adrian C Newland 50 Haematological aspects of systemic disease, 940

Atul B Mehta and A Victor Hoffbrand 51 Haematological aspects of tropical diseases, 956

Imelda Bates and Ivy Ekem 52 Neonatal haematology, 971

38 Stem cell transplantation, 722

Irene AG Roberts

Charles Craddock and Ronjon Chakraverty

Appendix I Normal values, 985

39 Normal haemostasis, 746

Keith Gomez, Edward GD Tuddenham and John H McVey

Appendix II World Health Organization classification

of tumours of the haematopoietic and lymphoid tissues, 986

40 The vascular function of platelets, 772

Index, 989

Stephen P Watson and Paul Harrison 41 Inherited bleeding disorders, 793

www.wiley.com/go/hoffbrand/postgraduate

Michael A Laffan and K John Pasi 42 Rare bleeding disorders, 813

Flora Peyvandi and Marzia Menegatti

Companion website This book has a companion website: www.wiley.com/go/hoffbrand/postgraduate with: • Figures and tables from the book for downloading • Interactive multiple-choice questions prepared by the authors

vi

Contributors

Paul RJ Ames

Clara Camaschella

Timothy JT Chevassut

Consultant Haematologist Department of Haematology Airedale General Hospital Steeton UK

Professor of Medicine Vita-Salut University; Division of Genetics and Cell Biology San Raffaele Scientific Institute Milan Italy

Senior Lecturer and Honorary Consultant in Haematology Brighton and Sussex Medical School Royal Sussex County Hospital Brighton UK

Dario Campana

Peter W Collins

Vice Chair for Laboratory Research Departments of Oncology and Pathology St Jude Children’s Research Hospital Memphis, TN USA

Senior Lecturer in Haematology Department of Haematology School of Medicine Cardiff University University Hospital of Wales Cardiff UK

Trevor Baglin Consultant Haematologist Department of Haematology Cambridge University Hospitals NHS Trust Addenbrookes Hospital Cambridge UK

Barbara J Bain

Peter J Campbell

Department of Haematology St Mary’s Hospital London UK

Cancer Genome Project Wellcome Trust Sanger Institute Cambridge UK

John A Barbara

Elias Campo

Emeritus Microbiology Consultant NHS Blood and Transplant Colindale London UK

Clinical Director and Professor of Pathology Center for Biomedical Diagnosis and Chief of Hematopathology Unit Hospital Clinic University of Barcelona Barcelona Spain

Imelda Bates Reader in Tropical Haematology Liverpool School of Tropical Medicine Liverpool UK

Joan Bladé Senior Consultant Haematologist Servicio de Hematología Hospital Clinic de Barcelona Barcelona Spain

Alan K Burnett Department of Haematology School of Medicine Cardiff University Cardiff UK

Marcela Contreras University College London and Blood Transfusion International London UK

Charles Craddock Centre for Clinical Haematology Leukaemia Unit Queen Elizabeth Hospital Birmingham UK

Daniel Catovsky

Kate Cwynarski

Emeritus Professor of Haematology Institute of Cancer Research Sutton Surrey UK

Consultant Haematologist and Honorary Senior Lecturer Department of Haematology Royal Free Hospital London UK

Ronjon Chakraverty Royal Free and University College Medical School London UK

Geoff Daniels Consultant Clinical Scientist Bristol Institute for Transfusion Sciences NHS Blood and Transplant Bristol UK

vii

Contributors

Inderjeet S Dokal

John M Goldman

Philip N Hawkins

Chair of Paediatrics and Child Health Centre Lead Barts and The London School of Medicine and Dentistry Queen Mary University of London Barts and The London Children’s Hospital London UK

Department of Haematology Imperial College School of Medicine Hammersmith Hospital London UK

Clinical Director National Amyloidosis Centre University College London Medical School Royal Free Hospital London UK

J Peter Donnelly Department of Haematology University Hospital Nijmegen Nijmegen The Netherlands

Mark T Drayson Department of Immunology University of Birmingham Medical School Birmingham UK

Elaine Dzierzak Department of Cell Biology Erasmus Stem Cell Institute Erasmus Medical Centre Rotterdam The Netherlands

Ivy Ekem Senior Lecturer and Head Department of Haematology University of Ghana Medical School Accra Ghana

Modupe O Elebute Consultant Haematologist King’s College Hospital London UK

Simon DJ Gibbs Clinical Academic Research Fellow (Haematology) National Amyloidosis Centre University College London Medical School; Royal Free Hospital London UK

Nicola Gökbuget Head of Study Center Goethe University Hospital Department of Medicine II Hematology/Oncology Frankfurt Germany

viii

Anthony H Goldstone Department of Haematology University College London London UK

Keith Gomez Senior Lecturer in Haematology Haemophilia Centre and Thrombosis Unit Royal Free and University College London Medical School London UK

Edward C Gordon-Smith Department of Haematology St George’s Hospital Medical School London UK

Michael Greaves Professor of Haematology Head of School of Medicine and Dentistry University of Aberdeen Aberdeen UK

Anthony R Green Department of Haematology University of Cambridge Cambridge Institute for Medical Research Cambridge UK

Torsten Haferlach MLL Munich Leukemia Laboratory GmbH Munich Germany

Paul Harrison Clinical Scientist Oxford Haemophilia and Thrombosis Centre The Churchill Hospital Headington Oxford UK

Chaim Hershko Department of Medicine Shaare Zedek Medical Centre; Professor Emeritus Hebrew U Hadassah Medical School Jerusalem Israel

Douglas R Higgs Professor of Molecular Haematology MRC Molecular Haematology Unit Weatherall Institute of Molecular Medicine John Radcliffe Hospital Oxford UK

Peter Hillmen Department of Haematology St James’s University Hospital Leeds UK

Dieter Hoelzer Professor of Internal Medicine Onkologikum Frankfurt Museum Embankment Frankfurt Germany

A Victor Hoffbrand Emeritus Professor of Haematology University College Medical School; Honorary Consultant Haematologist Royal Free Hospital London UK

Ronald Hoffman Albert A and Vera List Professor of Medicine Tisch Cancer Institute Mount Sinai School of Medicine New York, NY USA

Derralynn A Hughes Senior Lecturer in Haematology Department of Academic Haematology Royal Free and University College Medical School London UK

Contributors

Beverley J Hunt

Maurizio Margaglione

Emili Montserrat

Thrombosis and Haemostasis King’s College; Guy’s and St Thomas’ NHS Foundation Trust London UK

Associate Professor Medical Genetics Department of Biomedical Sciences University of Foggia Foggia Italy

Director, Institute of Hematology and Oncology Hospital Clinic University of Barcelona Barcelona Spain

Michael A Laffan Department of Haematology, Faculty of Medicine Imperial College School of Medicine Hammersmith Hospital London UK

Judith CW Marsh

Paul AH Moss

Department of Haematological Medicine King’s College Hospital London UK

Professor of Haematology and Head of School of Cancer Sciences University of Birmingham Birmingham UK

Ashutosh Lal

Steven GE Marsh

Ghulam J Mufti

Hematology/Oncology Children’s Hospital and Research Center at Oakland Oakland, CA USA

The Anthony Nolan Research Institute Royal Free Hospital London UK

Head, Department of Haematological Medicine King’s College Hospital and Kings College London London UK

John Mascarenhas

Tariq I Mughal

Assistant Professor of Medicine Division of Hematology/Oncology Tisch Cancer Institute Mount Sinai School of Medicine New York, NY USA

Guy’s Hospital London UK

David Linch Department of Haematology UCL Medical School London UK

Ann-Margaret Little Histocompatibility and Immunogenetics Service Gartnavel General Hospital Glasgow UK

John H McVey Weston Professor of Molecular Medicine Molecular Medicine Thrombosis Research Institute London UK

J Alejandro Madrigal The Anthony Nolan Research Institute Royal Free Hospital London UK

Pier M Mannucci Professor and Chairman of Internal Medicine A. Bianchi Bonomi Hemophilia and Thrombosis Center IRCCS Cà Granada Maggiore Hospital Foundation; Department of Internal Medicine University of Milan and Luigi Villa Foundation Milan Italy

Estella Matutes Reader and Consultant Haematologist Royal Marsden Hospital and Institute of Cancer Research London UK

Atul B Mehta Consultant Haematologist Department of Haematology University College London School of Medicine Royal Free Hospital London UK

Marzia Menegatti A. Bianchi Bonomi Hemophilia and Thrombosis Center IRCCS Cà Granada Maggiore Hospital Foundation; Department of Internal Medicine University of Milan and Luigi Villa Foundation Milan Italy

Narla Mohandas Vice President of Research New York Blood Center New York, NY USA

Adrian C Newland Professor of Haematology Department of Haematology Queen Mary University of London London UK

Roberta Palla A. Bianchi Bonomi Hemophilia and Thrombosis Center IRCCS Cà Granada Maggiore Hospital Foundation; Department of Internal Medicine University of Milan and Luigi Villa Foundation Milan Italy

K John Pasi Centre for Haematology Institute of Cell and Molecular Science Barts and The London School of Medicine and Dentistry London UK

Flora Peyvandi Associate Professor of Internal Medicine A. Bianchi Bonomi Hemophilia and Thrombosis Center IRCCS Cà Granada Maggiore Hospital Foundation; Department of Internal Medicine University of Milan and Luigi Villa Foundation Milan Italy

ix

Contributors

Stefano A Pileri

Jesús San-Miguel

George Vassiliou

Professor of Pathology Department of Haematology and Oncological Sciences; Director of the Haematopathology Unit Bologna University School of Medicine St Orsola Hospital Bologna Italy

Professor and Chairman of Haematology Department of Haematology Hospital Universitario de Salamanca Salamanca Spain

Wellcome Trust Sanger Institute Wellcome Trust Genome Campus Cambridge UK

Archibald G Prentice Department of Haematology Royal Free Hospital London UK

Drew Provan Senior Lecturer in Haematology Department of Haematology Queen Mary University of London London UK

Ching-Hon Pui Departments of Oncology and Pathology St Jude Children’s Research Hospital Memphis, TN USA

Farhad Ravandi Department of Leukaemia University of Texas MD Anderson Cancer Center Houston, TX USA

David Rees Senior Lecturer King’s College Hospital; Consultant Paediatric Haematologist Department of Haematological Medicine King’s College London School of Medicine London UK

Jonathan Sive Department of Haematology UCM Medical School London UK

Associate Professor Department of Haematology Policlinico Tor Vergata Rome Italy

Clare PF Taylor

Elliott P Vichinsky

Medical Director of SHOT Medical Directorate North London Blood Centre London UK

Hematology/Oncology Children’s Hospital and Research Center at Oakland Oakland, CA USA

Jecko Thachil

Stephen P Watson

Clinical Research Fellow in Haematology School of Clinical Sciences University of Liverpool Liverpool UK

BHF Chair in Cardiovascular Sceinces and Cellular Pharmacology Division of Medical Sciences Institute of Biomedical Research Centre for Cardiovascular Sciences University of Birmingham Birmingham UK

Swee Lay Thein Professor of Molecular Haematology Department of Haematological Medicine King’s College Hospital; Consultant Haematologist Division of Gene and Cell Based Therapy King’s College London School of Medicine London UK

William G Wood Professor in Haematology Weatherall Institute of Molecular Medicine John Radcliffe Hospital Oxford UK

Cheng-Hock Toh

Neal S Young

Professor of Haematology School of Clinical Sciences University of Liverpool Liverpool UK

National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, MD USA

Irene AG Roberts

Edward GD Tuddenham

Professor of Paediatric Haematology Centre for Haematology Hammersmith Campus Imperial College London London UK

Katharine Dormandy Chair of Haemophilia Haemophilia Centre and Thrombosis Unit Royal Free and University College London Medical School London UK

x

Adriano Venditti

Alberto Zanella Hematology 2 Unit IRCCS Cà Granada Maggiore Hospital Foundation Milan Italy

Preface to the sixth edition

Haematology continues to advance and change more rapidly than most areas of medicine. This sixth edition of Postgraduate Haematology includes much knowledge that has been gained in the five years since the previous edition. Professor Tony Green of the Department of Haematology, University of Cambridge has joined the editors of the last edition to help cover malignant diseases, where major changes have occurred in their classification (WHO, 2008) and in diagnostic procedures and treatment protocols. Haematological oncology now forms the major workload for many consultant haematologists. Nevertheless, benign conditions are a major source of work for the general haematologist and chapters concerning all these conditions have been extensively updated, often by new authors. Despite increased understanding of the molecular basis of haematological diseases and advances in their investigation and management, we have kept the size of the book unchanged by omitting ten chapters from the fifth edition and incorporating their essential information into the remaining chapters.

As for previous editions, this book is aimed at providing haematologists in training and consultants with up-to-date knowledge of the aetiology of blood diseases combined with a practical guide to their investigation and treatment. The views expressed are those of the individual authors but relevant literature is listed at the end of each chapter to provide additional reference material. Many of our authors are based outside the UK, particularly in Europe and the USA, and we hope this book will be used by haematologists practising in the UK, Europe and internationally. We are grateful to our publishers Wiley-Blackwell for their unstinting help during the publishing process and particularly to Rebecca Huxley and Jennifer Seward. We also thank Jane Fallows who, as previously, has with great expertise drawn all the scientific diagrams. London and Cambridge, 2011 AVH, DC, EGDT, ARG

xi

Preface to the first edition

In this book the authors combine an account of the physiological and biochemical basis of haematological processes with descriptions of the clinical and laboratory features and management of blood disorders. Within this framework, each author has dealt with the individual subjects as he or she thought appropriate. Because this book is intended to provide a foundation for the study of haematology and is not intended to be a reference book, it reflects, to some extent, the views of the individual authors rather than providing comprehensive detail and a full bibliography. For these the reader is referred to the selected reading given at the end of each chapter. It is hoped that the book will prove of particular value to students taking either the Primary or the Final Part of the examination for Membership of the Royal College of Pathologists and the Diplomas of Clinical Pathology. It should also prove useful to physicians wishing to gain special knowledge of haematology and to technicians taking the Advanced Diploma in Haematology of the Institute of Medical Laboratory Technology, or the Higher National Certificate in Medical Laboratory subjects. We wish to acknowledge kind permission from the editors and publishers of the British Journal of Haematology, the Journal

xii

of the Royal College of Physicians of London and the Quarterly Journal of Medicine for permission to reproduce figures 4.1, 4.5, 4.10, 4.11, 4.12, 9.4 and 9.10, also the publishers of Progress in Haematology for figure 7.2, and many other publishers who, together with the authors, have been acknowledged in the text. We are particularly grateful to Professor J.V. Dacie for providing material which formed the basis of many of the original illustrations in Chapters 4–8. We are greatly indebted to Mrs T. Charalambos, Mrs J. Cope and Mrs D. Haysome for secretarial assistance and to Mrs P. Schilling and the Department of Medical Illustration for photomicrography, art work and general photography. Finally, we are grateful for the invaluable help and forbearance we have received from Mr R. Emery and William Heinemann Medical Books. London, 1972 AVH SML

CHAPTER 1

Stem cells and haemopoiesis Elaine Dzierzak

1

Erasmus Stem Cell Institute, Erasmus Medical Centre, Rotterdam, The Netherlands

Introduction, 1 Hierarchical organization and lineage relationships in the adult haemopoietic system, 1 Sites of adult haemopoiesis, 3 Development of HSCs, 3 Waves of haemopoietic generation in embryonic development, 3

Embryonic haemopoietic sites and haemopoietic migration, 5 HSC quiescence, proliferation and ageing, 6 Haemopoietic supportive microenvironments, 6 Adult bone marrow microenvironment, 6 Microenvironments important for haemopoietic development in the conceptus, 7

Introduction Haemopoietic stem cells (HSCs) are the foundation of the adult blood system and sustain the lifelong production of all blood lineages. These rare cells are generally defined by their ability to self-renew through a process of asymmetric cell division, the outcome of which is an identical HSC and a differentiating cell. Through a series of proliferation and differentiation events, mature blood cells are produced. In health, HSCs provide homeostatic maintenance of the system through their ability to generate the hundreds of millions of erythrocytes and leucocytes needed each day. In trauma and physiological stress, HSCs are triggered to replace the lost or damaged blood cells. The tight regulation of HSC self-renewal ensures the appropriate balance of blood cell production. Perturbation of this regulation and unchecked growth of HSCs and/or immature blood cells results in leukaemia. Over the last 50 years, bone marrow transplantation, and more recently cord blood transplantation, have underscored the medical value of stem cell regenerative therapy. However, insufficient numbers of HSCs are still a major constraint in clinical applications. As the pivotal cells in this essential tissue, HSCs are the focus of intense research to further our understanding of their normal behaviour and the basis of their dysfunction in haemopoietic disease and leukaemia, and to provide insights and new strategies into improved clinical transplantation therapies. This chapter provides current and historical information on the organization of the adult haemopoietic cell differentiation hierarchy, the ontogeny of HSCs, the stromal microenvironment supporting these cells,

Postgraduate Haematology: 6th edition. Edited by A. Victor Hoffbrand, Daniel Catovsky, Edward G.D. Tuddenham, Anthony R. Green © 2011 Blackwell Publishing Ltd.

Haemopoietic regenerative and replacement therapies, 8 Stem cell transplantation, 8 New sources of HSCs for transplantation, 8 Selected bibliography, 10

and the molecular mechanisms involved in the regulation of HSCs.

Hierarchical organization and lineage relationships in the adult haemopoietic system The haemopoietic system is the best-characterized cell lineage differentiation hierarchy and, as such, has set the paradigm for the growth and differentiation of tissue-specific stem cells (Table 1.1). HSCs are defined by their high proliferative potential, ability to self-renew and potential to give rise to all haemopoietic lineages. HSCs produce immature progenitors that gradually and progressively, through a series of proliferation and differentiation events, become restricted in lineage differentiation potential. Such restricted progenitors produce the terminally differentiated functional blood cells. The lineage relationships of the variety of cells within the adult haemopoietic hierarchy (Figure 1.1) are based on results of in vivo transplantation assays in radiation chimeric mice and many in vitro differentiation assays that became available following the identification of haemopoietic growth factors. These assays facilitated measurement of the maturational progression of stem cells and progenitors, at or near the branch points of lineage commitment. Clonal analyses, in the form of colony-forming unit (CFU) assays, were developed to define the lineage differentiation potential of the stem cell or progenitor, and to quantitate the number/frequency of such cells in the population as a whole. In general, the rarer a progenitor is and the greater its lineage differentiation potential, the closer it is in the hierarchy to the HSC. In vitro clonogenic assays measure the most immature progenitor CFU-GEMM/Mix (granulocyte, erythroid, macrophage, megakaryocyte), bipotent progenitors 1

Postgraduate Haematology

Table 1.1 Transcription factors involved in haemopoietic and progenitor cell formation, survival and differentiation Cell

Transcription factors

Stem cells from mesoderm Generate and/or maintain stem cells Myelopoiesis

SCL/TAL-1; LMO2/RBTN-2 RUNX-1a/AML-1; TEL-1

Erythroid/megakaryocyte/mast cells Erythropoiesis, megakaryopoeisis Lymphopoiesis B Plasma cell T

MLL; GATA2a PU.1; C/EBPα, C/EBPε GFI-1, EGR-1, NAB2 GATA2 GATA2; GATA1; FOG1 SCL; EKLF; p45NF-E2 IKAROS1 E2A; EBF; PAX5 BLIMP1 CSL; GATA3, T-BET, NFATc

Immunophenotype of haemopoietic stem cells Positive

Negative

CD34 Thy1 AC133 cKIT

CD33 CD38 Lineage markers HLA DR

a

Based on mouse studies.

CFU-GM (granulocyte, macrophage) and restricted progenitors CFU-M (macrophage), CFU-G (granulocyte), CFU-E (erythroid) and BFU-E (burst forming unit-erythroid). While such in vitro clonogenic assays measure myeloid and erythroid potential, lymphoid potential is revealed only in fetal thymic organ cultures and stromal cell co-cultures in which the appropriate microenvironment and growth factors are present. Long-term culture assays (6–8 week duration), such as the cobblestone-area forming cell (CAFC) and the long-term culture-initiating cell (LTC-IC) assays, reveal the most immature of haemopoietic progenitors. In vivo, the heterogeneity of the bone marrow population of immature progenitors and HSCs is reflected in the time periods at which different clones contribute to haemopoiesis. Shortterm in vivo repopulating haemopoietic progenitor cells such as CFU-S (spleen) give rise to macroscopic erythromyeloid colonies on the spleen within 14 days of injection. Bona fide HSCs give rise to the long-term high-level engraftment of all haemopoietic lineages. Serial transplantations reveal the ability of the long-term repopulating HSCs to self-renew. The clonal nature 2

of engraftment and the multilineage potential of HSCs has been demonstrated through radiation and retroviral marking of bone marrow cells. Moreover, such studies suggest that, at steady state, only a few HSC clones contribute to the haemopoietic system at any one time. Further analyses of bone marrow HSCs show that this compartment consists of a limited number of distinct HSC subsets, each with predictable behaviours as described by their repopulation kinetics in irradiated adult recipients. In general, the bone marrow haemopoietic cell compartment as measured by in vitro clonogenic assays and in vivo transplantation assays shows a progression along the adult differentiation hierarchy from HSCs to progenitors and fully functional blood cells with decreased multipotency and proliferative potential, and an increased cell turnover rate. The use of flow cytometry to enrich for HSCs and the various progenitors in adult bone marrow has been instrumental in refining precursor–progeny relationships in the adult haemopoietic hierarchy. HSCs are characteristically small ‘blast’ cells, with a relatively low forward and side light scatter and low metabolic activity. Both mouse and human HSCs are negative for expression of mature haemopoietic lineage cell-surface markers, such as those found on B lymphoid cells (CD19, B220), T lymphoid cells (CD4, CD8, CD3), macrophages (CD15, Mac-1) and granulocytes (Gr-1). Positive selection for mouse HSCs relies on expression of Sca-1, c-kit, endoglin and CD150 markers and for human HSCs on expression of CD34, c-kit, IL-6R, Thy-1 and CD45RA markers. Similarly, cell types at lineage branch points have been identified, including the CMP (common myeloid progenitor), CLP (common lymphoid progenitor) and GMP (granulocyte macrophage progenitor). Recently, using the flt3 receptor tyrosine kinase surface marker along with many other well-studied markers, the LMPP (lymphoid primed multipotent progenitor) has been identified within the lineage negative, Sca-1 positive, c-kit positive (LSK) enriched fraction of HSCs. These cells have granulocyte/macrophage, B lymphoid and T lymphoid potential, but little or no megakaryocyte/erythroid potential. This suggests that the first lineage differentiation event is not a strict separation into common lymphoid and myeloid pathways. While these cellsurface marker changes and functional restriction events are represented by discrete cells in the working model of the haemopoietic hierarchy as depicted in textbooks and Figure 1.1, it is most likely that there is a continuum of cells between these landmarks. The currently identified progenitor cells in the hierarchy represent the cells present at stable and detectable frequencies and for which we currently have markers and functional assays. As more cell-surface markers are identified and sensitivity of detection is increased, more intermediate cell subsets are likely to be identified and it may be possible to determine, throughout the continuum, all the molecular events needed for the differentiation of the haemopoietic system and the transit times necessary for differentiation to the next subset.

Stem cells and haemopoiesis

B

pre-B

B lymphocyte T

CLP pre-T

HSC

CFU-S

MPP GMP

Figure 1.1 The adult haemopoietic hierarchy. Haemopoietic stem cells are at the foundation of the hierarchy. Through a series of progressive proliferation and differentiation steps the mature blood cell lineages are produced. Haemopoietic stem cells have the greatest proliferative and multilineage differentiation potential, while the mature blood cells are not proliferative and are lineage restricted. While large numbers of mature cells are found in the blood and turn over rapidly, the bone marrow contains long-lived quiescent haemopoietic stem cells at a very low frequency.

T lymphocyte

G-CFC

Granulocyte

GM-CFC

Macrophage

CFU-E

Erythrocytes

GMCFC

CMP BFU-E

Platelets MEP MegCFC

Megakaryocyte

Proliferative potential

Sites of adult haemopoiesis Bone marrow, spleen, thymus and lymph nodes are the haemopoietic sites in the adult, and each tissue plays a special role in supporting the growth and differentiation of particular haemopoietic cell lineages and subsets. Equally important is the blood itself, which is a mobile haemopoietic tissue, with mature blood cells travelling through the circulation to function in all parts of the body. Not only do the terminally differentiated cells, such as erythrocytes and lymphocytes, move by means of the circulation, but HSCs (at low frequency) also migrate through the circulation from the bone marrow to other haemopoietic tissues. HSCs are mostly concentrated in the bone marrow and are found in the endosteal and vascular niches (Figure 1.2). HSCs can be induced to circulate by administration of granulocyte colony-stimulating factor (G-CSF) and it is of great interest to determine whether these cells retain all the characteristics of stem cells. Recent improvements in confocal microscopy have allowed the visualization of the migration of circulating HSCs to the bone marrow endosteal niche by time-lapse imaging in the mouse. The estimated frequency of HSCs is 1 per 104–105 mouse bone marrow cells and 1 per 20 × 106 human bone marrow cells.

Frequency and turnover rate

HSCs are also found in the mouse spleen at approximately a 10-fold lower frequency and in the circulating blood at a 100fold lower frequency. The capacity for HSCs to migrate and also be retained in the bone marrow is of relevance to clinical transplantation therapies. HSCs injected intravenously in such therapies must find their way to the bone marrow for survival and effective haemopoietic engraftment. For example, stromalderived factor (SDF)-1 and its receptor CXCR4 (expressed on HSCs) are implicated in the movement of HSCs and the retention of HSCs in the bone marrow. Indeed, HSC mobilization can be induced through AMD3100, an antagonist of SDF-1, and by the administration of G-CSF. Mobilization strategies with G-CSF are used routinely to stimulate bone marrow HSCs to enter the circulation, allowing ease of collection in the blood rather than through bone marrow biopsy.

Development of HSCs Waves of haemopoietic generation in embryonic development Until the mid 1960s it was thought that blood cells were intrinsically generated in tissues such as the liver, spleen, bone marrow 3

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Endothelial niche

Expand

Differentiate Migrate

Haemangioblast

Apoptosis Haemopoietic

Self-renew

Haemogenic endothelium

HSC

Endothelial

Haemopoietic

Figure 1.3 Precursors to haemopoietic cells in embryonic stages. The mesodermal precursor to haemopoietic and endothelial lineages at early stages of development is the haemangioblast. Later, haemogenic endothelial cells are the precursors to haemopoietic stem cells and progenitor cells. These cells appear to exist during a short window of developmental time.

Endosteal niche

Figure 1.2 The bone marrow haemopoietic niches. Haemopoietic stem cells are found in the endosteal and endothelial niches of the bone marrow. These niches support the maintenance, self-renewal, expansion, differentiation, migration and survival of haemopoietic stem cells through local growth factor production, cell–cell interactions and more distance signals.

and thymus. Survival studies in which cells from unirradiated tissues were injected into lethally irradiated mice showed that it was the bone marrow that contains the potent cells responsible for rescue from haemopoietic failure. Later, through clonal marking studies, it was demonstrated that bone marrow harbours HSCs during the adult stages of life. But where, when and how are HSCs generated during ontogeny? In the 1970s, examination of mouse embryo tissues suggested that adult haemopoietic cells are generated in the yolk sac, migrate and colonize initially the fetal liver and subsequently the bone marrow, where they reside throughout adult life. However, studies in nonmammalian vertebrate models (avian and amphibian) demonstrated that the aorta region in the body of the embryo generates the long-lived adult blood system, while the yolk sac (or equivalent tissue) produces the transient embryonic haemopoietic system. In agreement with these studies, the aorta–gonad– mesonephros (AGM) region of the mouse embryo was later found to generate the first adult HSCs. The development of the mammalian adult haemopoietic system is complex and begins its development in the mouse embryo during mid-gestation. As a growing organism, the embryo itself needs rapid haemopoiesis to thrive before the adult system is generated. Thus, a simple transient haemopoietic system is generated during early development and rapidly produces primitive erythroid and myeloid cells. In the yolk sac 4

both haemopoietic and endothelial cells are simultaneously generated from a common mesodermal precursor cell, the haemangioblast (Figure 1.3). Thereafter, many haemopoietic progenitor and differentiated cell types are generated in both the yolk sac and the intraembryonic region of the dorsal aorta to create an intermediate haemopoietic system. It is likely that these cells also are derived from a haemangioblast-type precursor or from haemogenic endothelial cells, a specialized population of endothelial cells that have haemogenic potential. At both these early times in ontogeny, the mouse embryo contains no HSCs. Hence, in the absence of HSCs, the embryo generates a haemopoietic system that is short-lived and lacks the important qualitative characteristics (longevity and self-renewability) of the adult haemopoietic system. Independent and distinct waves of haemopoiesis supply the embryo and adult and do not arise from the same cohorts of mesodermal precursor cells (Figure 1.4). The adult system has its foundation in a cohort of initiating HSCs. The first HSCs are de novo generated in the AGM region, only after embryonic haemopoietic cells are differentiated directly from mesodermal precursors. The first adult HSCs are autonomously generated in the mouse AGM at E10.5 and in the human AGM beginning at week 4 of gestation. Recently, the process of HSC generation has been visualized in real time in the mouse embryo (Boisset et al., 2010). This remarkable observation demonstrating that HSCs are derived via a transdifferentiation event in which specialized endothelial cells lining the aorta bud into the lumen to form round cells with HSC fate, confirm the marking and static microscopic studies performed in avian embryos (Figure 1.5). The emerging mouse aortic HSCs are characterized by the loss of cell-surface markers for endothelium, such as Flk-1 and VE-cadherin, and the gain of expression of the haemopoietic markers c-kit, CD41 and CD45 and the HSC markers Sca1, c-kit and endoglin (Boisset et al., 2010). Expression of HSC markers confirms that the emerging

Stem cells and haemopoiesis

Long-lived adult haemopoiesis

Transient embryonic haemopoiesis

Definitive HSCs

Primitive Progenitors

Figure 1.4 Waves of haemopoietic cell emergence during embryonic stages. The earliest haemopoietic cells are produced during the first wave of haemopoietic fate determination. The onset of this wave occurs in the yolk sac blood islands and produces transient primitive erythroid cells. This wave continues with the production of transient haemopoietic progenitors in the absence of bona fide haemopoietic stem cells. True long-lived definitive haemopoietic stem cells (adult repopulating stem cells) are generated in the second wave of haemopoietic cell emergence in the AGM region. In this wave, haemogenic endothelial cells bud into the aortic lumen as these cells take on haemopoietic stem cell fate.

Haemopoietic cluster Aorta

Gonad

Mesonephros

Inductive microenvironment

Figure 1.5 Schematic diagram of the aorta–gonad–mesonephros (AGM) region and haemopoietic cell clusters emerging from the dorsal aorta. The haemopoietic stem cell inductive microenvironment is localized in the ventral aspect of the aorta.

AGM cells are HSCs as functionally potent as bone marrow HSCs, since these sorted cells can form a complete long-term haemopoietic system in irradiated adult recipient mice. Lineage tracing experiments in the mouse embryo have indicated that the adult haemopoietic system is generated during a short window of development, spanning E9–E11. Using Cre-lox

recombination (temporally and cell lineage controlled) to mark VE-cadherin expressing endothelial cells in the mid-gestation embryo, it was found that almost all the blood cells in the circulation and haemopoietic tissues of the adult mice contained the recombination marker, unequivocally demonstrating that adult HSCs are the progeny of embryonic endothelial cells. Moreover, these cells require the Runx1 transcription factor as demonstrated by Runx1 conditional deletion in this mouse model. Other lineage tracing experiments were also performed using Cre-lox technology so as to mark the earliest cells expressing the Runx1 and SCL transcription factors, both of which are known to be important for haemopoietic cell development. The progeny of marked SCL-expressing (endothelial and haemopoietic) cells and Runx1-expressing (definitive haemopoietic and haemogenic endothelial) cells also contributed to the bone marrow cells in the adult. Thus, the progeny of haemopoietic cells generated from haemogenic endothelium in the embryo contribute to a cohort of adult bone marrow HSCs that form the foundation of haemopoiesis throughout adult life.

Embryonic haemopoietic sites and haemopoietic migration The AGM and yolk sac are not the only sites where haemopoietic cells are found in the early conceptus. The placenta is a highly haemopoietic tissue and has recently been shown to generate haemopoietic cells de novo. Much like the early-stage yolk sac, the mouse placenta can produce erythromyeloid progenitors. Embryos deficient for the Ncx1 gene, lacking a heartbeat and circulation, have such progenitors in the yolk sac and placenta at early stages, suggesting that haemopoietic progenitors are generated in these tissues. Unfortunately, the embryos die before the onset of HSC generation at mid-gestation, precluding analysis of HSC production in the yolk sac and placenta. In normal embryos where the circulation is established between the embryo body and the extraembryonic tissues at E8.25, HSCs are detected in the placenta and yolk sac only beginning at E11, subsequent to the first HSC generation in the AGM at E10.5. Thus it is uncertain whether the placenta (or the yolk sac) can generate HSCs de novo. At present, there is no method by which cells can be uniquely marked in the specific developing tissues to examine this. Nonetheless, quantitative studies in which HSC numbers in each of these tissues was determined suggest that the AGM cannot generate all the HSCs that are eventually found in the fetal liver (a tissue that harbours haemopoietic cells but does not generate them) and later in the adult bone marrow (Figure 1.4). In particular, the placenta at mid-gestation contains an abundance of HSCs, suggesting that this highly vascularized tissue may generate HSCs from haemogenic endothelium and/or that the placenta is a highly supportive and expansive microenvironment for AGM-derived HSCs. Like the mouse placenta, the developing human placenta contains HSCs. Already at week 6 in gestation HSCs can be 5

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detected, as analysed by in vivo xenotransplantation into immunodeficient mice. Also, haemopoietic progenitors are found at these early stages. Phenotypic characterization shows that HSCs and progenitors are in both the CD34-positive and CD34-negative fractions at week 6 of gestation and are exclusively in the CD34-positive fraction by week 19. These cells are in close association with the placental vasculature. Taken together, the development of the haemopoietic system in the human conceptus closely parallels that in the mouse conceptus. Interestingly, together with the umbilical cord blood harvested at birth, the placenta may provide additional haemopoietic progenitors and HSCs for preclinical studies and potential clinical therapies.

HSC quiescence, proliferation and ageing Somatic stem cells undergo lifelong self-renewal and possess the potential to produce the differentiated cells of the tissue. HSCs are considered to be relatively dormant stem cells, dividing rather infrequently. They are enriched in the quiescent fraction of adult bone marrow and are resistant to antiproliferative drugs such as 5-fluorouracil. Recent studies using a label-retaining method for analysis of cycling versus non-cycling cells shows that dormant HSCs in homeostatic conditions cycle only once every 21 weeks. The adult mouse possesses approximately 600 of these dormant LSK CD150+CD48−CD34− HSCs. Interestingly, 38% of HSCs in G0, considered to be the dormant HSCs, can be activated by injury, 5-fluorouracil or G-CSF. These cells can return to the dormant state after the reestablishment of homeostasis. The maintenance of HSC dormancy is thought to be an important strategy for preventing stem cell exhaustion during adult life. It has been demonstrated by serial transplantation in the mouse that HSC self-renewal is limited to about six rounds of transplantation and that the ability of the transplanted stem cells to repopulate progressively decreases. Studies of chromosome shortening in human HSCs suggest that self-replication is limited to about 50 cell divisions. It has been suggested that accumulating DNA mutations and loss of telomere repeats affect HSC function. Recently, a set of experiments have demonstrated that HSCs are markedly reduced in number and/or function in ageing mice. Comparison of various inbred mouse strains has shown that the rate of haemopoietic cell cycling is inversely correlated with their mean lifespan. The decrease in HSC quantity or quality was due to cell-intrinsic genetic or epigenetic factors. Causative genes were identified by transcriptional profiling comparisons between the HSCs of the different strains. Of particular interest are chromatin modifiers involved in prevention of HSC exhaustion through maintenance of a stem cell-specific transcriptional programme. Changes in chromatin structure associated with high HSC turnover would result in stem cell senescence (which is thought to protect stem cells from malignant transformation by oncogenic events). 6

Haemopoietic supportive microenvironments Adult bone marrow microenvironment Most tissue-specific stem cells are maintained in a special microenvironment to support their long-term growth and selfrenewal. To provide the continuous production of human blood over many decades, HSCs are also maintained in a specialized microenvironment, the haemopoietic supportive niches of the adult bone marrow (see Figure 1.2). The importance the bone marrow haemopoietic niche and the interactions between supportive cells and HSCs was first demonstrated in mice. In transplantation studies of anaemic mouse strains naturally deficient in the c-kit receptor tyrosine kinase (W mice) or kit-ligand (KL; Steel mice) it was revealed that bone marrow from W mutant mice could not repopulate the haemopoietic system of wild-type irradiated recipient mice, while bone marrow from Steel mutant mice could. In contrast, W mutant mice could be repopulated by wild-type donor bone marrow cells, whereas Steel recipients were defective for repopulation by wild-type donor cells. Thus, it was proposed that a receptor–ligand interaction was involved in the support of HSCs within the bone marrow microenvironment and it was subsequently shown that HSCs express c-kit and bone marrow stromal cells express KL. The development of ex vivo culture systems to study this complex microenvironment allowed further dissection of the cellular and molecular aspects of the bone marrow microenvironment. These studies were aided by the isolation of mesenchymal stromal cells. Stromal cell lines have been derived from the adult mouse bone marrow and fetal liver tissues. These are generally of mesenchymal lineage as determined by cell-surface marker expression and their osteogenic and adipogenic potentials. Although widely heterogeneous in their ability to support haemopoiesis, some stromal lines (MS5 and AFT024, for example) have been shown to support the growth and/or maintenance of HSCs in co-cultures for long periods. Moreover, they have been instrumental in further characterization of these haemopoietic supportive niches. Comparative transcriptional profiling and database analysis of HSC supportive and non-supportive stromal cell lines has revealed a complex genetic programme involving a wide variety of known molecules and molecules whose function in haemopoiesis is unknown. The in vivo bone marrow microenvironment is very complex, containing osteoblastic niches and vascular niches localized within the trabecular regions of the long bones. HSCs are maintained in close association with the so-called ‘stromal cells’ of the niches. Some of the key molecular regulators within the bone marrow niches include N-cadherin, CD150 and the SDF1/ CXCR4, Notch, Wnt, Hedgehog, Tie2/angiopoietin, transforming growth factor (TGF), bone morphogenetic protein (BMP)

Stem cells and haemopoiesis

and fibroblast growth factor (FGF) signalling pathway molecules. These regulators are implicated in a variety of cellular processes, such as HSC maintenance, differentiation, selfrenewal and homing. Indeed, live tracking of haemopoietic progenitor/stem cells in the mouse model has shown the homing ability of these cells to bone marrow niches, and mouse models as well as in vitro culture systems are beginning to reveal the specific molecular mechanisms involved.

Microenvironments important for haemopoietic development in the conceptus Prior to the necessity for an adult haemopoietic supportive microenvironment, the embryo contains several haemopoietic inductive microenvironments. The extraembryonic tissues, yolk sac and placenta, and the intraembryonic AGM generate haemopoietic progenitor cells, while the AGM region generates HSCs (Figure 1.6). Little is known about the differences between the microenvironments of these three tissues. However, the AGM microenvironment is the most well characterized due to the simplicity of its structure, with the aorta at the midline and the laterally located gonads and mesonephroi (see Figure 1.5). It is known that the avian AGM region contains different types of mesenchymal stem/progenitor cells and a population of aorta-associated stem cells called ‘meso-angioblasts’ contributes to cartilage, bone and muscle tissues and also to blood. In

Yolk sac

AGM Fetal liver

Placenta

Bone marrow

Figure 1.6 Haemopoietic sites during development. The first haemopoietic stem cells arise in the AGM region. Other haemopoietic cells and progenitors are generated in the yolk sac and placenta. It is as yet undetermined whether the yolk sac and placenta can generate haemopoietic stem cells. Haemopoietic cells generated in these three tissues migrate and colonize the fetal liver. Subsequently, the long-lived haemopoietic cells (primarily the haemopoietic stem cells) migrate and colonize the bone marrow, where they reside in the adult stages of life.

the mouse AGM region, cells more typical of mesenchymal stem/progenitor cells have been found. Interestingly, mapping and frequency analysis of mesenchymal progenitors in the mouse conceptus show that mesenchymal progenitors, with the potential to differentiate into cells of the osteogenic, adipogenic and/or chondrogenic lineages, reside in most of the sites harbouring haemopoietic cells, suggesting that both the HSC and mesenchymal stromal cell microenvironment develop in parallel in the AGM region. Many stromal cell lines have been established from the AGM region, placenta and fetal liver. Stromal cell lines isolated from both the mid-gestation AGM and placenta can support immature haemopoietic progenitors. In vivo assays show that some of the AGM stromal clones are potent supporters of HSCs as compared with adult bone marrow and fetal liver cell lines. Indeed some of these lines can support the haemopoietic differentiation of embryonic stem (ES) cells. Although there is one report in the literature of an AGM stromal cell line with the capacity to induce HSC formation from early embryo cells, these results have not been reproduced. Phenotypic characterization of haemopoietic supportive AGM stromal lines places them in the vascular smooth muscle cell (VSMC) hierarchy, in between a mesenchymal stem cell and a VSMC. Thus, while AGM and other embryonic stromal cell lines can provide important signals for the maintenance of the first HSCs, the lack of firm evidence for HSC induction with such lines suggests that the AGM inductive microenvironment is likely to be complex with a variety of spatial and temporal cues emanating from several cell types. Within the normal physiology of the embryo, the AGM lies between the ventral tissue that includes the endoderm-derived gut and the dorsal tissue including the notochord and the ectoderm-derived neural tube (see Figure 1.5). Mouse AGM explant culture experiments have shown that dorsal tissues/ signals repress AGM HSC activity and ventral tissues/signals enhance HSC emergence. In both mouse and human AGM regions, cells expressing HSC markers are closely adherent to the vascular endothelium on the ventral aspect of the aorta. In the mouse, at precisely E10.5, single endothelial cells bud into the lumen as they take on HSC identity (see Figure 1.5). Importantly, HSC activity as determined by functional transplantation assays is localized exclusively to the ventral aspect of the mouse mid-gestation aorta. Thus there is a strong positive ventral positional influence on HSC generation in the AGM, and morphogens and local signals emanating from the ventral endodermal tissues may be responsible for establishing the HSC inductive microenvironment. Haemopoietic transcription factors required for HSC generation such as Gata2 and Runx1 are expressed in cells of the ventral aortic clusters and endothelium. Deficiency of Gata2 and Runx1 in mice leads to mid-gestation embryonic lethality, with complete absence of adult haemopoiesis (although embryonic haemopoiesis occurs), thus demonstrating that these two 7

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pivotal transcription factors promote the HSC genetic programme. Zebrafish and frog embryos have been useful models for dissecting the cascade of upstream events that lead to HSC induction. Developmental growth factor signalling pathways, such as the BMP, Hedgehog and Notch pathways, converge to activate expression of the two transcription factors in aortic haemopoietic cells and promote the HSC programme. In both the mouse and human embryo, BMP4 is expressed in the mesenchyme underlying the ventral aspect of the aorta at the time of haemopoietic cluster formation. Culture experiments have demonstrated the positive influence of BMP4 exposure to mouse and human HSC-containing cell populations. However, it remains to be determined whether BMP4 acts directly on HSCs or stimulates the microenvironment to produce HSC effectors. Similarly, Hedgehog signalling regulates HSCs in the AGM region. However, while Hedgehog signalling acts ventrally in zebrafish embryos, in the mouse embryo Hedgehogactivated cells surround the aorta. This lack of ventral restriction suggests a more complex pattern of regulation of this signalling pathway in the mouse embryo. Other ventrally localized HSC regulators include the Notch signalling molecules, as well as Wnt3a and interleukin (IL)-1. High-throughput chemical screens offer another means of identifying molecules involved in HSC growth, maintenance and expansion. Through such a screen in zebrafish embryos, prostaglandin E2 (PGE2) was recently identified as a regulator of HSC number. When tested in the murine transplantation model, ex vivo exposure of bone marrow cells to PGE2 enhanced short-term repopulation by haemopoietic progenitors and increased the frequency of long-term repopulating bone marrow HSCs. It has been shown that PGE2 modifies the Wnt signalling pathway, which in turn is thought to control HSC self-renewal and bone marrow repopulation. The zebrafish chemical screen also identified chemical blood flow modulators as regulators of HSC development. Nitric oxide synthetase inhibition or deficiency has also been shown to reduce transplantable murine bone marrow HSCs. Thus, these types of modulators hold promise for clinical treatments of bone marrow HSCs and the bone marrow haemopoietic niche. Together with more general physiological cues, such as the haemopoietic growth factors, KL, IL-3, Flt3 and thrombopoietin, these developmental regulators may be useful for expansion of HSC number and enhancement of HSC function for therapeutic purposes.

Haemopoietic regenerative and replacement therapies Stem cell transplantation For over 50 years, HSC transplantation has been the most successful and significant clinical cell regenerative therapy. Initially, whole bone marrow was the source of cells used in clinical 8

transplantation, but through experience and much research new and/or improved sources of transplantable HSCs were found. These now include the CD34+CD38− fraction of adult bone marrow, mobilized peripheral blood HSCs and the CD34+CD38− fraction of umbilical cord blood. The cumulative data from the large number of patients worldwide receiving a bone marrow transplant provide valuable information on the success of autologous versus allogeneic transplantation, the number of human leucocyte antigen (HLA) differences that are tolerated by the recipient, the incidence of graft-versus-host disease (GVHD), and the unexpected and advantageous graftversus-leukaemia effect. Interestingly, umbilical cord blood (UCB) appears to offer an advantageous source of HSCs for several reasons: UCB HSCs are young, being harvested at the neonatal stage of development, thus circumventing concerns about the ageing of HSCs. UCB transplantation induces less frequent and less severe GVHD, since UCB contains many fewer activated T cells than adult bone marrow. Also, UCB HSCs are highly proliferative. However, only relatively small numbers of cells are harvested (approximately 10-fold lower than those in adult bone marrow) and this limits their use to paediatric patients, unless multiple UCB units are transplanted. Nonetheless, the large number of UCB units (400 000) in cord blood banks (> 50) around the world (catalogued and recorded by EUROCORD and other coordinating efforts) offer greater availability and HLA donorcell selection, especially for rare haplotypes.

New sources of HSCs for transplantation The ability to expand HSCs ex vivo is a theoretically practical and attractive means to obtain an accessible and limitless source of HSCs for transplantation therapies. Unfortunately, despite many years of research using different culture systems and combinations of haemopoietic growth factors and proliferation stimulating agents, ex vivo expansion of HSCs has not been achieved. However, HSC developmental studies have begun to provide new insights into the processes directing the generation and growth of HSCs. Haemogenic endothelial cells As described in this chapter, the temporally and spatially limited production of HSCs in the embryonic aorta, examination of the specific microenvironment, and knowledge of the precursors to these stem cells has yielded insight into how HSCs may be induced and/or expanded without undergoing differentiation. If cells such as the haemogenic endothelial cells lining the ventral wall of the embryonic dorsal aorta are present in the adult vasculature, they could provide a novel source of inducible HSC precursors, particularly if they can be sustained and expanded to large numbers in culture. Alternatively, if haemogenic endothelial cells do not exist in the adult but it is possible to direct endothelium to be haemogenic, potential

Stem cells and haemopoiesis

therapeutic interventions could include the in vivo site-specific stimulation of HSC induction in the vasculature using the same developmental modulators and small molecules that affect the generation of HSCs in the embryonic aortic haemogenic endothelium. Some recent studies have suggested the presence of such cells in the human embryonic liver and fetal bone marrow. Embryonic stem cells and induced pluripotent stem cells Pluripotent ES cells have been used to generate differentiated cells in many tissue systems, including the haemopoietic system. Such haemopoietic-directed differentiation of human ES cells towards HSCs would be a potentially attractive alternative to conventional sources of HSCs. ES cells differentiated into embryoid bodies can be induced to differentiate into haemopoietic progenitors in cultures containing BMP4 and a cocktail of haemopoietic growth factors. These haemopoietic cells arise from haemangioblasts and/or primitive endothelial-like cells that express PECAM-1, FLK-1 (KDR) and VE-cadherin and are thought to represent the types of precursors, progenitors and differentiated cells found normally in the yolk sac. However, although ES cells can be induced to produce haemopoietic progenitors and differentiated cells of all haemopoietic lineages (and HoxB4 expression in mouse ES cells can promote granulocytic engraftment of adult irradiated mice), there are no convincing data showing the production of HSCs that are fully

potent in adult transplantation scenarios. This could suggest that the relevant haemopoietic inductive environment such as the AGM region is missing. Bone marrow, fetal liver and AGM stromal cell lines have been used to promote human ES cell haemopoietic differentiation in co-cultures. An AGM stromal cell line appears to significantly enhance spontaneous haemopoietic differentiation in high-density human ES cell cocultures and provide cells capable of primary and secondary haemopoietic engraftment into immunocompromised NOD/ LtSz-Scid IL2Rγ null recipients. Together, the induction of haemogenic endothelial cells from ES cells followed by haemopoietic induction with factors and/or cells of the AGM microenvironment may yield cells with the functions expected for definitive HSCs. Recent reports of somatic cell reprogramming by means of induced pluripotency makes the human ES cell differentation approach an exciting prospect for future cell-based therapies. The ability to produce patient-specific pluripotent cells (induced pluripotent stem or iPS cells) will eliminate all rejection issues that surround transplantation of HSCs from allogeneic donors. Indeed, a proof-of-principle study using gene-corrected mouse iPS cells from a thalassaemic mouse demonstrates the ability of such cells to form after transplantation normal functioning erythroid cells (Figure 1.7). Unfortunately, the presence of definitive adult HSCs was not demonstrated and hence future studies are needed to prove that HSCs can be generated in vitro from iPS/ES cells.

Transplantation

Sickle cell anaemia mouse model

iPS cell haemopoietic differentiation

Culture tail tip fibroblasts

Figure 1.7 Experimental approach in which iPS cells were used to treat sickle cell anaemia in a mouse model. (From Hanna et al. 2007 with permission of the American Association for the Advancement of Science.)

Correct sickle mutation in iPS cells

Oct4, Sox2, Klf4, Myc transduction

9

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Selected bibliography Abramson S, Miller RG, Phillips RA (1977) The identification in adult bone marrow of pluripotent and restricted stem cells of the myeloid and lymphoid systems. Journal of Experimental Medicine 145: 1567–79. Adolfsson J, Mansson R, Buza-Vidas N et al. (2005) Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment. Cell 121: 295–306. Arai F, Suda T (2007) Maintenance of quiescent haematopoietic stem cells in the osteoblastic niche. Annals of the New York Academy of Sciences 1106: 41–53. Blank U, Karlsson G, Karlsson S (2008) Signalling pathways governing stem-cell fate. Blood 111: 492–503. Boisset JC, Cappellen G, Andrieu C, Galjart N, Dzierzak E, Robin C (2010) In vivo imaging of hematopoietic stem cells emergence. Nature 464: 116–20. Breems DA, Blokland EA, Neben S et al. (1994) Frequency analysis of human primitive haematopoietic stem cell subsets using a cobblestone area forming cell assay. Leukaemia 8: 1095–104. Broudy VC (1997) Stem cell factor and hematopoiesis. Blood 90: 1345–64. Broxmeyer HE, Hangoc G, Cooper S et al. (2007) AMD3100 and CD26 modulate mobilization, engraftment, and survival of haematopoietic stem and progenitor cells mediated by the SDF-1/ CXCL12-CXCR4 axis. Annals of the New York Academy of Sciences 1106: 1–19. Charbord P, Oostendorp R, Pang W et al. (2002) Comparative study of stromal cell lines derived from embryonic, fetal, and postnatal mouse blood-forming tissues. Experimental Hematology 30: 1202–10. Chen MJ, Yokomizo T, Zeigler BM et al. (2009) Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter. Nature 457: 887–91. Cumano A, Dieterlen-Lievre F, Godin I (1996) Lymphoid potential, probed before circulation in mouse, is restricted to caudal intraembryonic splanchnopleura. Cell 86: 907–16. De Haan G, Gerrits A (2007) Epigenetic control of haematopoietic stem cell ageing the case of Ezh2. Annals of the New York Academy of Sciences 1106: 233–9. Durand C, Robin C, Bollerot K et al. (2007) Embryonic stromal clones reveal developmental regulators of definitive haematopoietic stem cells. Proceedings of the National Academy of Sciences USA 104: 20838–43. Dzierzak E, Speck NA (2008) Of lineage and legacy: the development of mammalian haematopoietic stem cells. Nature Immunology 9: 129–36. Gekas C, Dieterlen-Lievre F, Orkin SH et al. (2005) The placenta is a niche for haematopoietic stem cells. Developmental Cell 8: 365–75. Gluckman E, Rocha V (2009) Cord blood transplantation: state of the art. Haematologica 94: 451–4. Goessling W, North TE, Loewer S et al. (2009) Genetic interaction of PGE2 and Wnt signalling regulates developmental specification of stem cells and regeneration. Cell 136: 1136–47.

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Gothert JR, Gustin SE, Hall MA et al. (2005) In vivo fate tracing studies using the Scl stem cell enhancer: embryonic haematopoietic stem cells significantly contribute to adult hematopoiesis. Blood 105: 2724–32. Hackney JA, Charbord P, Brunk BP et al. (2002) A molecular profile of a haematopoietic stem cell niche. Proceedings of the National Academy of Sciences USA 99: 13061–6. Hanna J, Wernig M, Markoulaki S et al. (2007) Treatment of sickle cell anaemia mouse model with iPS cells generated from autologous skin. Science 318: 1920–3. Harrison DE, Astle CM (1982) Loss of stem cell repopulating ability upon transplantation. Effects of donor age, cell number, and transplantation procedure. Journal of Experimental Medicine 156: 1767–79. Jordan CT, Lemischka IR (1990) Clonal and systemic analysis of long-term hematopoiesis in the mouse. Genes and Development 4: 220–32. Kaufman DS. (2009) Toward clinical therapies using hematopoietic cells derived from human pluripotent stem cells. Blood 114: 3513–23. Kaufman DS, Hanson ET, Lewis RL et al. (2001) Haematopoietic colony-forming cells derived from human embryonic stem cells. Proceedings of the National Academy of Sciences USA 98: 10716–21. Kennedy M, D’Souza SL, Lynch-Kattman M et al. (2007) Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures. Blood 109: 2679–87. Kumaravelu P, Hook L, Morrison AM et al. (2002) Quantitative developmental anatomy of definitive haematopoietic stem cells/long-term repopulating units (HSC/RUs): role of the aorta–gonad–mesonephros (AGM) region and the yolk sac in colonization of the mouse embryonic liver. Development 129: 4891–9. Ledran MH, Krassowska A, Armstrong L et al. (2008) Efficient haematopoietic differentiation of human embryonic stem cells on stromal cells derived from haematopoietic niches. Cell Stem Cell 3: 85–98. Lo Celso C, Fleming HE, Wu JW et al. (2009) Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche. Nature 457: 92–6. Lux CT, Yoshimoto M, McGrath K et al. (2008) All primitive and definitive hematopoietic progenitor cells emerging before E10 in the mouse embryo are products of the yolk sac. Blood 111: 3435–8. Medvinsky A, Dzierzak E (1996) Definitive hematopoiesis is autonomously initiated by the AGM region. Cell 86: 897– 906. Mendes SC, Robin C, Dzierzak E (2005) Mesenchymal progenitor cells localize within haematopoietic sites throughout ontogeny. Development 132: 1127–36. Metcalf D (1984) The Hemopoietic Colony Stimulating Factors. Elsevier Science Publishers, Amsterdam. Minasi MG, Riminucci M, De Angelis L et al. (2002) The mesoangioblast: a multipotent, self-renewing cell that originates from the dorsal aorta and differentiates into most mesodermal tissues. Development 129: 2773–83.

Stem cells and haemopoiesis

Moore KA, Ema H, Lemischka IR (1997) In vitro maintenance of highly purified, transplantable haematopoietic stem cells. Blood 89: 4337–47. Morrison SJ (ed.) (2002) The Purification of Mouse Haematopoietic Stem Cells at Sequential Stages of Maturation. Humana Press, Totowa, NJ. North TE, Goessling W, Peeters M et al. (2009) Haematopoietic stem cell development is dependent on blood flow. Cell 137: 736–48. Oostendorp RA, Harvey KN, Kusadasi N et al. (2002) Stromal cell lines from mouse aorta–gonads–mesonephros subregions are potent supporters of haematopoietic stem cell activity. Blood 99: 1183–9. Orkin SH, Zon LI (2008) Hematopoiesis: an evolving paradigm for stem cell biology. Cell 132: 631–44. Ottersbach K, Dzierzak E (2005) The murine placenta contains haematopoietic stem cells within the vascular labyrinth region. Developmental Cell 8: 377–87. Ottersbach K, Smith A, Wood A et al. (2009) Ontogeny of haematopoiesis: recent advances and open questions. British Journal of Haematology 148: 343–55. Peeters M, Ottersbach K, Bollerot K et al. (2009) Ventral embryonic tissues and Hedgehog proteins induce early AGM haematopoietic stem cell development. Development 136: 2613–21. Rhodes KE, Gekas C, Wang Y et al. (2008) The emergence of haematopoietic stem cells is initiated in the placental vasculature in the absence of circulation. Cell Stem Cell 2: 252–63. Robin C, Bollerot K, Mendes S et al. (2009) Human placenta is a potent hematopoietic niche containing hematopoietic stem and progenitor cells throughout development. Cell Stem Cell 5: 385–95. Samokhvalov IM, Samokhvalova NI, Nishikawa S (2007) Cell tracing shows the contribution of the yolk sac to adult haematopoiesis. Nature 446: 1056–61. Spooncer E, Boettiger D, Dexter TM (1984) Continuous in vitro generation of multipotential stem cell clones from src-infected cultures. Nature 310: 228–30. Szilvassy SJ, Humphries RK, Lansdorp PM et al. (1990) Quantitative assay for totipotent reconstituting haematopoietic stem cells by a competitive repopulation strategy. Proceedings of the National Academy of Sciences USA 87: 8736–40. Taichman RS, Reilly MJ, Emerson SG (2000) The hematopoietic microenvironment: osteoblasts and the hematopoietic microenvironment. Hematology (Amsterdam, Netherlands) 4: 421–6. Takahashi K, Okita K, Nakagawa M, Yamanaka S (2007) Induction of pluripotent stem cells from fibroblast cultures. Nature Protocols 2: 3081–9.

Taoudi S, Medvinsky A (2007) Functional identification of the haematopoietic stem cell niche in the ventral domain of the embryonic dorsal aorta. Proceedings of the National Academy of Sciences USA 104: 9399–403. Tavian M, Peault B (2005) Embryonic development of the human haematopoietic system. International Journal of Developmental Biology 49: 243–50. Tavian M, Zheng B, Oberlin E et al. (2005) The vascular wall as a source of stem cells. Annals of the New York Academy of Sciences 1044: 41–50. Till JE, McCulloch EA (1961) A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiation Research 14: 213–22. Trevisan M, Yan XQ, Iscove NN (1996) Cycle initiation and colony formation in culture by murine marrow cells with long-term reconstituting potential in vivo. Blood 88: 4149–58. Vaziri H, Dragowska W, Allsopp RC et al. (1994) Evidence for a mitotic clock in human haematopoietic stem cells: loss of telomeric DNA with age. Proceedings of the National Academy of Sciences USA 91: 9857–60. Wilkinson RN, Pouget C, Gering M et al. (2009) Hedgehog and Bmp polarize haematopoietic stem cell emergence in the zebrafish dorsal aorta. Developmental Cell 16: 909–16. Wilson A, Trumpp A (2006) Bone-marrow haematopoieticstem-cell niches. Nature Reviews Immunology 6: 93–106. Wilson A, Laurenti E, Oser G et al. (2008) Haematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell 135: 1118–29. Xie Y, Yin T, Wiegraebe W et al. (2009) Detection of functional haematopoietic stem cell niche using real-time imaging. Nature 457: 97–101. Yokomizo T, Dzierzak E (2010) 3 dimensional cartography of hematopoietic clusters in the vasculature of whole mouse embryos. Development, in press. Zambidis ET, Oberlin E, Tavian M et al. (2006) Blood-forming endothelium in human ontogeny: lessons from in utero development and embryonic stem cell culture. Trends in Cardiovascular Medicine 16: 95–101. Zeigler BM, Sugiyama D, Chen M et al. (2006) The allantois and chorion, when isolated before circulation or chorio-allantoic fusion, have haematopoietic potential. Development 133: 4183–92. Zhang Y, Li C, Jiang X et al. (2004) Human placenta-derived mesenchymal progenitor cells support culture expansion of long-term culture-initiating cells from cord blood CD34+ cells. Experimental Hematology 32: 657–64.

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CHAPTER 2

2

Erythropoiesis Douglas R Higgs and William G Wood Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK

Introduction, 12 The origins of blood during development, 12 Differentiation of HSCs to form erythroid progenitors, 13 The transcription factor programme underlying erythropoiesis, 15 Terminal maturation of committed erythroid cells, 16

Changes in the cell-surface phenotype that accompany erythroid differentiation and maturation, 17 Changes in gene expression in erythroid differentiation and maturation, 18 The regulation of erythropoiesis by signalling pathways, 19 Sensing hypoxia, 19

Introduction The process of erythropoiesis includes all steps of haemopoiesis, starting with the initial specification of haemopoietic stem cells (HSCs) from mesoderm during embryogenesis. HSCs either undergo self-renewal or, through the process of lineage specification, differentiate and proliferate to form committed erythroid progenitors. Finally, they undergo terminal differentiation through a series of erythroblastic maturation stages to develop into red blood cells. In a normal adult, the numbers of circulating red blood cells and their precursors remain more or less constant with a balance between the continuous loss of mature cells by senescence and new red cell production in the marrow. There also needs to be adequate reserves to cope rapidly with increased demand as a result of physiological or pathological circumstances. This balance is maintained by an oxygen-sensing system that is affected by the red cell mass and responds via the production of erythropoietin (Epo), which in turn controls red cell production by binding and signalling to committed erythroid progenitors. Many other cytokines, growth factors and hormones also influence erythroid proliferation, differentiation and maturation. Over the past 20 years, key transcription factors controlling the internal programmes of erythroid progenitors have been identified and some insights into their roles in lineage specification and erythroid differentiation have been discovered. Understanding the basic biology of erythropoiesis provides a

Postgraduate Haematology: 6th edition. Edited by A. Victor Hoffbrand, Daniel Catovsky, Edward G.D. Tuddenham, Anthony R. Green © 2011 Blackwell Publishing Ltd.

12

Erythropoietin and the erythropoietin receptor, 20 Other signalling pathways, 22 Apoptosis during normal erythropoiesis, 22 Erythropoiesis in clinical practice, 23 Selected bibliography, 24

logical basis for the diagnosis and treatment of the inherited and acquired anaemias that are so frequently encountered in clinical practice.

The origins of blood during development Primitive haemopoiesis in humans (predominantly erythropoiesis) first appears in the blood islands of the extraembryonic yolk sac at around day 21 of gestation. About 1 week later (days 28–40), definitive HSCs emerge from the aorta–gonad– mesonephros (AGM) region, within the ventral wall of the dorsal aorta and are also found in the vitelline and umbilical arteries and the placenta. Both primitive (embryonic) and definitive (fetal/adult) HSCs arise in close association with endothelial cells. Several lines of evidence now suggest that haemopoietic and endothelial cells may emerge from a common progenitor, the haemangioblast, giving rise to both blood cells and blood vessels (see Chapter 1). At about 30–40 days, definitive haemopoiesis starts to occur in the fetal liver and definitive erythroid cells are released into the circulation at about 60 days. By 10–12 weeks, haemopoiesis starts to migrate to the bone marrow, where eventually erythropoiesis is established during the last 3 months of fetal life (Figure 2.1). Primitive and definitive erythropoietic cells are distinguished by their cellular morphology, cell-surface markers, cytokine responsiveness, growth kinetics, transcription factor programmes and more general patterns of gene expression. In particular, the types of haemoglobin produced are quite distinct in embryonic (Hb Gower I ζ2ε2, Gower II α2ε2 and Hb Portland ζ2γ2), fetal (HbF α2γ2) and adult (HbA α2β2 and HbA2 α2δ2) erythroid cells. These specific patterns of globin expression have provided critical markers for identifying the developmental

Erythropoiesis

Fetal liver (30 days)

Embryo (20 days) Ectoderm

Bone marrow (70 days)

Yolk sac

Primitive red cells Blood islands ζ2γ2 α2ε2 ζ2ε2

Ma Dorsal aortae

AGM Liver

α2γ2 α2β2 α2δ2

Neural tube A

Somite Dorsal aorta

Endothelium Developing haematopoietic cells

Hindgut

Mesonephros Genital ridge/ gonad

Figure 2.1 An outline of the origin and development of erythropoiesis during embryogenesis. Although both primitive (blood islands) and definitive (AGM, liver and bone marrow) haemopoiesis are derived from mesoderm, probably via a haemangioblast, the true origin of these early cells is not yet clear. The figure shows the formation of embryonic blood islands in the extraembryonic yolk sac and the formation of definitive haemopoiesis initially in the AGM region, with subsequent migration to the liver and bone marrow. ‘A’ denotes a magnified

image of the early embryonic aortic region. Ma denotes a macrophage. The specific types of haemoglobin formed at each stage of erythropoiesis are indicated. The approximate times at which CD34+ cells first appear at each site are given in days of gestation. (Adapted from Dzierzak E, Medvinsky A, de Bruijn M (1998) Qualitative and quantitative aspects of haematopoietic cell development in the mammalian embryo. Immunology Today 19: 228–36 with permission.)

stages of erythropoiesis. Nevertheless, it is still not clear whether primitive and definitive haemopoiesis in mammals have entirely separate origins or if they are both derived from common stem cells that arise during early development. Accurately defining the embryological origins of these cells continues to be of considerable importance for understanding the normal mechanisms that establish and maintain HSCs and how these programmes are subverted in common haematological disorders.

ulocytes). To prevent depletion of the haemopoietic cells requires a system that not only maintains a self-renewing stem cell pool, but also has the potential to differentiate into all types of highly specialized mature blood cells through a process referred to as lineage specification. At present, the mechanisms underlying self-renewal and the early events committing multipotential HSCs to an increasingly restricted repertoire of lineage(s) are not fully understood. The probability of commitment to any particular lineage may be influenced by a complex interplay between the internal transcriptional programmes and epigenetic patterns (e.g. changes in nuclear position, replication timing, chromatin modification, DNA methylation) with external signals from the microenvironment (e.g. cytokines, growth factors and cell–cell interactions) acting via signal transduction pathways. Microarray analyses of HSCs and their progeny consistently show a very wide range of gene expression in the earliest cell populations. Furthermore, many of the genes that are specific to individual lineages (e.g. erythroid, myeloid or lymphoid) are already transcribed, albeit at low levels, in HSCs. In other

Differentiation of HSCs to form erythroid progenitors At all stages of development there is a continuous need to renew senescent blood cells that are ultimately lost from the peripheral blood days, weeks or months after undergoing terminal differentiation. For example, throughout adult life approximately 1011 senescent red cells must be replaced every day, and there are similar requirements for other mature blood cells (e.g. gran-

13

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In human adult bone marrow, approximately 1 per 104–106 nucleated cells are long-lived, multipotential HSCs that can be enriched on the basis of their cell-surface markers (e.g. CD33+ and CD34+ and lack of lineage-specific markers; see Figures 2.2 and 2.3), but such markers do not exclusively select stem cells

words, HSCs appear to show ‘multilineage priming’ and, as their progeny become committed to one pathway of differentiation, that lineage-specific gene expression programme becomes reinforced, whereas those of other lineages are suppressed.

Lymphoid lineages

Self-renewal

Other myeloid lineages HSC

CFU-GEMM

BFU-E

CFU-E

SCL/tal-1 Runx1 Key Tel transcription LMO2 factors MLL GATA-2 Bmi-1

GATA-1 FOG-1 Gfi-1b

Figure 2.2 Summary of some steps in self-renewal, lineage specification and differentiation of haemopoietic stem cells to red cells. Some of the key transcription factors involved in this process are summarized beneath the diagram.

NF-E2 EKLF

188 h 60 h 1

30 h 2

4

50 h 8

48 h

16

Erythron Progenitors HSC CFU-GEMM BFU-E Early

BFU-E Late

Precursors Pro Bas

50–60

Frequency per 104 nucleated bm cells

~1

~1

4–10

20–60

% cells in cycle

Low

15–20

30–40

60–70

CD34 CD71 (TfR) EpoR GPA Globin mRNA Hb

++ ± – – – –

+++ ± – – – –

++ ±

± ++ ++ + – –

– – –

±

± – –

Figure 2.3 The specification and terminal differentiation of erythroid cells from haemopoietic stem cells. At the top, the estimated times for maturation of terminally differentiating cells are shown. The precursors are as follows: pronormoblasts (Pro), basophilic erythroblasts (Bas), polychromatic erythroblasts (Pol), 14

CFU-E

400

60–70 – +++ ++ ++++ + ±

– +++ ++ ++++ + +

Early Pol

Late Ort

500

1000

80

0

– +++++ ± ++++ +++ +++

– ++ ± ++++ +++++ +++++

Retic

RBC

+ – ++++ ++ +++++

orthochromatic erythroblasts (Ort), reticulocytes (Retic), mature red blood cells (RBCs). The number of divisions from pronormoblasts to orthochromatic normoblasts (1–16) are also shown. Some examples of the expression patterns of key cell-surface markers are shown below.

Erythropoiesis

(see Chapter 1). The only rigorous assay for bona fide HSCs is to measure their ability to contribute, throughout life, to all haemopoietic lineages in vivo. This has been amply demonstrated in mice, and the repeated, predictable success of human bone marrow transplantation clearly demonstrates the existence of such cells in humans. The pathway of differentiation from HSCs to committed erythroid progenitors is still the topic of some debate. One model (proposed by Weissman) posits a common myeloid progenitor from which the granulocyte/monocyte, erythroid and megakaryocyte lineages develop. In a second model (Jacobsen) erythroid/megakaryocytic progenitors split before the separation of lymphoid and granulocyte/monocyte lineages. As stem cells differentiate, they form multipotential progenitor cells that have short-term repopulating ability but have lost long-term repopulating ability. Such cells can be assayed in vitro by their ability to form ‘cobblestone’ areas under stromal cells in longterm marrow cultures. Further differentiation progressively restricts the lineage potential of these cells as well as reducing their proliferative capacity, resulting in tripotential, bipotential and unipotential progenitors. These progenitor cells are functionally defined by their ability to produce clonal colonies in semisolid medium supplemented with a cocktail of haemopoietic cell growth factors permissive for the growth of all lineages. Erythroid cells can be found in multilineage colonies (CFUGEMM), which include granulocytes, macrophages and megakaryocytes, and in bipotential colonies with megakaryocytes (CFU-E/Mk). The earliest progenitors that are restricted to the erythroid lineage produce large colonies in vitro, consisting of several subunits, known as erythroid bursts (BFU-E, containing from several hundred up to 30 000 cells) after 12–14 days of growth. Their frequency in bone marrow is approximately 4–10 per 104 nucleated cells. Late erythroid progenitors form colonies (CFU-E) of 8–64 cells after about 7 days in vitro and constitute 20–60 per 104 bone marrow cells. CFU-Es defined in these culture systems most closely correspond in vivo to pronormoblasts (also known as proerythroblasts), the earliest morphologically recognizable erythroid precursor in the bone marrow. Once formed, these cells are destined to undergo terminal differentiation to form mature red cells, as discussed later. Erythroid differentiation and maturation within the adult bone marrow in vivo is dependent on the microenvironment provided by the stromal cells (fibroblasts, fat cells, endothelial cells, macrophages and smooth muscle cells). There are also immunoregulatory cells (monocytes, macrophages and lymphocytes) that contribute to local cytokine production. Erythroblasts are not randomly distributed in the bone marrow but are organized into erythroblastic islands containing one or two central macrophages, surrounded by layers of erythroblasts at different stages of maturation (Figure 2.1). A number of techniques have been described for the production of erythroblasts in liquid cultures. The great advantage of

these techniques is that they allow the production of large numbers of erythroblasts from peripheral blood samples, enabling functional analyses of normal or abnormal erythropoiesis without the need for bone marrow sampling.

The transcription factor programme underlying erythropoiesis As discussed above in the stochastic model of cell differentiation, many factors must be integrated for a cell to make the decision to undergo self-renewal or differentiation, become quiescent, proliferate or undergo apoptosis. Over the past few years, it has emerged that key transcription factors play a major role in regulating the formation, survival, proliferation and differentiation of multipotent stem cells as they undergo the transition to erythroid cells. These transcription factors may operate on their own or as members of multicomponent complexes involved in activation and/or repression. Many of the key transcription factors were originally identified because they are associated with chromosomal translocations found in leukaemia. This supports a model in which dysregulation of the normal transcriptional programme plays a causal role in haematological malignancies. At present, the key transcription factors known to be involved in specifying HSCs as they develop during embryogenesis and in maintaining them throughout life include Runx1 (AML-1), SCL (tal-1), LMO2 (rhombotin), Tel (ETV6), MLL and GATA2 (see Figure 2.2). In addition, the homeobox (Hox) genes and proteins that modify their expression (e.g. Bmi-1) have also been shown to play a role in haemopoiesis. Many of these factors (e.g. SCL, Runx1) appear to act quite differently in primitive as opposed to definitive haemopoiesis. Furthermore, not only is their importance in early definitive progenitors well established, but also many of these transcription factors play additional roles, later in differentiation, in specific haemopoietic lineages, including erythropoiesis. Once progenitor cells have been committed to become erythroid cells, the most important transcription factors that enable them to proceed through terminal differentiation are GATA-1 and its cofactor FOG-1 (friend of GATA-1). GATA-1 was first identified by its ability to bind functionally important regulatory sequences in the globin genes. Since then, GATA-binding motifs have been found in the promoters and/or enhancers of virtually all erythroid-specific genes studied, including haem biosynthetic enzymes, red cell membrane proteins (including blood group antigens) and erythroid transcription factors such as erythroid Kruppel-like factor (EKLF) and GATA-1 itself. GATA-1 expression is restricted to erythroid, megakaryocytic, eosinophilic, mast cell and multipotential progenitors of the haemopoietic system. However, GATA-1 expression is highly upregulated in pronormoblasts and basophilic erythroblasts (see Figures 2.2 and 2.3). 15

Postgraduate Haematology

Gene targeting studies in mice have shown that GATA-1 is essential for normal erythropoiesis. Mice that produce no GATA-1 die from severe anaemia. Although they produce adequate numbers of erythroid colonies (CFU-E), there is an arrest in erythroid maturation at the pronormoblast stage of differentiation. In vitro differentiated mouse embryonic stem cells lacking GATA-1 also fail to mature past the pronormoblast stage and undergo rapid apoptosis, indicating a role for GATA1 in survival and maturation of erythroblasts. GATA-1 may protect mature erythroblasts from apoptosis by directly or indirectly inducing expression of the anti-apoptotic protein Bcl-XL. GATA-1 almost certainly regulates gene expression working as part of multiprotein complexes interacting, for example, with FOG-1, LMO2, SCL and a variety of ubiquitously expressed transcription factors. FOG-1 is a protein containing multiple zinc fingers, four of which interact with GATA-1. Like GATA-1, FOG-1 is expressed in erythroid and megakaryocytic cells and is coexpressed and directly interacts with GATA-1 during development. Genetically modified mice that express no FOG-1 also die in mid-gestation as a result of severe anaemia with arrest in erythroid maturation at the pronormoblast stage. GATA-2 is a second member of the GATA family of proteins that is involved in haemopoiesis. Both GATA-1 and GATA-2 are particularly relevant for erythropoiesis. Both are expressed in multipotent progenitors, although GATA-2 appears to be more important than GATA-1 at this stage, when GATA-2 plays an important role in the expansion and maintenance of haemopoietic progenitors. During erythroid differentiation the level of GATA-2 declines as GATA-1 increases. In mouse embryos lacking GATA-2, erythrocytes are present, but in severely reduced numbers. There appears to be some overlap and redundancy between the roles of GATA-1 and GATA-2; in the absence of GATA-1 increased levels of GATA-2 may fulfil some, but not all, of the normal roles of GATA-1. Furthermore, there is evidence that the level of GATA-2 is regulated by the level of GATA-1. During normal erythroid development, it appears that GATA-2 may initiate the erythroid programme to be replaced later by GATA-1 during terminal erythroid maturation. Expression of the two related zinc-finger DNA-binding proteins Gfi-1 and Gfi-1b is restricted to haemopoietic cells. Gfi-1b is expressed only in multipotent progenitors, megakaryocytes and erythroblasts, in which its pattern of expression mimics that of GATA-1. Gfi-1b-deficient mouse embryos die with a failure to produce mature red cells, although early precursors are formed normally. This is very similar to the phenotype observed in GATA-1-deficient embryos. However, from in vitro colony assays in which Gfi-1b is overexpressed, it appears that its effect is not on erythroid commitment, but rather it promotes the proliferation of erythroid progenitors as they undergo the transition from late BFU-Es to CFU-Es. EKLF is a zinc finger-like protein of the Kruppel family, which binds the consensus sequence 5′-NCNCNCCCN-3′ and 16

is mainly restricted to erythroid cells. These binding sites are found in the regulatory elements of several erythroid-specific genes, including the β-globin gene. Disruption of binding at this site gives rise to β thalassaemia. Mice in which EKLF is absent die from severe anaemia at the fetal liver stage caused in part by β thalassaemia, but also due to the failure to synthesize correctly other EKLF-regulated proteins (e.g. the red cell membrane protein Band3 and the α-globin stabilizing protein AHSP) required for red cell maturation. Therefore, EKLF may play a wider role than originally predicted in coordinating erythroid cell maturation and globin gene regulation. Sequence motifs of the general class (T/C)GCTGA(G/C) TCA(T/C), called Maf recognition elements (MAREs), have been found in the enhancers of many erythroid-specific genes (e.g. globins, haem synthesis enzymes), and it was shown that they bind the transcription factor NF-E2. Purification of NF-E2 revealed that it consists of two subunits, p45NF-E2 and p18NFE2 (now known as MafK). Both proteins contain basic zipper (B-ZIP) domains through which they form heterodimers and bind DNA. p45 is expressed mainly in erythroid cells, whereas p18 is widely expressed, although it is the predominant small Maf family member in erythroid cells. Furthermore, it is now known that both p45 and p18 are members of larger groups of proteins with overlapping functions. Other p45-like molecules include Nrf1, 2 and 3 and Bach1 and 2. All these proteins bind as obligate heterodimers with Maf proteins. It seems likely that binding to MARE elements is an important aspect of erythroidspecific activation, but it is not clear which proteins in this family bind the key sites or whether there is redundancy in the need for specific members of this family. At present it is not fully understood how these transcription factors combine to commit cells to the erythroid lineage and terminal erythroid differentiation. However, this could involve the presence or absence of specific transcription factors, changes in the levels of the proteins and/or protein modification. One principle that seems to be emerging is that factors affiliated with different lineages such as GATA-1 (erythroid) and PU1 (lymphocytes and granulocytes) are both present in uncommitted progenitors, reflecting the potential to develop along alternative different pathways (so-called multilineage priming). It is now known that GATA-1 and PU1 interact and cross-antagonize each other. Therefore, as cells differentiate, reinforcement of the transcriptional programme of one lineage may actively suppress an alternative lineage.

Terminal maturation of committed erythroid cells After the erythroid programme has been specified, the final phase of erythropoiesis involves the maturation of committed erythroid progenitors to fully differentiated red cells. The earliest recognizable erythroid precursor cell in the bone marrow is

Erythropoiesis

(i)

(ii)

(iii)

(iv)

(a) Orthochromatic (Late)

Polychromatic (Intermediate)

GPA

Pronormoblasts (early)

CD71 (TfR)

(b)

Figure 2.4 (a) Examples of pronormoblasts (i), basophilic and polychromatic erythroblasts (ii) and polychromatic and orthochromatic erythroblasts (iii and iv). All these different cell types can also be conveniently viewed at http://hsc.virginia.edu/ medicine/clinical/pathology/educ/innes/text/nh/mature.html. (b) An example of early (pronormoblasts), intermediate (polychromatic erythroblasts) and late (orthochromatic erythroblasts) erythroid precursors separated on the basis of their cell-surface markers (CD71 and GPA).

the pronormoblast (Figure 2.4a, i) which, as discussed above, corresponds to the CFU-Es identified in vitro. The pronormoblast is a relatively large cell (12–20 µm) with a non-granular deep-blue cytoplasm and a large nucleus occupying about three-quarters of the cell that has a finely stippled chromatin pattern containing one or more prominent nucleoli. The cytoplasm contains numerous ribosomes, several mitochondria, centrioles, a prominent Golgi apparatus and a few strands of rough endoplasmic reticulum. Division of these

cells leads to smaller (10–16 µm) basophilic normoblasts (Figure 2.4a, ii). Again, the cytoplasm stains deep blue and the nucleus occupies a large proportion of the cell but has a coarser reticular chromatin pattern with a few small masses of condensed chromatin adjacent to the nuclear membrane. Further divisions form early polychromatic and late polychromatic normoblasts (10–12 µm), with increasing development of a pink cytoplasm and condensed (6 µm) nuclei (Figure 2.4a, iii). Late polychromatic/orthochromatic normoblasts (Figure 2.4a, iv) are non-dividing cells with deeply staining structureless nuclei. As the cell proceeds through terminal differentiation, nucleoli disappear and the nucleus condenses further and is eventually extruded. Such nuclei are phagocytosed and degraded by the macrophages of the bone marrow. Ultrastructural studies have shown that nuclear extrusion usually occurs outside the sinusoids of the bone marrow, and that newly formed reticulocytes usually pass through pre-existing gaps in the walls of these sinusoids by diapedesis. Thus, the mature reticulocyte has no nucleus but has a few mitochondria and ribosomes and its cytoplasm stains predominantly pink because of the high concentration of haemoglobin. The cytoplasm still has a greyish tint due to the presence of ribosomes. When stained supravitally, the ribosomes precipitate into basophilic granules or a reticulum. Reticulocytes continue to synthesize haemoglobin for 24–48 hours after release from the bone marrow. On average, these cells are about 20% larger than mature red cells, which are circular, flat, biconcave discs with a mean diameter of 8.5 µm. It has been estimated that, on average, four divisions occur within the morphologically recognizable proliferating precursor pool, so that each newly formed pronormoblast develops into 16 red cells (see Figure 2.3). As a small amount of cell death (ineffective erythropoiesis) normally occurs, the average amplification is slightly less than 16-fold. The majority (60–80%) of pronormoblasts, basophilic normoblasts and early polychromatic normoblasts are in cell cycle, mostly in S phase, with G1 and G2 stages lasting only a few hours. At any time, about 3% pronormoblasts, 5% basophilic erythroblasts and 6% polychromatic normoblasts are undergoing mitosis, which has been variously estimated to last for between 60 and 100 min. Erythroid cells eventually exit the cell cycle and, consistent with this, late polychromatic/orthochromatic erythroblasts are post-mitotic, non-dividing cells.

Changes in the cell-surface phenotype that accompany erythroid differentiation and maturation The cell-surface phenotypes of erythroid progenitors and precursors are quite distinctive, reflecting the different signalling programmes of the cells as they differentiate. These markers are 17

Postgraduate Haematology

also of value in the analysis of erythroid progenitors and precursors as they can be used to identify and purify subpopulations of cells. CD34 is present on nearly all multipotent progenitors and committed BFU-Es but is lost on later erythroid progenitors (CFU-E) and all precursors. A similar pattern of expression is shown for the receptor c-Kit. Epo receptor (see below) first appears in small numbers (20–50 copies per cell) on late BFU-Es, increases in CFU-Es and pronormoblasts (∼1000 copies per cell) and subsequently declines and disappears in later erythroid precursors. CD71 (transferrin receptor, TfR) allows transferrin-bound iron to be taken into the cell and is present on early haemopoietic cells but is considerably upregulated on cells that are actively synthesizing haemoglobin, reaching a peak of 800 000 molecules per cell on polychromatic normoblasts. CD71 levels diminish in the late phase of terminal differentiation and the receptor is not detectable on mature erythrocytes. Glycophorin A (GPA) is a membrane sialoglycoprotein whose expression is highly upregulated as erythroid progenitors mature from pronormoblasts. Combinations of these cell-surface markers can be used to distinguish early, intermediate and late erythroid precursors (Figure 2.4b). Developing erythroid cells express cell-surface adhesion molecules that interact with the extracellular matrix; these include ICAM-1 (a member of the immunoglobulin superfamily) and integrin α4β1 VLA4 (CD29/CD49d), which interacts with fibronectin. These adhesion molecules are most highly expressed in the early precursors and lost as maturation proceeds, freeing erythroid cells to exit the bone marrow.

Iron storage

Transferrin receptor

Changes in gene expression in erythroid differentiation and maturation As cells go through the final divisions of erythropoiesis and post-mitotic maturation there is progressive condensation of chromatin accompanied by complex changes in gene expression. When assessed by microarray analysis, many mRNAs are downregulated as multipotent progenitors enter terminal differentiation, reflecting the commitment of multipotent cells to a single specialized lineage. A subset of general mRNAs associated with proliferation, replication and cell cycle control show alterations as the growth characteristics of the cells change. mRNAs encoding proteins that characterize the red cell phenotype are, in general, upregulated. Examples include blood group antigens, red cell membrane proteins (e.g. spectrin, ankyrin, actin, protein 4.1), red cell glycolytic pathway enzymes, carbonic anhydrase and enzymes of the haem synthesis pathway such as δ-aminolaevulinic acid synthase (ALAS). A full catalogue of these changes in gene expression can be found at http:// hembase.niddk.nih.gov. The main purpose of erythropoiesis is to synthesize large amounts of haemoglobin (Figures 2.3–2.5). Globin mRNA sequences are first expressed in pronormoblasts and early basophilic erythroblasts. Globin chain synthesis parallels accumulation of globin mRNA, increasing at the polychromatic and orthochromatic stages. The amount of globin mRNA reaches 20 000 molecules per cell in late polychromatic and

IRE/IRP

Haemoglobin α-Globin

IRE/IRP

HRI

Fe2+

IRE/IRP

β-Globin Haem

HRI

Haem

Succinyl CoA Glycine

Ferrochelatase Protoporphyrin

ALA synthase δ-Aminolaevulinic acid

Protoporphyrinogen Coproporphyrinogen

δ-Aminolaevulinic acid

PBG deaminase

Porphobilinogen

Figure 2.5 Coordination of globin synthesis, haem synthesis and iron regulation. Blue lines indicate some of the known regulatory feedback systems. The red shaded box indicates reactions

18

Coproporphyrinogen

Uroporphyrinogen

occurring in the mitochondria. Rate-limiting controls of haem synthesis are shown in black boxes. ALA, δ-aminolaevulinic acid; PBG, porphobilinogen.

Erythropoiesis

orthochromatic erythroblasts. During the later stages of erythroid cell maturation, the amount of RNA per cell and the rate of total protein synthesis declines, but the relative stability of globin mRNA ensures that globin becomes the predominant polypeptide made in late erythroblasts and reticulocytes. The individual components of the haemoglobin synthetic pathway (iron, free porphyrins, haem and monomeric globin chains) are all extremely toxic to the cell, and consequently many positive and negative feedback loops have evolved and been incorporated into this process. The synthesis of globin must be very accurately matched with the synthesis of haem in which some steps occur in the cytoplasm and others in the mitochondria (Figure 2.5). mRNAs encoding many components of the haem biosynthetic pathway (e.g. ALAS and porphobilinogen deaminase) are coordinately upregulated in terminal erythroid differentiation and their genes contain similar cis-regulatory elements. Continued translation of globin chains from mRNA only occurs in the presence of adequate haem. Reduced levels of haem rapidly trigger the formation of the haem-regulated inhibitor (HRI), a kinase that interacts with the translation initiating factor eIF-2α and prevents translation of α- and βglobin mRNA. The synthesis of haem itself is also regulated at many points and is particularly sensitive to the levels of available iron. Via a well-characterized pathway involving the ironregulatory proteins (IRP1 and IRP2), binding to iron response elements (IREs) in the mRNA transcripts of ferritin, TfR and ALAS, the level of intracellular iron thus controls the translation of RNAs involved in iron storage, iron transport and haem synthesis (see Chapter 3). The discovery of hepcidin, which controls the uptake of iron from the gut, iron transport across the placenta and iron release from macrophages, adds another level of control to this complex system (see Chapter 3). Not surprisingly, diseases affecting the supply of iron (iron deficiency and the anaemia of chronic disease), the synthesis of haem (sideroblastic anaemia, lead poisoning, alcohol ingestion) or the synthesis of globin (thalassaemia) have interrelated effects on globin synthesis, haem synthesis and iron metabolism (Figure 2.5). No mechanistic connection between haemoglobin synthesis and erythroid proliferation or differentiation has yet been established. However, it has been postulated that haemoglobin content and/or haemoglobin concentration per se may be a negative regulator of cell division. When haemoglobin synthesis is reduced or delayed, as in iron deficiency, the cells may undergo an extra division, yielding smaller hypochromic cells. Alternatively, when haemoglobin synthesis exceeds DNA synthesis, as in megaloblastic anaemias, the cells may skip a division and nuclear extrusion may occur early, resulting in macrocytosis. Although plausible, these hypotheses remain unproven.

The regulation of erythropoiesis by signalling pathways The normal red cell lifespan is 120 days and therefore, to maintain equilibrium, approximately 1% of the circulating red cell pool must be replaced daily. For a total of about 3 × 1013 circulating erythrocytes and a lifespan of 120 days, the erythrocyte production rate needs to be maintained at approximately 1010/ hour in the steady state. Erythropoiesis accounts for about 20% of the nucleated cells in a normal bone marrow reflected in the myeloid/erythroid ratio (usually about 4 : 1). As committed erythroid cells become late BFU-E and CFU-E, they upregulate expression of the receptor for erythropoietin (EpoR). It is estimated that in the steady state, with low levels of circulating Epo, a high proportion of erythroid cells die through apoptosis. This provides a reserve that can be rescued by the increase in Epo levels that accompany anaemia. Signalling through EpoR not only prevents apoptosis but also stimulates proliferation. It is at the late progenitor/early precursor stages (CFU-E/pronormoblast) that there is considerable proliferative potential for expanding the overall level of erythropoiesis. Soon after reaching the CFU-E stage, erythroid cells enter the phase of terminal differentiation, after which there is only limited potential for further expansion. The two major components regulating erythropoiesis include sensing hypoxia and regulating the supply of erythroid precursors, mainly by controlling the numbers of erythroid progenitors via the Epo–EpoR signalling pathway.

Sensing hypoxia Tissue hypoxia induces a variety of physiological responses in addition to activation of the Epo–EpoR pathway (see below). Parallel responses include the stimulation of new blood vessels by vascular endothelial growth factor (VEGF) and metabolic changes (e.g. in glycolytic pathway enzymes) that enable continued energy production despite inadequate oxygen availability. In addition, expression of TfR is upregulated. Over the past 20 years the mechanisms by which cells sense hypoxia and orchestrate their response have been discovered. It has been shown that the most important mediator of this cellular response is a transcription factor called HIF (hypoxia-inducible factor), which activates the genes that influence the adaptive responses to hypoxia including those encoding Epo, glycolytic pathway enzymes, TfR and VEGF (Figure 2.6). HIF is a heterodimer constituting one of three α-subunits (HIF1-α, HIF2-α or HIF3-α) bound to the aryl hydrocarbon receptor nuclear translocator (ARNT), also known as HIF1-β. HIF1-α is a member of the basic helix–loop–helix (bHLH) family of transcription factors in which the HLH domains mediate subunit dimerization, whereas the basic domains bind

19

Postgraduate Haematology

Hypoxia

High O2 Ub

Prolyl hydroxylase oxygen sensor

Ub

vHL HIF1-α HIF1-β

EpoR Erythropoiesis

Ub

OH

VEGF Angiogenesis

Glycolytic enzymes Supply of energy

Oxygenation

Ub Ub

HIF1-α

TfR Supply of iron

Ub

Degradation via proteasome

Figure 2.6 The oxygen-sensing system. Ub, ubiquitination; vHL, von Hippel–Lindau protein.

DNA. HIF binds to hypoxia-response elements (HREs, 5′TACGTG-3′) located in the regulatory regions of hypoxiainducible genes such as the gene encoding Epo (see below). Whereas changes in oxygen levels do not affect the levels of HIF1-β, which is expressed constitutively, hypoxia elevates the levels of HIF1-α subunits by increasing protein stability. The oxygen sensor is a ferrous iron prolyl hydroxylase that requires molecular oxygen as a cosubstrate to hydroxylate specific proline residues in the α-subunits of HIF. Once hydroxylated, HIF1-α subunits become targets for ubiquitination by the widely expressed von Hippel–Lindau (vHL) protein and are thus targeted for proteosomal degradation. Under normal circumstances the α-subunits are undetectable but, when cells are exposed to hypoxic stimuli, the oxygen sensor can no longer hydroxylate the α-subunits of HIF. In this situation, α-subunits accumulate as they are no longer polyubiquitinated and degraded. This allows the α-subunits to heterodimerize with HIF1-β and activate the hypoxia-response genes. If the vHL protein is mutated (vHL syndrome), there is prolonged stimulation by HIF, leading to the development of polycythaemia (Epo stimulation) and vascular tumours (VEGF stimulation).

Erythropoietin and the erythropoietin receptor The main initial site of Epo production is the fetal liver, with production largely switching to the kidney shortly after birth. Under normoxic conditions, little or no Epo mRNA is detectable in the kidneys but hypoxia results in its accumulation within 30 min in the peritubular interstitial cells and levels can increase 200-fold over baseline. Epo is a 166-aminoacid 34.4-kDa glycoprotein, found in serum at baseline levels 20

of 1–30 mU/mL that can be elevated 1000-fold by severe anaemia. It contains about 40% carbohydrate, rich in sialic acid residues, and has a half-life of 7–8 hours in plasma, whereas non-glycosylated Epo is cleared rapidly from the circulation. There are no preformed stores of Epo and, normally, 90% of the hormone is produced in the kidney and 10% in the liver and elsewhere. The Epo gene contains a hypoxia-response element at its 3′-end, and in the kidney and liver a cooperative interaction of HIF with HNF-4, including recruitment of the coactivator CBP/p300, leads to a significant increase in transcription and increased serum levels of the protein. The binding of Epo to EpoR results in signal transduction to the nucleus, and this constitutes the most important pathway for controlling definitive erythropoiesis. Careful clinical and haematological studies, together with the analysis of experimental animal models, have been important in establishing exactly which aspects of erythropoiesis are regulated by the Epo–EpoR system. Fetal livers of genetically modified mice in which Epo or EpoR has been deleted are devoid of late erythroid cells but contain normal numbers of BFU-E, demonstrating that this signalling system is not required for lineage specification but is essential for proliferation and differentiation of erythroid precursors into mature cells. When tissue oxygenation is compromised owing to reduced ambient oxygen tension, blood loss, shortened red cell survival or any uncompensated need for increased oxygen delivery, the level of Epo rises, stimulating red cell production. The low numbers (20–50) of EpoRs on BFU-E explain the relative Epo non-responsiveness of these cells, and much higher levels (∼ 1000) are found in CFU-E, pronormoblasts and basophilic erythroblasts. When circulating Epo increases, apoptosis of

Erythropoiesis

these erythroid progenitors decreases and CFU-Es rapidly respond by proliferating and differentiating. Therefore, the most important effect of Epo is to increase the number of progenitor cells that develop into viable pronormoblasts. It has also been suggested that Epo is able to speed up the rate of terminal differentiation by shortening the cell cycle and maturation times of erythroblasts, thereby explaining the macrocytosis that often accompanies ‘stress’ erythropoiesis; this remains to be confirmed. The erythropoietin signalling system is relatively well understood. EpoR belongs to the cytokine receptor superfamily. Like other members of this family (growth hormone, prolactin and G-CSF), Epo was thought to induce dimerization of cell-surface receptors (EpoRs), triggering autophosphorylation and activation of the Janus family of protein tyrosine kinases (JAK2). More recent data suggest an alternative model in which unliganded EpoR dimers exist in a conformation that prevents activation of JAK2, but the receptor may undergo a ligand-induced conformational change that allows JAK2 to be activated. JAK2 and/or other kinases then phosphorylate specific tyrosine residues in EpoR, creating docking sites for the SH2 domains of several signal transduction proteins, which eventually results in the activation of at least three signal transduction pathways: STAT5, Ras/MAP kinase and phosphatidylinositol 3-kinase (PI3-K) (Figures 2.7 and 2.8). Considerable interest has concentrated on the JAK2–STAT5 pathway. JAK2 is essential for erythropoiesis, and genetically modified mice in which JAK2 expression has been eliminated

Dimerization or ligand-induced conformational change

P JAK2

Epo

die as embryos, with a phenotype similar to mice deficient in Epo or EpoR. However, the numbers of erythroid progenitors are more severely diminished, suggesting that JAK2 is required earlier in erythropoiesis than Epo–EpoR. JAK2 is rapidly phosphorylated in response to Epo stimulation. Dimerization or conformational changes of EpoR brings the associated JAK2 molecules into close proximity, enabling them to transphosphorylate and activate each other (Figures 2.7 and 2.8). STAT5 is phosphorylated and activated by EpoR. Phosphorylated STAT5 dissociates from the receptor, dimerizes and moves to the nucleus, where it activates gene expression and is thought to be important as an anti-apoptotic signal. Both fetal and adult mice defective in STAT5 have a defect in regulating survival of early erythroblasts, leading to a persistent anaemia. Activation of the PI3-K and Ras-MAP kinase pathway (Figure 2.8) may be sufficient for normal erythroid differentiation, although they may not be essential as other pathways can compensate for loss of signalling through PI3-K. Therefore, in erythroid cells, activation of several, apparently redundant intracellular signalling pathways can support differentiation. Nevertheless, it is thought at present that these pathways may converge by activating a few important anti-apoptotic proteins, including Bcl-2, Bcl-XL and protein kinase B (also known as Akt) (Figure 2.8). The full control of this system is complex and it should be noted that the EpoR pathway can also be activated by other mechanisms. For example, activation of c-Kit by its ligand, stem cell factor (SCF), causes tyrosine phosphorylation of EpoR and

Epo

JAK2 P

P JAK2 P P P STAT P

Epo

JAK2 P P P P STAT P

P JAK2 P P P

Epo

JAK2 P P P P

P JAK2 P P P SHP1

JAK2 P P P P SHP1

P STAT STAT P

P STAT STAT P Target gene

Figure 2.7 A summary of signalling via the erythropoietin (Epo) receptor as described in the text. P denotes regions of phosphorylation. The diagram shows Epo-induced dimerization or conformational change with transphosphorylation of JAK2,

followed by phosphorylation of the Epo receptor. This is followed by binding and phosphorylation of STAT5. Binding of SHP1 (far right) to the Epo receptor activates its phosphatase activity, which can then dephosphorylate JAK2 and terminate signalling. 21

Postgraduate Haematology

Kidney

Epo

P Pl3-K

P

Oxygenation

JAK2 P

P

STAT P

P

Ras MAPK

GATA-1

Bcl-XL Anti-apoptotic

Fas ligand

Fas

Figure 2.8 A summary of the apoptotic pathways (Epo and Fas) in erythroid progenitors. These cells (BFU-E and CFU-E) undergo apoptosis in the absence of Epo signalling or in the

presence of Fas signalling. Bcl-XL may be the key pathway through which these effects are mediated.

a functional interaction between the two receptors is essential for normal erythropoiesis. Mechanisms for switching off the Epo–EpoR signalling pathway also exist. Specific phosphorylated tyrosines that occur on the Epo-stimulated dimerized EpoR provide docking sites for the SH2 domains of protein tyrosine phosphatases such as SHP1. Binding activates the phosphatase, which removes the activating phosphates from JAK2, terminating the positive signal from this pathway (Figure 2.7).

In addition to SCF and Epo, recent observations have shown that stimulation of the nuclear hormone receptors for dexamethasone (glucocorticoid receptor) and estrogen (estrogen receptor) produces sustained proliferation of erythroid progenitors. Furthermore, the nuclear hormone receptors for thyroid hormone (c-ErbA/thyroid hormone receptor), all-trans retinoic acid (retinoic acid receptor) and 9-cis- retinoic acid (RXR) were found to promote erythroid differentiation. Such observations are consistent with previous reports showing that patients with a wide range of endocrine disorders (hypothyroidism, hypopituitarism, Addison’s disease and male hypogonadism) all have variable degrees of normochromic normocytic anaemia. It appears, therefore, that many hormones of the endocrine system can modify erythropoiesis.

Other signalling pathways Erythropoiesis is also influenced by pathways other than Epo– EpoR. Erythroid progenitors express receptors for SCF, insulinlike growth factor (IGF-1) and insulin. After Epo, the second most important signalling system for erythropoiesis involves SCF (Kit ligand) and its receptor (c-Kit). Activation by SCF induces tyrosine phosphorylation of its own receptor. SCF was originally identified by its ability to stimulate proliferation of multipotent haemopoietic progenitors, but it is also effective in supporting growth of committed progenitors, including erythroid progenitors, acting synergistically with Epo. 22

Apoptosis during normal erythropoiesis Programmed cell death (apoptosis) plays an important role in normal erythropoiesis (Figure 2.8), helping to regulate the accumulation of erythroid precursors to match the need for new mature red cells. Excess erythroid precursors are removed

Erythropoiesis

by apoptosis, and at least two pathways seem to be involved. First, it appears that late BFU-E, CFU-E and pronormoblasts may all require continuous signalling via EpoR, which is highly expressed on the surface of these cells, to prevent apoptosis. In the absence of Epo, these cells rapidly undergo programmed cell death in culture. It has been shown that, in part, this reflects a need for signals from EpoR, via the JAK2–STAT5 pathway, to induce or stabilize expression of the anti-apoptotic protein Bcl-XL. Apoptosis of erythroid precursors may also occur as a result of activation of the Fas receptor (FasR, known as CD95) that is present on both early and late erythroid precursors, although its activating ligand (FasL, known as CD95L) appears only on late erythroblasts. Binding of FasL to FasR activates proteolytic caspases that cleave intracellular proteins, possibly including the erythroid transcription factor GATA-1, with subsequent loss of Bcl-XL. This regulation of erythropoiesis by negative feedback is thought to take place in the erythropoietic islands of the bone marrow, where the number of mature erythroblasts may control the expansion and differentiation of their less mature precursors. As well as extracellular anti-apoptotic signals, erythroblasts also use internal programmes to ensure their own survival. The transcription factor GATA-1 is essential for maturation of erythroblasts; the absence of GATA-1 leads to apoptosis and a block in maturation. Some of the target genes regulated by GATA-1 are likely to be important for cell survival. Although many of the targets are unknown, it is clear that GATA-1 strongly induces expression of Bcl-XL and may therefore cooperate with EpoR signalling. In this way, Epo signalling, Fas-mediated signalling and GATA-1 converge on Bcl-XL, which represents a key target of the erythroid cell survival programme.

Erythropoiesis in clinical practice Erythropoiesis is disturbed to a greater or lesser extent in almost all multisystem diseases and so the reader is referred to other

chapters and references for specific examples. The aim of this chapter is to provide a framework for thinking about the process of erythropoiesis in clinical practice. The first stage involves the production of committed erythroid progenitors. The second involves controlling red cell production, which is mainly achieved via the oxygen sensor influencing the level of Epo, which in turn controls the numbers of late BFU-Es and CFU-Es, although many other hormones, cytokines and growth factors may modify the response. The third phase requires terminal erythroid differentiation to mature red cells containing large amounts of specific proteins such as haemoglobin. This phase makes significant demands on a variety of nutritional factors and cofactors, particularly iron, vitamin B12 and folate, but also manganese, cobalt, vitamin C, vitamin E, vitamin B6 (pyridoxine), thiamine, riboflavin, pantothenic acid and amino acids. Absolute or relative deficiencies of these cofactors can negatively regulate erythropoiesis. The output from this process (red cell mass) is required to meet the demands for adequate tissue oxygenation, which itself has a major influence on the production of Epo, thus completing the regulatory loop (Figure 2.9). Simple diagnostic tools are available to test the circuit in a logical manner (see also Chapter 6). First, one can evaluate the overall level of erythropoiesis by estimating the ratio of myeloid precursors to erythroid precursors in the marrow (normally about 4 : 1, but with a very broad normal range). Total erythropoiesis can be measured accurately using radioactive (59Fe) ferrokinetic assays. The plasma iron turnover measures the total (i.e. effective and ineffective) amount of erythropoiesis, whereas the red cell iron utilization assay measures effective erythropoiesis. To a large extent, these two parameters can now be assessed much more easily by measuring the levels of soluble TfR and the reticulocyte count. Soluble TfR is a truncated form of the receptor that circulates in a complex with transferrin. The erythroblasts rather than the reticulocytes are the main source of soluble TfR and, when iron stores are adequate and available, measuring the level of soluble TfR (normal range 5.0 ± 1.0 mg/ mL) is a good guide to the total level of erythropoiesis. Soluble TfR levels are increased when erythropoiesis is stimulated and

Epo

HIF

BFU-E CFU-E

Figure 2.9 Summary of the regulation of erythropoiesis with the key points for assessment boxed in blue. ME denotes assessment of the myeloid/ erythroid ratio in the bone marrow. Hct, haematocrit.

Haematinics Fe, B12, folate

ME

Oxygenation

Retic Red cell mass

Hct

Tfr-R

23

Postgraduate Haematology

decreased when diminished. The interpretation of soluble TfR levels is complicated in iron deficiency as this condition independently raises the level of soluble TfR. The reticulocyte count (0.5–2.0% or 25–75 × 109/L) is raised in proportion to the degree of anaemia when erythropoiesis is effective (e.g. uncomplicated response to bleeding), but is relatively low when erythropoiesis is ineffective (e.g. β thalassaemia) or an abnormality prevents a normal response (e.g. nutritional deficiency). The output of the system, the red cell mass, can be accurately measured by radioactive dilution techniques using 51Cr, but can often be reliably estimated from the haematocrit or concentration of haemoglobin. Changes in red cell size, shape and haemoglobin content, often reflected in the red cell morphology, may provide important guides to specific abnormalities in red cell maturation (e.g. haemoglobinopathies, thalassaemia, nutritional deficiencies). If the red cell mass is appropriate to meet the demands for oxygenation, then Epo production will be suppressed and the serum level will be in the normal range (∼ 25–50 mU/mL in cord blood and ∼10–30 mU/mL in adults). If there is inadequate oxygenation, the level of Epo will generally be raised in proportion to the degree of anaemia (e.g. up to 3–10 U/mL after severe blood loss) unless there is some impediment to Epo production (e.g. chronic renal failure, anaemia of chronic diseases). For any given degree of anaemia the level of Epo in the blood may vary depending on the underlying conditions. For example, levels tend to be very high in aplastic anaemia and less than anticipated in thalassaemia. This may reflect the different numbers of precursors in the marrow that are able to bind available Epo molecules, thus altering the number of free Epo molecules that are measured. These apparently straightforward assessments may be more difficult to interpret when there are multiple causes of abnormal erythropoiesis, and in particular when complicated by nutritional deficiencies, which should always be evaluated in parallel with these studies. In addition to the common nutritional anaemias, the vast number of specific diagnostic tests to determine the inherited or acquired disorders that may perturb each phase of erythropoiesis are described elsewhere in this book. Proper oxygen delivery to the tissues requires sufficient circulating mature red cells, and any appropriate therapy should be aimed at correcting this. An important caveat is that excessive red cells may cause a sluggish circulation that can cause ischaemia, leading to serious complications (e.g. myocardial infarction and stroke). The simplified circuit presented here to describe the process of erythropoiesis (Figure 2.9) indicates three potential routes for therapeutic intervention. The first is to correct nutritional deficiencies, usually iron and less commonly folate or vitamin B12. The discovery of the role of the iron-regulatory peptide hepcidin in the anaemias of chronic disorders suggests that some remaining common forms of anaemia related to this class (caused by inability to use stored iron) may be amenable to rational treatment in the not too 24

distant future. A second frequently used approach is to correct anaemia, of any cause, with red cell transfusion, and the criteria for such treatment are set out in other sections of this book. The final approach is to increase erythropoiesis by administering recombinant human erythropoietin (rHuEpo). Following its considerable benefit to patients with the anaemia of chronic renal failure who are not capable of producing normal levels of Epo, rHuEpo has been assessed in a wide range of disorders (e.g. aplastic anaemia, red cell aplasia, thalassaemia intermedia, cancer of all types, haematological malignancy, myelodysplastic syndrome, rheumatoid arthritis, autologous blood donors, after stem cell transplantation and more). Modified forms of Epo, with a higher carbohydrate content and longer half-lives in vivo, have been developed and approved for clinical use, while smallmolecule Epo mimetics with higher affinity for EpoR are under investigation. A review of the effectiveness of these therapeutic alternatives is beyond the scope of this chapter, but the considerable expense involved in treating patients, often over relatively long periods of time, with a hormone that does not always directly address the known pathophysiology of the anaemia requires careful consideration. Finally, there are some conditions in which hormonal deficiency is known to contribute to anaemia (e.g. hypothyroidism, Addison’s disease). In these cases appropriate correction of the hormonal deficiency logically helps correct the anaemia. Some rare forms of anaemia respond to a variety of therapies for unexplained reasons. For example, some cases of Diamond– Blackfan anaemia respond to corticosteroids, and some cases of congenital dyserythropoietic anaemia respond to interferon alfa, suggesting that there are still many unknown aspects to this clinically important and intellectually fascinating process of erythropoiesis.

Selected bibliography Erythropoiesis in the context of general haemopoiesis Cross MA, Enver T (1997) The lineage commitment of haemopoietic progenitor cells. Current Opinion in Genetics and Development 7: 609–13. Dzierzak E, Speck NA (2008) Of lineage and legacy: the development of mammalian haematopoietic stem cells. Nature Immunology 9: 129–36. Joshi C, Enver T (2003) Molecular complexities of stem cells. Current Opinion in Hematology 10: 220–8. Murre C (2007) Defining the pathways of early adult hematopoiesis. Cell Stem Cell 1: 357–8. Orkin SH, Zon LI (2008) Hematopoiesis: an evolving paradigm for stem cell biology. Cell 132: 631–44. Palis J (2008) Ontogeny of erythropoiesis. Current Opinions in Haematology 15: 155–61. Tavian M, Hallais M-F, Péault B (1999) Emergence of intraembryonic haematopoietic precursors in the pre-liver human embryo. Development 126: 793–803.

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Wickramasinghe SN (1975) Erythropoiesis. In: Human Bone Marrow (SN Wickramasinghe, ed.), pp. 162–232. Blackwell Scientific Publications, Oxford.

Regulation and differentiation of erythroid cells Beguin Y (2003) Soluble transferrin receptor for the evaluation of erythropoiesis and iron status. Clinica Chimica Acta 329: 9–22. Bunn HF (2007) New agents that stimulate erythropoiesis. Blood 109: 868–73. De Maria R, Zeuner A, Eramo A et al. (1999) Negative regulation of erythropoiesis by caspase-mediated cleavage of GATA-1. Nature 401: 489–93. Panzenbock B, Bartunek P, Mapara MY et al. (1998) Growth and differentiation of human stem cell factor/erythropoietindependent erythroid progenitor cells in vitro. Blood 92: 3658– 68. Schofield CJ, Ratcliffe PJ (2004) Oxygen sensing by HIF hydroxylases. Nature Reviews. Molecular Cell Biology 5: 343–54. Transcription factors controlling erythropoiesis Bungert J, Engel JD (1996) The role of transcription factors in erythroid development. Annals of Medicine 28: 47–55. Cantor AB, Orkin SH (2002) Transcriptional regulation of erythropoiesis: an affair involving multiple partners. Oncogene 21: 3368–76.

Gubin AN, Njoroge JM, Bouffard GG et al. (1999) Gene expression in proliferating human erythroid cells. Genomics 59: 168–77. Shivdasani RA, Orkin SH (1996) The transcriptional control of hematopoiesis. Blood 87: 4025–39. Sieweke MH, Graf T (1998) A transcriptional factor party during blood cell differentiation. Current Opinion in Genetics and Development 8: 545–51.

Erythropoiesis in clinical practice Beguin Y (2003) Soluble transferrin receptor for the evaluation of erythropoiesis and iron status. Clinica Chimica Acta 329: 9–22. Eschbach JW (2000) Current concepts of anaemia management in chronic renal failure: impact of NKF-DOQI. Seminars in Nephrology 20: 320–9. Muirhead N, Bargman JA, Burgess E et al. (1995) Evidencebased recommendations for the clinical use of recombinant human erythropoietin. American Journal of Kidney Disease 26: S1–S24. Samol J, Littlewood TJ (2003) The efficacy of rHuEPO in cancerrelated anaemia. British Journal of Haematology 121: 3–11. Unger FE, Thompson AM, Blank MJ et al. (2010) Erythropoiesissimulating agents – time for a reevaluation. The New England Journal of Medicine 362: 189–192.

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CHAPTER 3

Iron metabolism, iron deficiency and disorders of haem synthesis

3

A Victor Hoffbrand1, Chaim Hershko2 and Clara Camaschella3 1

University College Medical School and Royal Free Hospital, London, UK Shaare Zedek Medical Center, Jerusalem, Israel 3 Vita-Salute University, Milan, Italy 2

Introduction, 26 Distribution of body iron, 26 Proteins important in iron metabolism, 27 Haemoglobin, 27 Ferritin and haemosiderin, 27 Transferrin and transferrin receptors, 28 Divalent metal transporter 1, 28 Ferroportin (SLC40A1), 28 Growth differentiation factor and twisted gastrulation protein, 28 Other proteins, 28 Hepcidin, 28 Matriptase-2 (TMPRSS6), 32 Intracellular iron homeostasis, 32 Normal iron balance, 33

Iron absorption, 33 Dietary and luminal factors, 34 Mucosal factors: molecular aspects of iron absorption and its regulation, 34 Iron uptake by erythroid cells, 34 Haem synthesis and mitochondrial iron metabolism, 35 Intracellular transit iron and plasma non-transferrin-bound iron, 36 Breakdown of haemoglobin, 36 Diagnostic methods for investigating iron metabolism, 36 Storage iron, 36 Iron supply to the tissues, 36

Introduction Iron (atomic weight 55.85) is essential for many metabolic processes. It shares with other transition metals two properties of particular importance in biology: the ability to exist in more than one relatively stable oxidation state and the ability to form many complexes. Its ability to exist in both ferric and ferrous states underlies its role in critical enzyme reactions concerned with oxygen and electron transport and the cellular production of energy. As well as physiologically active iron compounds, many of which are haem proteins, there are also specialized proteins of iron transport and storage. The latter are necessary to enable iron to remain in solution at neutral pH, at which ferric iron is insoluble, and to limit the potential toxicity of this reactive metal. The insolubility of ferric iron also means that although the earth’s crust contains approximately 4% iron and iron may be plentiful in the diet, much of this is unavailable. As

Postgraduate Haematology: 6th edition. Edited by A. Victor Hoffbrand, Daniel Catovsky, Edward G.D. Tuddenham, Anthony R. Green © 2011 Blackwell Publishing Ltd.

26

Iron deficiency anaemia, 38 Sequence of events, 38 Causes of iron deficiency, 39 Management of iron deficiency, 40 Iron refractory iron deficiency anaemia, 41 Pathological alterations in haem synthesis, 41 Porphyrias, 41 Lead poisoning, 42 Sideroblastic anaemia, 43 Selected bibliography, 45

a result, the body is limited in the adjustments it can make to excessive loss of iron, which frequently occurs due to haemorrhage, and iron deficiency is the most common cause of anaemia throughout the world. The general need to conserve the metal is reflected in the absence of any physiological mechanism for excretion of iron, control of iron balance being at the level of iron absorption. This is important in the rarer but potentially fatal disorders of iron overload (see Chapter 4).

Distribution of body iron The concentration of iron in the adult human body is normally about 50 mg/kg in males and 40 mg/kg in females. The largest component is circulating haemoglobin, with 450 mL (1 unit) of whole blood containing about 200 mg of iron (Figure 3.1). Much of the remainder is contained in the storage proteins ferritin and haemosiderin. These are found mainly in the reticuloendothelial cells of the liver, spleen and bone marrow (which gain iron from breaking down red cells), and in parenchymal liver cells (which normally gain most of their iron from the plasma iron-transporting protein transferrin).

Iron metabolism, iron deficiency and disorders of haem synthesis

Iron-donating tissues and iron stores Macrophages 500 mg

Hepatocytes 250 mg

RE stores

Liver stores

Gut

Absorption 1 mg/day Ineffective erythropoiesis

Plasma (transferrin) 4 mg Loss

Figure 3.1 The major compartments of iron in a 70-kg man. Iron supply for erythropoiesis and release of iron from senescent red cells dominate internal iron exchange. RE, reticuloendothelial.

Red cell haemoglobin 2500 mg

Bone marrow Erythroblasts 150 mg

1 mg/day

Tissues Iron-containing enzymes 150 mg Myoglobin 300 mg

Iron receptor tissues and functional iron compounds

Proteins important in iron metabolism Haemoglobin Haemoglobin (molecular weight 64 500) contains four haem groups linked to four globin chains, and can bind four molecules of oxygen. Myoglobin (molecular weight 17 000) accounts for 4–5% of body iron and has a single haem group attached to its one polypeptide chain. It has a higher affinity for oxygen than haemoglobin and behaves as an oxygen reserve in muscles. The mitochondria contain a series of haem and non-haem iron proteins (including the cytochromes a, b and c, succinate dehydrogenase and cytochrome oxidase) that form an electron transport pathway responsible for the oxidation of intracellular substrates and the simultaneous production of adenosine triphosphate (ATP). Haem is an essential component of microsomal and mitochondrial cytochrome P450, which is concerned with hydroxylation reactions (including drug detoxification by the liver), and of cyclooxygenase, involved in prostaglandin synthesis. Other haem proteins include the enzymes catalase and lactoperoxidase, which are concerned with peroxide breakdown, and tryptophan pyrrolase, involved in the oxidation of tryptophan to formylkynurenine. There is a smaller group of iron sulphur proteins (e.g. xanthine oxidase, reduced nicotinamide adenine dinucleotide dehydrogenase and aconitase). Iron is also necessary for the function of ribonucleotide reductase, a key enzyme in DNA synthesis.

Ferritin and haemosiderin Ferritin is the primary iron storage protein and provides a reserve of iron. It consists of an approximately spherical apo-

protein shell (molecular weight 480 000) enclosing a core of ferric hydroxyphosphate (up to 4000 iron atoms). Human ferritin is made up from 24 subunits (molecular weight about 20 000) of two immunologically distinct types: H and L. There are multiple gene copies, which are mostly pseudogenes, on 12 different chromosomes. The coding loci are located at 11q12–q13 for the heavy chain and 19q13.3–q13.4 for the light chain. An intronless gene on chromosome 5 (q23.1) codes for mitochondrial ferritin, a novel H-type ferritin. The internal cavity of the ferritin molecule communicates with the exterior via six channels, through which ferrous iron may enter (to interact with a ferroxidase centre on the ferritin H subunit) or leave (after reduction, e.g. by dihydroflavins or ascorbic acid). The way in which ferritin iron is mobilized is poorly understood, and a process in which the entire ferritin molecule is degraded within lysosomes prior to iron release has also been suggested. Variation in the proportion of H to L subunits explains the heterogeneity of ferritin from different tissues on isoelectric focusing: L-rich ferritins (from spleen and liver) are more basic than H-rich ferritins (from heart and red cells). The small amount of ferritin normally present in serum contains little iron and consists almost exclusively of L subunits. It is also heterogeneous, owing to glycosylation. This glycosylation and the direct relationship of serum concentration to storage iron in macrophages suggest that serum ferritin is secreted by macrophages in response to changing iron levels. Haemosiderin, unlike ferritin, is a water-insoluble, crystalline, protein–iron complex that is visible by light microscopy when stained by the Prussian blue (Perls’) reaction. It has an amorphous structure, with a higher iron/protein ratio than ferritin, and is probably formed by the partial digestion of ferritin aggregates by lysosomal enzymes. In normal subjects, the 27

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majority of storage iron is present as ferritin, and haemosiderin is predominantly found in macrophages rather than hepatocytes. In iron overload, the proportion present as haemosiderin increases considerably in both cell types.

Transferrin and transferrin receptors Transferrin is a single-chain polypeptide (molecular weight 79 500) present in plasma (1.8–2.6 g/L) and extravascular fluid (Table 3.1). It has a plasma half-life of 8–11 days. The protein is synthesized predominantly by the liver, synthesis being inversely related to iron stores. Two atoms of ferric iron bind to each molecule. Although transferrin contains only about 4 mg of body iron at any time, it is vital to iron transport, with over 30 mg iron passing through this compartment each day (Figure 3.1). The binding sites (N-terminus and C-terminus) contain three tyrosine and two histidine residues and an arginine group. The uptake of iron from transferrin requires that the protein is attached to specific receptors on the cell surface. The transferrin receptor gene (TFRC) codes for TFR1, a transmembrane protein (identified as CD71), each molecule of two subunits binding one transferrin molecule. A second receptor, TFR2, also binds transferrin (Table 3.1). Through their binding with HFE, TFR1 and TFR2 are involved in regulating hepcidin synthesis (see Figure 3.2). Lactoferrin is a glycoprotein (molecular weight 77 000) that is structurally related to transferrin. It is found in milk and other secretions and in neutrophils. It is thought to have a bacteriostatic action at secreting surfaces by depriving microorganisms of the iron needed for their growth.

which triggers its tyrosine phosphorylation, internalization and degradation by ubiquitin in lysosomes.

Growth differentiation factor and twisted gastrulation protein Growth differentiation factor (GDF)-15 is a member of the transforming growth factor (TGF)-β superfamily of proteins, which also includes the bone morphogenetic proteins (BMPs). GDF-15 exerts different functions according to the cell context, inhibiting hepcidin synthesis, macrophage activation, proliferation of immature haemopoietic progenitors and growth of tumour cell lines. It is strongly expressed during erythroblast maturation with lower levels in the other tissues. It is inducible by iron depletion and by hypoxia but is independent of hypoxiainducible transcription factors (HIFs). A second erythroid regulator of hepcidin expression, twisted gastrulation protein (TWSG1), has been identified. It is produced during the early stages of erythropoiesis and interferes with BMP-mediated hepcidin synthesis.

Other proteins The roles in iron metabolism of hemojuvelin (HJV), BMP-6, SMADs, ferrioxidative and reduction enzymes, and caeruloplasmin are discussed under the headings of hepcidin regulation, iron absorption, iron uptake by erythroid cells and haem synthesis.

Hepcidin Divalent metal transporter 1 Divalent metal transporter (DMT)1 is an electrogenic pump that requires proton cotransport in order to transfer Fe2+ across cell membranes. This occurs at the apical membrane and subapical endosomes of the duodenal enterocyte and the transferrin-cycle endosome, both of which have a low pH. The intestinal DMT is produced by different mRNA splicing from that which produces endosomal DMT1. DMT1 expression is upregulated in iron deficiency (see later) and may be involved in absorption of other divalent metal cations including Mn2+, Co2+, Zn2+, Cu2+ and Pb2+, although this is not established as a major function.

Ferroportin (SLC40A1) This transmembrane domain protein is the basolateral transporter of iron, essential for iron release from macrophages, the intestinal absorptive enterocyte and placental syncytiotrophoblasts. It is also present in intracellular compartments. Caeruloplasmin is required for the cell surface localization of ferroportin, whose concentration is controlled by hepcidin, 28

Hepcidin has a central role in the regulation of iron metabolism and absorption (Figure 3.2). A product of the HAMP gene (Table 3.1), it is a small peptide (25 amino acids) with several isoforms and is released from a large prepropeptide of 84 amino acids. It is predominantly expressed in the liver. It regulates iron homeostasis by binding to cell-surface ferroportin, causing its tyrosine phosphorylation, internalization, ubiquitination and degradation in lysosomes. It therefore acts to inhibit iron absorption, iron release from macrophages and iron transport across the placenta. It is bound in plasma to α2-macroglobulin and the major route of clearance is the kidney. Hepcidin can be measured in serum or urine by ELISA or mass spectrometrybased techniques. These have shown low or undetectable levels in iron deficiency and extremely high levels in inflammatory conditions, with inappropriately low levels in haemochromatosis and iron-loading anaemias. Regulation of hepcidin expression The regulation of hepcidin expression is transcriptional. Hepcidin expression is increased in response to raised serum iron, iron overload and inflammation, and is suppressed by

Table 3.1 Iron transport proteins, oxidoreductases, storage proteins and regulators. Protein (gene)

Chromosome location

Tissue expression

Structure

Function

Regulation

Mutations and disease

Duodenal cytochrome b1 (CYBRD1) DMT1 (SLC11A2)

2q31

Enterocyte +

TMP, 6TMD

Ferric reductase

Fe (hepcidin)

–

12q13

Widespread

TMP, 568 aa

Fe uptake

Fe (3′-IRE)

Hemojuvelin (HFE2)

1q21.2

Liver, heart, muscle

Regulator of hepcidin synthesis

?

Frataxin (FXN)

9q21.11

FLVCR (FLVCR1)

1q32.3

Heart, spinal cord, cerebellum Erythroid

Membrane-bound receptor or secreted protein Mitochondrial protein, 210 aa Major facilitator family

Mk mouse, Belgrade rat Human microcytic anemia Juvenile HC

Ferroportin 1 (SLC11A3) Hepcidin (HAMP)

2q32 19q13.1

Liver, spleen, enterocyte Plasma (liver)

Hephaestin (HEPH)

Xq11–q12

Enterocyte

Haemochromatosis (HFE)

6p21.3

Widespread

Mitoferrin (SLC25A37) STEAP3

8p21.2

Erythroid

2q14.2

Erythroid, placenta (with TFR1)

Transferrin receptor (TFR)

3q26.2–qter

Widespread: highest number in erythroblasts

20–25 aa TMP, 1TMD, copper protein with homology to caeruloplasmin HLA class I heavy chain

Mitochondrial inner membrane Six-transmembrane epithelial antigen of the prostate-3 TMP dimer of 90 kDa polypeptide

Friedreich ataxia

Fe (5′-IRE)

Human HC, autosomal dominant Juvenile HC (digenic HC) Sla mouse

Regulator of iron homeostasis Fe2+ oxidase

Fe (HFE)

Regulates TFRC, iron uptake and hepcidin expression Mitochondrial iron importer Ferric reductase (also reduces copper)

?

Human HC, autosomal recessive

?

Erythropoietic protoporphyria

Binds transferrin

–

?

Fe (3′-IRE)

(Lethal in knockout mouse)

29

Iron metabolism, iron deficiency and disorders of haem synthesis

TMP, 571 aa, 9TMD

Mitochondrial iron donor Receptor for feline leukaemia virus C; haem export Fe export

Protein (gene)

Chromosome location

Tissue expression

Structure

Function

Regulation

Mutations and disease

Transferrin receptor 2 (TFR2)

7q22

Liver, erythroid cells

60% similarity in extracellular domain to TFRC

No IRE

Human HC, autosomal recessive

Transferrin (TF)

3q21

Atransferrinaemia

11q13

Single-chain polypeptide, glycoprotein Subunit of ferritin

Iron stores

Ferritin heavy chain (FTH1)

Plasma, extravascular space Widespread, cytosolic

Binds transferrin, iron homeostasis, regulator of hepcidin synthesis Iron transport

Fe (IRE)

Ferritin light chain (FTL)

19q13.3– q13.4

Widespread, cytosolic

Subunit of ferritin

Iron storage (catalytic subunit for iron incorporation) Iron storage

IRP1 (ACO1)

9p21.1

Widespread

Cytoplasmic, 98 kDa with 4Fe–4S cluster

Cell iron

IRP2 (IREB2)

15

Widespread

Cell iron

Not known

Matriptase-2 (TMPRSS6)

22q13.1

Mainly liver

Cytoplasmic, 105 kDa, no 4Fe–4S cluster Type II transmembrane protease from plasma membrane

Regulation of synthesis of FTH, FTL, TFRC, DMT1, ferroportin 1, ALAS2 As IRP1

Autosomal dominant Fe overload (very rare) Hyperferritinaemia and cataract syndrome Neuroferritinopathy* Not known

Cleaves hemojuvelin

Unknown

Homozygous mutation leads to IRIDA

Fe (IRE)

*In hyperferritinaemia/cataract syndrome, mutations affect the 5′-UTR IRE. In neuroferritinopathy, mutations affect FTL coding sequences. aa, amino acid; HC, haemochromatosis; IRIDA, iron refractory iron deficiency anaemia; IRE, iron response element; IRP, iron regulatory protein; TMD, transmembrane domain; TMP, transmembrane protein.

Postgraduate Haematology

30 Table 3.1 Continued

Iron metabolism, iron deficiency and disorders of haem synthesis

Erythropoiesis Other tissues

Iron absorption

Duodenum

FPN

Transferrin saturation Iron release from macrophage FPN

Figure 3.2 Stimulatory and inhibitory signals of hepcidin regulation. Hepcidin, as well as hemojuvelin (HJV), transferrin receptor 2 (TFR2) and HFE, are all produced in the hepatocyte. High plasma iron and inflammation stimulate hepcidin synthesis. This is mediated by SMADs and STAT3 respectively. Conversely, low plasma iron, increased rates of erythropoiesis (including ineffective erythropoiesis) and hypoxia inhibit hepcidin production. This is mediated by matriptase, growth differentiation factor (GDF)-15, twisted gastrulation protein (TWSG1) and HIF1-α respectively. Hepcidin binds ferroportin (FPN), causing its destruction and so inhibits iron absorption and iron release from macrophages into plasma and from intracellular compartments. BMP, bone morphogenetic protein.

? TFR1 TF/HFE

TFR2 HFE

HJV BMP6

SMAD1, 5, 8 +4

Matriptase 2

Hepcidin Hepatocyte

STAT3

IL-6

iron deficiency, hypoxia and increased erythropoietic activity. Under basal conditions, expression depends on signalling through the BMP/SMAD pathway (Figure 3.2). HJV is a member of the repulsive guidance molecules (RGM) family that is highly expressed in liver, skeletal muscles and the heart. It is either associated with cell membranes through a glycosylphosphatidylinositol anchor or released as a soluble form. Membrane-bound HJV participates in the pathway regulating hepcidin expression as a BMP coreceptor, whereas soluble HJV antagonizes BMP-6. BMP-6 is the master hepcidin activator in vivo. HFE and TFR2 are also involved in hepcidin expression (Figure 3.2). HFE is able to bind TFR1 and TFR2. During low

TWSG1 GDF15

HIF Hypoxia

Erythroblasts Low iron

Erythropoietin

or basal serum iron conditions, HFE and TFR1 exist as a complex at the plasma membrane, TFR1 serving to sequester HFE to silence its activity. Diferric serum transferrin (Fe2+-TF) competes with HFE for binding to TFR1. Increased serum transferrin saturation therefore results in dissociation of HFE from TFR1. Acting as an iron sensor, HFE then binds to TFR2 and conveys the Fe2+-TF status to the signal transduction effector complex. HJV binds to BMP, then phosphorylates SMADs to form an SMAD-1/-5/-8–SMAD-4 complex, which translocates to the nucleus and stimulates hepcidin production by activating its promoter. In keeping with this model, genetic mutations of HFE, TFR2, HJV and hepcidin all result in haemochromatosis with low serum hepcidin levels (see 31

Postgraduate Haematology

Chapter 4). Iron levels also seem to control BMP-6 production but whether this is through liver iron or circulating transferrin is unclear. A second type of transcriptional hepcidin regulation occurs in inflammation. Interleukin (IL)-6 and IL-1β induce transcription of the hepcidin gene by activating STAT3 (signal transducer and activator of transcription 3) and its binding to a regulatory element in the hepcidin promoter. It may converge on a final shared SMAD-4-dependent pathway. The hepcidin response is remarkably rapid. In humans, iron ingestion results in a sharp increase in urinary hepcidin excretion within 12–24 hours of starting treatment. Likewise, infusion of recombinant IL-6 results in significant increase in urinary hepcidin and decreased serum iron and transferrin saturation within 2 hours of infusion. These observations imply that hepcidin expression is directly controlled by serum iron (probably by transferrin saturation) and IL-6 and not by longterm gradual accumulation of iron in tissues. Response to anaemia and hypoxia Hepcidin levels can be measured in patient serum or urine by ELISA or mass spectrometry. Hepcidin levels are reduced or undetectable in iron deficiency anaemia and extremely high in inflammatory conditions. Iron absorption is accelerated in iron deficiency, ineffective erythropoiesis and hypoxia (Figure 3.2). Erythroid precursors secrete GDF-15 and TWSG1, which inhibit hepcidin production by the liver. Serum concentrations of GDF-15 are grossly increased in thalassaemia major and other conditions associated with ineffective erythropoiesis and in iron deficiency, resulting in hepcidin suppression and so inability to turn off intestinal iron absorption. In addition, other studies indicate that the von Hippel–Lindau hypoxia inducible factors (HIFs), which stimulate erythropoietin synthesis, control iron homeostasis by the downregulation of hepcidin, repressing its promoter with the upregulation of ferroportin. Under normal conditions, HIFs play a useful role by mobilizing iron and supporting erythrocyte production in response to anaemia/hypoxia. However, the same mechanism may contribute to the harmful accumulation of iron in response to chronic anaemia associated with ineffective erythropoiesis in thalassaemia and other dyserythropoietic anaemias. In addition, soluble HJV in vitro and in vivo and erythropoietin itself in cell models are able to inhibit hepcidin transcription.

Matriptase-2 (TMPRSS6) This is a type 2 member of the transmembrane serine protease family mainly expressed in the liver. Membrane-bound matriptase-2 regulates hepcidin expression by cleaving membrane-bound HJV, releasing soluble HJV fragments. The reduction in membrane-bound HJV and the presence of soluble HJV both result in reduced hepcidin transcription. The factors

32

which regulate matriptase-2 expression need to be elucidated. Matriptase-2 activity overrides all known activating stimuli of hepcidin synthesis. Homozygous TMPRSS6–/– mutations in mice and humans result in marked upregulation of hepcidin and blockade of intestinal and macrophage iron transport into plasma, leading to a refractory hypochromic microcytic anaemia (see pp. 41, 51).

Intracellular iron homeostasis Synthesis of several of the proteins involved in iron metabolism is regulated at the level of RNA translation by two cytoplasmic iron-dependent proteins, namely IRP1 and IRP2 (Table 3.1). These are capable of binding to mRNAs that contain a sequence forming a stem-and-loop structure called an iron-responsive element or IRE (Figure 3.3). IRP1 (molecular weight 98 000) contains an iron–sulphur (4Fe–4S) cluster and functions as a cytoplasmic aconitase with low affinity for the IRE when intracellular iron is abundant. When iron is scarce, however, the iron–sulphur cluster is no longer present and IRP1 binds to the IRE with high affinity. IRP2 (molecular weight 105 000) is expressed ubiquitously but is less abundant than IRP1. IRP2 has an extra section of 73 amino acids rich in proline, serine and cysteine that mediates IRP2 degradation in iron-replete cells. Activation of IRP2 requires accumulation of the protein as a result of new synthesis. Degradation takes place in the proteosome after addition of iron. The 3′-untranslated region (3′-UTR) of TFR1 contains five IREs, whereas the 5′-UTR region of ferritin mRNA contains a single IRE. Binding of IRP when there are low levels of intracellular iron protects TFR1 mRNA from cytoplasmic degradation but inhibits translation of ferritin mRNA by interfering with the binding of initiation factors. In contrast, when intracellular iron is increased, the opposite effects occur. Thus, coordinated regulation of TFR1 and ferritin acts to maintain a constant intracellular iron content over the short term by balancing cellular iron uptake and storage. Erythroid δ-aminolaevulinic acid synthase (ALAS2) mRNA also has an IRE in its 5′-UTR region, whereas ‘housekeeping’ ALAS1 mRNA does not. The IRP–IRE system is therefore involved in matching iron supply to haem synthesis, with repression of protoporphyrin synthesis in iron-deficient erythroblasts (see p. 38). Mitochondrial aconitase interconverts citrate and isocitrate and has a putative IRE at the 5′-UTR of its mRNA. DMT1 like TFR1 has a 3′-IRE and is upregulated in iron deficiency. Ferroportin 1 has a 5′-IRE (Table 3.1) but binding of IRP is weak compared with the TFR1, ferritin and DMT1 IREs. However, as occurs for DMT1, ferroportin 1 may be translated in a non-IRE isoform that escapes the IRP control in some tissues, for example in duodenal cells.

Iron metabolism, iron deficiency and disorders of haem synthesis

Table 3.2 Daily iron losses and requirements.

Iron regulatory protein (IRP1) – 4Fe–4S

Low iron

High iron

+ 4Fe–4S

IRE-binding protein

Cytoplasmic aconitase Iron regulatory protein (IRP2)

Ubiquitination and degradation (haem-induced oxidation) Transcription inhibited

5'

Ferritin coding ALA-S coding Ferroportin coding IRE

AAAA 3'

mRNA stabilized

5'

TfR coding DMT-1 coding

AAAA 3' IREs(5)

Figure 3.3 Coordinate regulation of expression of ferritin and transferrin receptor: the role of the iron response element (IRE)–iron regulatory protein (IRP) mechanism. When cellular iron levels are low, IRP binds to the IRE stem and loop structures of mRNA to inhibit translation of ferritin and ALA-S, but increases translation of transferrin receptors (TFR) and DMT1 by preventing degradation of the mRNA. When iron levels are high, the IRP functions as a cytoplasmic aconitase and no longer binds to the IREs. Ferritin synthesis can thus proceed, while TFR synthesis is reduced. IRP2 binds to IREs when iron levels are low, but is degraded after ubiquitination (initiated by haem-induced oxidation) when iron levels are high. The IRPs therefore provide two ways of sensing iron requirements either involving Fe–S proteins or haem proteins.

Normal iron balance The amount of iron in the body at birth depends on the blood volume and haemoglobin concentration, the birth weight (which determines blood volume) being particularly important. Delay in clamping the cord leads to an increased red cell mass by placental transfusion. The level of maternal iron stores has little effect on fetal iron. The newborn contains about 80 mg/kg at full term. Neonatal iron reserves are utilized for growth, and from 6 months to 2 years virtually no iron stores are present. Thereafter, iron stores gradually accumulate during childhood to around 5 mg/kg. In men, there is a further increase between 15 and 30 years to about 10–12 mg/kg (total up to approximately 1 g), whereas iron stores remain lower in women (average

Group (age, years)

Daily loss (mg)

Requirement for growth (mg)

Total (mg)

Urine, skin, faeces, etc.

Menses

Children 0.5–1 1–3 4–6 7–10

0.17 0.19 0.27 0.39

– – – –

0.55 0.27 0.23 0.32

0.72 0.46 0.50 0.71

Males 11–14 15–17 18+

0.62 0.90 1.05

– –

0.55 0.60 –

1.17 1.50 1.05

Females 11–14* 11–14 15–17 18+ Post menopause Lactating‡

0.65 0.65 0.79 0.87 0.87 1.15

– 0.48† 0.48† 0.48† – –

0.55 0.55 0.35 – – –

1.20 1.68 1.62 1.35 0.87 1.15

*Non-menstruating. Median loss. ‡ Average dietary requirement during pregnancy is 3–4 mg. Source: WHO (2001). †

300 mg) until the menopause. It would take 4 years or more for a man to deplete body iron stores and start developing iron deficiency anaemia solely due to lack of dietary intake or malabsorption. Requirements are higher in menstruating women and during periods of rapid growth in infancy and adolescence (Table 3.2). Menstrual blood loss has a median value of 30 mL, but the 95th centile value is 118 mL per month (equivalent to 1.9 mg iron per day), which has been found to be significantly associated with iron deficiency. Requirements are highest of all in pregnancy.

Iron absorption Iron absorption depends not only on the amount of iron in the diet but also, and more importantly, on the bioavailability of that iron, as well as the body’s needs for iron. A normal Western diet provides approximately 15 mg of iron daily. Of that iron, digestion within the gut lumen releases about half in a soluble form, from which about 3 mg may be taken up by mucosal cells and only about 1 mg (or 5–10% of dietary iron) transferred to

33

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Gut lumen

Mature enterocyte

Plasma

Haem oxygenase

Haem 25%

Hepcidin

Non-haem Fe 0–20%

Labile iron pool Fe2+

Fe3+

FPN

DCytb Hp

Dietary factors Fe2+

Fe2+

Fe3+

DMT1 Transferrin

Ferritin

Mitochondria

the portal blood in a healthy man. Iron absorption can thus be influenced at several different stages.

Dietary and luminal factors Much of dietary iron is non-haem iron derived from cereals (commonly fortified with additional iron in the UK), with a lesser component of haem iron from meat and fish. Even in iron deficiency, the maximum iron absorption from a mixed Western diet is no more than 3–4 mg daily. This figure is much less with the predominantly vegetarian, cereal-based diets of most of the world’s population. Iron is better absorbed from animal than vegetable sources. Iron is released from protein complexes by acid and proteolytic enzymes in the stomach and small intestine, and haem is liberated from haemoglobin and myoglobin. Iron is maximally absorbed from the duodenum and less well from the jejunum, probably because the increasingly alkaline environment leads to the formation of insoluble ferric hydroxide complexes. Acid pH, vitamin C and some low-molecular-weight chelates (e.g. sugars, amino acids) enhance absorption. Therapeutic ferrous iron salts are well absorbed on an empty stomach, but when taken with a meal absorption is reduced as a result of the same ligand-binding processes that affect dietary non-haem iron; phytates, tannates in tea and bran inhibit absorption.

Mucosal factors: molecular aspects of iron absorption and its regulation A variety of mechanisms for the binding of non-haem iron to the mucosal membrane have been described. Specific, saturable and receptor-mediated mechanisms, and passive diffusion at higher doses, may occur. 34

Figure 3.4 Molecular pathways of iron absorption. The area enclosed in the dotted box refers to the uptake of iron from the plasma in the developing enterocyte in the intestinal crypt. Otherwise, the diagram refers to iron absorption by the villous epithelial cell. DMT1, divalent metal transporter 1; FPN, ferroportin; Hp, hephaestin; TF, transferrin; TFR, transferrin receptor. For further details see text and Table 3.1.

The proposed process is illustrated in Figure 3.4. Non-haem iron is released from food as Fe3+ and reduced by duodenal cytochrome b1 (DCytb) to Fe2+. This is transported across the brush border membrane by DMT1, which is upregulated in iron deficiency. It is assumed that iron enters the labile pool and some may be incorporated into ferritin and lost when the cells are exfoliated. Iron destined for retention by the body is transported across the serosal membrane by ferroportin before uptake by transferrin as Fe3+. The regulation of duodenal cell iron release by hepcidin through its action on ferroportin is rapid and occurs for all absorptive cells, including those at the tip of the villus. Hephaestin is a copper-containing ferroxidase expressed predominantly in villous cells of the small intestine that converts Fe2+ to Fe3+ in the basolateral transfer step of iron absorption. Haem iron is initially bound by haem receptors at the brush border membrane and released intracellularly by haem oxygenase before entering the labile iron pool and following a common pathway with iron of non-haem origin. Iron absorption is regulated both at the stage of mucosal uptake and at the stage of transfer to the blood. DMT1 levels increase when intracellular iron is low and ferroportin concentration is also high due to low plasma hepcidin levels. The amount of iron transported to the plasma through ferroportin is hepcidin-dependent.

Iron uptake by erythroid cells About 85% of transferrin iron normally enters developing red cells for incorporation into haemoglobin. This tissue distribution of transferrin-bound iron reflects the expression of transferrin receptors, which are present in high concentration on

Iron metabolism, iron deficiency and disorders of haem synthesis

cells with a high iron requirement. The latter includes any rapidly dividing cells but is normally dominated by the cells of the erythron. A soluble truncated form of the transferrin receptor derived from these cell surfaces is detectable in serum. Transferrin receptors have the highest affinity for diferric transferrin. The transferrin–receptor complex is taken up by a process of receptor-mediated endocytosis (Figure 3.5). The iron is released at the low pH of the endosome, reduced from Fe3+ to Fe2+ by STEAP3, a ferrireductase, before the apotransferrin and receptor are recycled to the plasma and the cell membrane respectively. Iron release from the endosome is via DMT1 (Figure 3.5) and the iron is transported into mitochondria by mitoferrin or enters ferritin. Recently, direct endosome–mitochondrial iron transfer has also been suggested. A mitochondrial version of ferritin exists, coded by a gene on chromosome 5. Its physiological role is unknown but it is elevated in sideroblastic anaemias due to ALAS2 defects or myelodysplasia. Direct transfer of storage iron from macrophages to erythroblasts (rhopheocytosis) may also occur. Some 80–90% of iron taken into developing erythroblasts is converted to haem within 1 hour. Any iron taken up in excess of the requirement for haem synthesis is incorporated in ferritin (Figure 3.5). The red cell ferritin content is therefore increased when haemoglobin synthesis is impaired, as in thalassaemia syndromes or sideroblastic anaemia. Excess iron may be seen in the cytoplasm of mature red cells as one or more siderotic granules. These are composed of haemosiderin and stain blue with Perls’ reaction and purplish blue with Romanowsky stains,

Mitochondrion Ribosomes

Globin

α β

Haem Ferritin

Mitoferrin Haemoglobin Steap3

Fe2+ Endosome (acidic)

Transferrin receptor Diferric transferrin

Transferrin receptor Apotransferrin

Figure 3.5 Incorporation of iron from plasma transferrin into haemoglobin in developing red cells. Uptake of transferrin iron is by receptor-mediated endocytosis.

when they are called Pappenheimer bodies. The spleen removes these granules by its pitting action.

Haem synthesis and mitochondrial iron metabolism Haem consists of a protoporphyrin ring with an iron atom at its centre. Haem is synthesized from the precursors succinylCoA and glycine, which condense to form δ-aminolaevulinic acid (ALA) under the action of ALAS, with pyridoxal phosphate as a coenzyme (see Chapter 2; see also p. 39). Housekeeping ALAS (ALAS1) is coded by a gene on chromosome 3, but in erythroid cells erythroid-specific ALAS2 predominates and is encoded on the X chromosome. ALA can be utilized for the formation of both purines and haem. Four molecules of porphobilinogen condense under the influence of porphobilinogen deaminase and uroporphyrinogen cosynthase to form the tetrapyrrole ring compound uroporphyrinogen III. The latter is converted to protoporphyrin IX. Finally, iron in the ferrous form is incorporated under the influence of the enzyme ferrochelatase. Iron in haem has six coordinating valencies: four link the iron to nitrogen atoms in each pyrrole ring, whereas the remaining two link haem to histidine residues in the globin chain, the distal bond being unstable and easily replaced by oxygen to form oxyhaemoglobin. The mitochondria play a major role in haem synthesis as they contain ALAS, coproporphyrinogen oxidase and ferrochelatase, the enzyme sequence from ALA to coproporphyrinogen being situated in the cytoplasm. Besides haem, mitochondria utilize iron in the synthesis of iron–sulphur clusters, prosthetic groups essential for the function of several mitochondrial (respiratory chain complexes, ferrochelatase) and cytosolic (aconitase) proteins. A connection exists between haem synthesis and sulphur cluster biogenesis, because the activity of the iron regulatory protein IRP1, which controls ALAS2, is iron–sulphur clusterdependent and because ferrochelatase, the last enzyme in haem synthesis, has an iron–sulphur cluster. The assembly of iron–sulphur clusters is a complex, incompletely understood process that requires multiple proteins. The best known is frataxin, a likely iron donor in this pathway. The gene encoding frataxin is mutated in Friedreich ataxia (see pp. 43, 44, 53). An enzyme active in the pathway, whose function is still unclear, is glutaredoxin-5, which has been found mutated in a rare recessive form of sideroblastic anaemia (see pp. 43, 44). ABCB7, a member of a family of transmembrane proteins characterized by the ABC domain that binds and hydrolyses ATP, transfers iron–sulphur clusters from mitochondria to the cytosol. Mutations of ABCB7 cause an X-linked form of sideroblastic anaemia with ataxia (see pp. 43, 44). The mitochondria are also the site of the citric acid cycle, which supplies succinate. The mature red cell, which lacks mitochondria, is therefore unable to synthesize haem. 35

Postgraduate Haematology

A number of porphyrins are formed by side reactions during the synthesis of protoporphyrin. In the porphyrias (see p. 41), many of these compounds accumulate in the major sites of haem synthesis, the liver and the red cells. A haem exporter, FLVCR (Table 3.1), is present in erythroid cells and rids the cell of any haem made in excess.

Intracellular transit iron and plasma non-transferrin-bound iron It has been suggested but not proved that there is a transit pool of ‘metabolically active’ or ‘labile’ iron within cells, which receives iron from degraded haem or ferritin, exchanges with transferrin and is incorporated into newly synthesized iron-containing proteins. This iron is considered to be sensed by iron regulatory proteins and available for chelation. Within cells, low-molecular-weight chelates (e.g. with citrate) may be present. However, recent work questions the existence of these chelates and a model of iron transfer requiring direct protein–protein interaction that could involve interaction of organelles has been proposed. It is suggested that transferrincontaining endosomes may transfer iron directly to mitochondria. Within plasma in iron overload, non-transferrin-bound iron may also exist as oligomeric iron oxide, either free or bound to albumin, and is particularly toxic to various organisms (see Chapter 4).

Breakdown of haemoglobin After phagocytosis by macrophages, haem from senescent red cells is broken down by haem oxygenase (HMOX1) to release iron (see Chapter 8). As ferrous iron, it can then either enter ferritin (where it is oxidized to ferric iron by the ferritin protein) or be released into plasma (via ferroportin 1), where its binding to transferrin (also as the ferric form) may be facilitated by a plasma ferrous oxidase (e.g. caeruloplasmin). The release of macrophage iron is controlled by hepcidin, with high levels, as in inflammation or iron overload, reducing iron release. Changes in the release of iron from macrophages are thought to account for the diurnal rhythm of serum iron concentration, which is highest in the morning and lowest in the evening. A diurnal increase in serum hepcidin at noon and 8 p.m. is observed in healthy volunteers.

Diagnostic methods for investigating iron metabolism The large amount of iron present as haemoglobin means that the degree of any anaemia must always be considered in assessing iron status. Reduced amounts of haemoglobin accompany 36

an overall reduction in body iron in iron deficiency anaemia or after acute blood loss. In other anaemias, including the anaemia of chronic disease and most haemolytic and megaloblastic anaemias, iron is redistributed from the red cells to macrophage iron stores, with a corresponding increase in marrow-stainable iron and serum ferritin. The various measurements of iron status are listed in Table 3.3 and described below. No single measurement is ideal for all clinical circumstances, as all are affected by confounding factors (Table 3.3) and changes may develop sequentially (as in progressive negative iron balance) or may affect particular body iron compartments. Reference ranges for haemoglobin and the various measures of iron status are given in Appendix 1. Table 3.4 summarizes the changes in measures of iron status accompanying various types of hypochromic anaemia. The assessment of iron overload is discussed in Chapter 4.

Storage iron Serum ferritin In healthy subjects, the serum ferritin concentration correlates with iron stores, as assessed by quantitative phlebotomy or tissue biopsy. Normal concentrations of serum ferritin range from about 15 to 300 µg/L, and are higher in men (median about 90 µg/L) than in premenopausal women (median 30 µg/L). In neonates, the concentration in cord blood (median approximately 100 µg/L) rises further over the first 2 months of life as fetal haemoglobin is broken down, and thereafter falls to low levels (median 20–30 µg/L) throughout childhood and adolescence. Serum ferritin concentrations below 15 µg/L are virtually specific for storage iron depletion, but normal values do not exclude this and values above 300 µg/L do not necessarily, or even usually, indicate iron overload. This is because ferritin synthesis is influenced by factors other than iron (in particular, it behaves as an acute-phase reactant in many inflammatory diseases). For this reason, serum ferritin concentrations below 50 µg/L may be associated with a lack of storage iron in patients with the anaemia of chronic disease. A ferritin concentration above 100 µg/L suggests the presence of storage iron. Bone marrow aspiration Staining the bone marrow for iron gives an indication of reticuloendothelial iron stores as well as erythroblast iron (Figure 3.6a). In iron deficiency anaemia, reticuloendothelial iron and erythroblast iron are absent (Figure 3.6b).

Iron supply to the tissues Serum iron and iron-binding capacity The serum iron and, more particularly, the saturation of the total iron-binding capacity of transferrin (TIBC) give a measure of the iron supply to the tissues. A serum transferrin saturation

Iron metabolism, iron deficiency and disorders of haem synthesis

Table 3.3 Potential confounding factors in the interpretation of measures of iron status. Measurement

Confounding factors

Iron stores Serum ferritin

Increased: as an acute-phase protein (e.g. in infection, inflammation or malignancy) and by release of tissue ferritins by damage, especially to iron-rich organs (e.g. with hepatic necrosis, chronic liver disease, splenic or bone marrow infarction in sickle cell disease) Decreased: by ascorbate deficiency

Tissue iron supply Serum iron and transferrin saturation Serum transferrin receptor Red cell protoporphyrin/red cell ferritin/% hypochromic red cells/ reticulocyte haemoglobin content

Functional iron Haemoglobin concentration

Labile measures: normal short-term fluctuations mean that a single value may not reflect iron supply over a longer period Directly related to extent of erythroid activity as well as being inversely related to iron supply to cells Stable measures: reduced iron supply at time of red cell formation leads to increases in protoporphyrin and reticulocyte haemoglobin content and hypochromic red cells, and reduced red cell ferritin. However, values may not reflect current iron supply. May be affected by other causes of impaired iron incorporation into haem (e.g. lead poisoning, sideroblastic anaemias) Other causes for anaemia besides iron deficiency; a reciprocal relationship with iron stores should be expected in all anaemias except in iron deficiency anaemia May be reduced in other disorders of haemoglobin synthesis (e.g. thalassaemia, sideroblastic anaemias) in addition to iron deficiency

Red cell MCV, MCH

MCH, mean corpuscular haemoglobin; MCV, mean corpuscular volume.

Table 3.4 Differential diagnosis of hypochromic anaemia. Iron deficiency

Chronic disease

Thalassaemia trait (α or β)

Sideroblastic anaemia

IRIDA

MCV/MCH

↓

↓ or N

↓

↓

Serum iron TIBC Transferrin saturation Serum ferritin Serum TFR Serum hepcidin Bone marrow iron stores Erythroblast iron

↓ ↑ ↓ ↓ ↑ ↓ ↓ ↓

↓ ↓ or N ↓ N or ↑ N ↑ N or ↑ ↓

N N N N N N N N

↓ (congenital) ↑N (acquired) ↑ N ↑ ↑ N or ↑ ↓ N or ↑ Ring forms

↓ ↓ N ↑ N or ↑ ↑

IRIDA, iron refractory iron deficiency anaemia; MCH, mean corpuscular haemoglobin; MCV, mean corpuscular volume; N, normal; TFR, transferrin receptor; TIBC, total iron-binding capacity.

less than 15% is insufficient to support normal erythropoiesis. A rise in TIBC is characteristic of iron deficiency. A reduced serum iron concentration with a normal or reduced TIBC is a characteristic response to inflammation (see below).

Serum transferrin receptors Plasma concentrations reflect both the number of erythroid precursors and iron supply to the bone marrow. In clinical practice, these two factors must be considered in interpreting 37

Postgraduate Haematology

Red cell protoporphyrin When iron supply to the erythron is limited, iron incorporation into haem is restricted, leading to accumulation of the immediate precursor, protoporphyrin IX. This is lost only slowly from circulating red cells; concentrations greater than the normal upper limit of 80 µmol/mol haemoglobin therefore indicate that a reduction in iron supply has been present over the previous few weeks. Protoporphyrin levels may also increase in patients with sideroblastic anaemias and lead poisoning. Analysers that are portable and easy to operate are useful in large-scale field studies of iron deficiency anaemia as an initial screening test. (a)

Percentage of hypochromic red cells As iron supply to the erythron diminishes, the new red cells produced are increasingly hypochromic. Assessment of the haemoglobin content of individual red cells, which is possible using some automated cell counters, allows measurement of the percentage of hypochromic cells. Values rising to above 6% may help in the early identification of impaired iron supply in patients with chronic renal failure who are receiving treatment with recombinant erythropoietin, when associated inflammatory disease means that other measures of iron status can be misleading. (b)

Reticulocyte haemoglobin content Reticulocyte haemoglobin content (CHr) is useful in screening iron status, particularly in dialysis patients. A CHr cut-off value of 32 pg is appropriate for the assessment of iron-deficient erythropoiesis. Moreover, CHr may serve as a predictor of the response of anaemia to iron treatment. When response to treatment is favourable, an increase in CHr may be discerned within a few days of starting treatment before overall mean corpuscular haemoglobin (MCH) has changed.

Iron deficiency anaemia Sequence of events (c)

Figure 3.6 (a) Normal bone marrow showing plentiful iron in macrophages (Perls stain) with iron granules in erythroblasts (insets). (b) Iron deficiency: bone marrow showing absence of stainable iron (Perls stain). (c) Iron deficiency: peripheral blood film showing hypochromic microcytic red cells.

transferrin receptor levels. In the anaemia of chronic disease, the assay provides a valuable indicator of deficiency of body iron stores. Serum transferrin receptor levels only increase in this situation in the absence of storage iron. 38

Depletion of iron stores When the body is in a state of negative iron balance, the first event is depletion of body stores, which are mobilized for haemoglobin production. Iron absorption is increased when stores are reduced, before anaemia develops and even when the serum iron level is still normal, although the serum ferritin will have already fallen. Iron-deficient erythropoiesis With further iron depletion, manifested by a serum ferritin below 15 µg/L and fall in serum transferrin saturation to less than 15%, iron-deficient erythropoiesis develops with increasing concentrations of serum transferrin receptor and red cell

Iron metabolism, iron deficiency and disorders of haem synthesis

protoporphyrin. At this stage, the haemoglobin, mean corpuscular volume (MCV) and MCH may still be within the reference range, although they may rise significantly when iron therapy is given. Iron deficiency anaemia If the negative balance continues, frank iron deficiency anaemia develops. The red cells become obviously microcytic and hypochromic (Figure 3.6c), and poikilocytosis becomes more marked. The MCV and MCH are reduced, and target cells may be present. The reticulocyte count is low for the degree of anaemia. The serum TIBC rises and the serum iron falls, so that the percentage saturation of TIBC is usually less than 10%. The number of erythroblasts containing cytoplasmic iron (sideroblasts) is reduced at an early stage in the development of deficiency, and siderotic granules are entirely absent from these cells when iron deficiency anaemia is established. The erythroblasts have a ragged vacuolated cytoplasm and relatively pyknotic nuclei. The bone marrow macrophages show a total absence of iron, except where very rapid blood loss outstrips the ability to mobilize the storage iron. Platelets are frequently increased. Tissue effects of iron deficiency When iron deficiency is severe and chronic, widespread tissue changes may be present, including koilonychia (ridged nails, breaking easily), hair thinning, angular stomatitis (especially in those with badly fitting dentures), glossitis and pharyngeal webs (Paterson–Kelly syndrome). Partial villous atrophy, with minor degrees of malabsorption of xylose and fat, reversible by iron therapy, has been described in infants suffering from iron deficiency, but not in adults. Pica is sometimes present; in some who eat clay or chalk, this may be the cause rather than the result of iron deficiency. Iron-dependent enzymes in the tissues are usually better preserved than other iron-containing compounds. In severe iron deficiency, however, these enzymes are not inviolate and their levels may fall. This may be partly responsible for the general tissue changes, with mitochondrial swelling in many different cells (including, in the experimental animal, hepatic and myocardial cells), poor lymphocyte transformation and diminished cell-mediated immunity, and impaired intracellular killing of bacteria by neutrophils. A particular concern has been the finding that infants with iron deficiency anaemia may have impaired mental development and function, and that this deficit may not be completely restored by iron therapy. There is recent evidence that premature labour is more frequent in mothers with iron deficiency anaemia. It remains controversial whether impaired work performance seen in adults results from the anaemia or from depletion of mitochondrial iron-containing enzymes. It is also unclear to what extent some of the other tissue effects of iron deficiency can occur even in the absence of anaemia.

Table 3.5 Causes of iron deficiency. Blood loss Uterine: menorrhagia, post-menopausal bleeding, parturition Gastrointestinal: oesophageal varices, hiatus hernia, Helicobacter pylori, peptic ulcer, aspirin ingestion, hookworm, hereditary telangiectasia, carcinoma of the stomach, caecum or colon, ulcerative colitis, angiodysplasia, Meckel diverticulum, diverticulosis, haemorrhoids, etc. Renal tract: haematuria (e.g. renal or bladder lesion), haemoglobinuria (e.g. paroxysmal nocturnal haemoglobinuria) Pulmonary tract: overt haemoptysis, idiopathic pulmonary haemosiderosis Widespread bleeding disorders Self-inflicted Malabsorption Gluten-induced enteropathy (child or adult), gastrectomy, atrophic gastritis, chronic inflammation, clay eating, etc. Dietary Especially vegetarian diet

Causes of iron deficiency (Table 3.5) Diet Defective intake of iron is rarely the sole or major cause of iron deficiency in adults in Western communities. The diet may contain insufficient or poorly available iron as a result of poverty, religious tenets or food faddism. Iron deficiency is more likely to develop in subjects taking a largely vegetarian diet – the majority of the world’s population – who also have increased physiological demands for iron. Increased physiological iron requirements Iron deficiency is common in infancy, when demands for growth may be greater than dietary supplies. It is aggravated by prematurity, infections and delay in mixed feeding. It is also frequent in adolescence, in females and in pregnancy (Table 3.2). The fetus acquires about 280 mg of iron and a further 400–500 mg is required for the temporary expansion of maternal red cell mass. Another 200 mg of iron is lost with the placenta and with bleeding at delivery. Although iron absorption increases throughout pregnancy and increased requirements are partly offset by amenorrhoea, this may not be sufficient to meet the resultant net maternal outlay of over 600 mg iron. Blood loss Blood loss is the most common cause of iron deficiency in adults. A loss of more than about 6–8 mL of blood (3–4 mg iron) daily becomes of importance, as this equals the maximum amount of iron that can be absorbed from a normal diet. The 39

Postgraduate Haematology

loss is usually from the genital tract in women or from the gastrointestinal tract in either sex. The most common cause on a worldwide basis is infestation with hookworm, in which anaemia is related to the degree of infestation. In the UK, menorrhagia, haemorrhoids and peptic ulceration are common, as well as gastric bleeding because of salicylates or other nonsteroidal anti-inflammatory drugs, hiatus hernia, colonic diverticulosis and bowel tumours (Table 3.5). Some unusual causes of blood loss deserve mention. Cow’s milk intolerance in infants may lead to gastrointestinal haemorrhage. Self-induced haemorrhage may occur as an unusual form of Munchausen syndrome. Chronic intravascular haemolysis, such as that in paroxysmal nocturnal haemoglobinuria or mechanical haemolytic anaemia, may be a serious source of urinary iron loss. Malabsorption Malabsorption may be the primary cause of iron deficiency or it may prevent the body adjusting to iron deficiency from other causes. Dietary iron is poorly absorbed in gluten-induced enteropathy, in both children and adults. Gluten-induced enteropathy is encountered in about 5% of patients presenting with unexplained iron deficiency anaemia and, conversely, about 50% of patients with newly diagnosed coeliac disease have coexistent iron deficiency anaemia. Patients with this disease often show decreased or no response to oral therapy with inorganic iron. Helicobacter pylori gastritis appears to be a common cause of iron deficiency, responding favourably to eradication with triple therapy. Helicobacter pylori gastritis inhibits gastric hydrochloric acid secretion, interfering with the solubilization and absorption of inorganic food iron but it is also possible that gastrointestinal blood loss plays a significant role in the causation of iron deficiency associated with H. pylori infection. Lastly, achlorhydria associated with autoimmune gastritis, an entity preceding and closely related to pernicious anaemia, is an important cause of iron malabsorption due to impaired food iron solubilization. It is encountered in about 20% of patients with unexplained or refractory iron deficiency anaemia, mostly women of fertile age in whom achlorhydria aggravates the consequences of menstrual blood loss. Iron deficiency occurs in congestive heart failure due to malabsorption and iron loss and these patients may also have the features of the anaemia of chronic disorders. Intravenous iron benefits functional capacity and quality of life even in the absence of anaemia.

Management of iron deficiency Management entails (i) identification and treatment of the underlying cause and (ii) correction of the deficiency by therapy with inorganic iron. Iron deficiency is commonly due to blood loss and, wherever possible, the site of this must be identified and the lesion treated. 40

Oral therapy In most patients, body stores of iron can be restored by oral iron therapy. Iron is equally well absorbed from several simple ferrous iron salts, and as ferrous sulphate is the cheapest, this is the drug of first choice – 200 mg of ferrous sulphate contains 67 mg of iron. Where smaller doses are required, 300 mg of ferrous gluconate provides 36 mg of iron. It is usual to give 100–200 mg of elemental iron each day to adults and about 3 mg/kg per day as a liquid iron preparation to infants and children. The side-effects of oral iron, such as nausea, epigastric pain, diarrhoea and constipation, are related to the amount of available iron they contain. If iron causes gastrointestinal symptoms, these can usually be ameliorated by reducing the dose or taking the iron with food, but this also reduces the amount absorbed. Enteric-coated and sustained-release preparations should not be used, as much of the iron is carried past the duodenum to sites of poor absorption. Iron reduces absorption of tetracyclines (and vice versa) and of ciprofloxacin. The minimum rate of response should be a 20 g/L rise in haemoglobin every 3 weeks, and the usual rate is 1.5–2.0 g/L daily. This will be slower when the dose tolerated is less than 100 mg/day, but this is seldom of clinical importance. It is usually necessary to give iron for 3–6 months to correct the deficit of iron in circulating haemoglobin and in stores (shown by a rise in serum ferritin to normal). Failure to respond to oral iron is most commonly due to the patient not taking it, although there may be continued haemorrhage or malabsorption. In non-responding patients it is important to reassess the diagnosis to exclude other causes of microcytic anaemia such as iron-loading anaemias. For instance, many patients with thalassaemia trait, sideroblastic anaemia or other anaemias have been treated with iron before haemoglobin studies, bone marrow examination or other tests have revealed the correct diagnosis. A poor response may also be obtained if the patient has an infection, renal or hepatic failure, an underlying malignant disease or anaemia of inflammation due to high hepcidin levels (which inhibits absorption of therapeutic oral iron) and any other cause of anaemia in addition to iron deficiency. Parenteral iron therapy This is usually unnecessary, but it may be given if subjects genuinely cannot tolerate oral iron, particularly if gastrointestinal disease, such as inflammatory bowel disease, is present. It is also occasionally necessary in gluten-induced enteropathy and when it is essential to replete body stores rapidly (e.g. where severe iron deficiency anaemia is first diagnosed in late pregnancy) or when oral iron cannot keep pace with continuing haemorrhage (e.g. in patients with hereditary haemorrhagic telangiectasia). Patients with chronic renal failure who are being treated with recombinant erythropoietin are also likely to require parenteral iron therapy. In this situation, the demand for iron by the expanded erythron may outstrip the ability to

Iron metabolism, iron deficiency and disorders of haem synthesis

mobilize iron from stores, leading to a ‘functional’ iron deficiency. Increased red cell loss at dialysis contributes to iron needs and oral iron therapy is usually inadequate to prevent an impaired response to erythropoietin. Serum ferritin in this setting is, as in other chronic inflammatory conditions, an unreliable indicator of iron deficiency. The use of transferrin iron saturation, percentage of hypochromic red cells or reticulocyte haemoglobin content for detection of functional iron deficiency is discussed on p. 38. A transferrin saturation of less than 20% indicates functional iron deficiency. Intravenous ferric carboxymaltose has been shown to benefit patients with congestive heart failure and iron deficiency with or without anaemia. From all parenteral preparations, the iron complex is taken up by macrophages of the reticuloendothelial system, from which iron is released to circulating transferrin, which then transports it to the marrow. In the UK, three preparations are available. Iron dextran (CosmoFer) is given intravenously by slow injection or infusion or deep intramuscularly into the gluteal muscle. An iron–sucrose complex, Venofer, is given by slow intravenous infusion or injection. The deficit in body iron should be calculated from the degree of anaemia; it is usually 1–2 g. In patients receiving erythropoietin treatment in chronic renal failure, smaller intravenous doses of Venofer (25–150 mg/ week) may be used, with regular monitoring of serum ferritin to avoid iron overload. Ferrinject is a macromolecular iron(III)hydroxide carbohydrate complex (molecular weight approximately 150 000). It can be administered as an intravenous bolus (maximum single dose 200 mg) or slow infusion (maximum single dose 1000 mg). Newer intravenous preparations including ferumoxytol and ferrous gluconate (Ferrlecit) may become available. Parenteral iron should not be used if there is a history of allergy as anaphylaxis occasionally occurs. For iron dextran, a test dose should therefore be given slowly, followed by close medical supervision of the rest of the infusion. Headache, flushing, nausea, skin rashes, urticaria, shivering, general aches and pains, dyspnoea and syncope are possible immediate adverse effects. Delayed reactions, including arthralgia, fever and lymphadenopathy, are well described and can persist for several days. An exacerbation of rheumatoid arthritis may also be precipitated.

Iron refractory iron deficiency anaemia Homozygous or doubly heterozygous germline frameshift, splice junction or missense mutations of matriptase-2 (TMPRSS6) are a cause of iron refractory iron deficiency anaemia. The mutations may affect different conserved domains of the protein, including a trypsin-like serine protease domain. The patients show a microcytic hypochromic anaemia with normal or raised serum and urine hepcidin levels and low serum iron and percentage saturation of iron-binding capacity.

The patients absorb iron poorly and are refractory to oral iron therapy but are partially responsive to parenteral iron. A microcytic hypochromic anaemia with liver iron overload has also been described in a few patients with homozygous or doubly heterozygous mutations of DMT1. Liver iron stores are increased but erythroid iron utilization is impaired and serum hepcidin levels are low for the degree of iron overload. These patients may respond to erythropoietin injections. The patients are susceptible to infections. Deficiency of serum transferrin due to mutations of the transferrin gene causes a hypochromic microcytic anaemia with tissue iron overload caused by increased plasma nontransferrin-bound iron and low hepcidin levels. Treatment has been with infusions of fresh frozen plasma or apotransferrin. Deficiency of caeruloplasmin also causes a mild hypochromic microcytic anaemia with iron overload in the liver and progressive neurodegeneration. There is failure of ferroxidase activity, which impairs iron mobilization from stores.

Pathological alterations in haem synthesis Porphyrias These are a group of inherited or acquired diseases, each characterized by a partial defect in one of the enzymes of haem synthesis (see Chapter 2). Increased amounts of the intermediates of haem synthesis accumulate, the disorders being classified by whether the effects are predominantly in the liver or the erythron (Table 3.6). A full discussion of these disorders is beyond the scope of this chapter, but those with a particular haematological overlap are mentioned briefly. Congenital erythropoietic porphyria This is a very rare autosomal recessive disorder that is due to reduced uroporphyrinogen III synthase activity. Most patients are heteroallelic for mutations in the uroporphyrinogen III synthase gene. A single case with a germline mutation of the X-linked erythroid-specific transcription factor GATA binding protein (GATA-1) has been described. Large amounts of porphyrinogens accumulate, and their conversion by spontaneous oxidation to photoactive porphyrins leads to severe, and disfiguring, cutaneous photosensitivity and dermatitis, as well as a haemolytic anaemia with splenomegaly. Increased amounts of uroporphyrin and coproporphyrin, mainly type I, are found in bone marrow, red cells, plasma, urine and faeces. Ring sideroblasts have been found in the marrow in some cases but rarely in large numbers. The age of onset and clinical severity of the disease are highly variable, ranging from non-immune hydrops fetalis to a later onset in which there are only cutaneous lesions. Treatment, including avoidance of sunlight and splenectomy to improve red cell survival, is only partially effective. High-level 41

Postgraduate Haematology

Table 3.6 Human porphyrias. Form Hepatic Acute intermittent porphyria Hereditary coproporphyria Porphyria variegate Porphyria cutanea tarda Erythropoietic Congenital erythropoietic porphyria Erythropoietic protoporphyria

Inheritance

Enzyme defect

Clinical features*

Autosomal dominant Autosomal dominant Autosomal dominant Acquired or (rare) autosomal dominant

Porphobilinogen deaminase Coproporphyrinogen oxidase Protoporphyrinogen oxidase Uroporphyrinogen decarboxylase

A A+P A+P P

Autosomal recessive

Uroporphyrinogen cosynthase

P

Autosomal dominant

Ferrochelatase

P

*Acute attacks (A) of the gastrointestinal and/or nervous system are related to the accumulation of porphyrin precursors (δaminolaevulinic acid and porphobilinogen). Photosensitive skin lesions (P) are seen when the level of the enzyme defect in the haem synthetic pathway leads to the accumulation of formed porphyrins.

blood transfusions to suppress erythropoiesis (combined with iron chelation therapy) have been used to reduce porphyrin production sufficiently to abolish the clinical symptoms. Allogeneic bone marrow transplantation has been successful. Erythropoietic protoporphyria This is the most common erythropoietic porphyria and is usually caused by an autosomal dominant inherited deficiency of ferrochelatase, which results in increased free (not Zn) protoporphyrin concentrations in bone marrow, red cells, plasma and bile. Bone marrow reticulocytes are the primary source of the excess protoporphyrin. This leaks from cells and is excreted in the bile and faeces. Molecular analysis of the ferrochelatase gene has revealed a variety of missense, nonsense and splicing mutations as well as deletions and insertions. The onset of the disease is usually in childhood. Expression of the gene is variable, and photosensitivity and dermatitis range from mild or absent to moderate in degree. There is little haemolysis, but a mild hypochromic anaemia may occur, and accumulation of protoporphyrins can occasionally lead to severe liver disease. Treatment is by the avoidance of sunlight; β-carotene may also diminish photosensitivity. Iron deficiency should be avoided as this may increase the amount of free protoporphyrin. Other groups of patients with a variant of erythropoietic protoporphyria have been discovered to have mutations of mitoferrin or gain-of-function mutation of ALAS2. Porphyria cutanea tarda This is the most common of the hepatic porphyrias and occurs worldwide. The incidence in the UK has been estimated at 2–5 per million. Type I or ‘sporadic’ porphyria cutanea tarda (PCT) accounts for 80% of cases of PCT. The underlying metabolic 42

abnormality is decreased activity of uroporphyrinogen decarboxylase (UROD) in the liver. Type II disease is an autosomal dominant disorder caused by mutations in the UROD gene. Type III disease is a rare familial form and appears to result from unknown inherited defects that affect hepatic UROD activity. There is a marked increase in porphyrins in liver, plasma, urine and faeces. In the urine, uroporphyrin and heptacarboxylporphyrin predominate, with lesser amounts of coproporphyrin and pentacarboxylporphyrin and hexacarboxylporphyrin. The disease is characterized by photosensitivity and dermatitis. It is precipitated in middle or later life, more often in men than women, by factors such as liver disease, alcohol excess or estrogen therapy. A modest increase in liver iron is a common feature. Either the homozygous or heterozygous presence of the C282Y and H63D mutations in the HFE gene may predispose to the development of PCT. Prevalence of the C282Y mutation is increased in both sporadic (type I) and familial (type II) PCT. In the UK, only homozygosity for C282Y (found in about 25% of patients) is significantly more common than in the general population (0.7%). In southern Europe, where C282Y is much less common, the H63D mutation is associated with PCT. Iron is known to inhibit UROD. Removal of the iron by repeated phlebotomy is standard treatment, usually leading to remission.

Lead poisoning Chronic ingestion of lead in humans causes an anaemia that is usually normochromic or slightly hypochromic. Red cell lifespan is shortened and there is a mild rise in reticulocytes, but jaundice is rare. Basophilic stippling is a characteristic, though not universal, finding and it is thought to be due to

Iron metabolism, iron deficiency and disorders of haem synthesis

precipitation of RNA, resulting from inhibition of the enzyme pyrimidine 5′-nucleotidase. Siderotic granules, and occasionally Cabot rings, are found in circulating red cells. The bone marrow shows increased sideroblasts, in some patients with ring sideroblasts. Red cell protoporphyrin and coproporphyrin are raised, as is urinary excretion of ALA, coproporphyrin III and uroporphyrin I. The cause of the anaemia appears to be multifactorial. Haemolysis, probably due to the blocking of sulphydryl groups with consequent denaturation of structural proteins, and damage to mitochondria, with defective haemoglobin production due to inhibition of the enzymes of haem synthesis, are the major factors.

roblasts in the primary forms of anaemia contain iron granules arranged in a ring around the nucleus (Figure 3.7). These ring sideroblasts (Table 3.8) (more than four perinuclear granules per cell and covering one-third or more of the nuclear circumference) are the diagnostic feature of the anaemia. Ring sideroblasts may comprise a small (< 15%) population of the erythroblasts in a wide variety of clinical disorders.

Sideroblastic anaemia The sideroblastic anaemias comprise a group of refractory anaemias (Table 3.7) in which there are variable numbers of hypochromic cells in the peripheral blood, with an excess of iron in the bone marrow; at least 15% of the developing eryth-

Table 3.7 Sideroblastic anaemias. Hereditary X-linked Erythroid-specific ALAS2 (Xp11.21) mutations Associated with spinocerebellar ataxia: mitochondrial ATPbinding cassette (ABCB7) mutations Autosomal Thiamine responsive, THTR-1 (SLC19A2) mutations: DIDMOAD syndrome Glutaredoxin-5 (GLRX5) mutations Associated mitochondrial myopathy: pseudouridine synthase-1 mutations SLC25 family transporter mutations

Figure 3.7 Sideroblastic anaemia. Erythroblasts showing perinuclear rings of iron (Perls stain). Table 3.8 Siderocytes and sideroblasts. Siderocyte

Normal sideroblast

Mitochondrial Pearson (marrow–pancreas) syndrome Kearns–Sayre syndrome Acquired Primary Refractory anaemia with ring sideroblasts Secondary Drugs: isoniazid, pyrazinamide, cycloserine, chloramphenicol, penicillamine Mitochondrial toxicity: alcohol, lead poisoning Copper deficiency systemic disease: carcinoma, rheumatoid arthritis DIDMOAD, diabetes insipidus, diabetes mellitus, optic atrophy and deafness.

Abnormal sideroblasts

Mature red cell containing one or more siderotic granules (Pappenheimer bodies) Nucleated red cell containing one or more siderotic granules, granules few, difficult to see, randomly distributed in the cytoplasm, reduced proportion of sideroblasts in iron deficiency and anaemia of chronic disorders Cytoplasmic iron deposits (ferritin aggregates): increased granulation, granules larger and more numerous than normal, easily visible and randomly distributed, proportion of sideroblasts usually parallels the percentage saturation of transferrin (e.g. haemolytic anaemia, megaloblastic anaemia, iron overload, thalassaemia disorders) Mitochondrial iron deposits (non-ferritin iron): ring sideroblasts in inherited and acquired sideroblastic anaemias

43

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Inherited sideroblastic anaemias These are rare disorders manifesting mainly in males, usually in childhood or adolescence, but occasionally presenting late in life, when they need to be distinguished from the more common acquired form termed ‘refractory anaemia with ring sideroblasts’.

X-linked ALAS2 mutations In most reported families, inheritance has followed an X-linked pattern. More than 25 different mutations of the gene for erythroid-specific ALAS2, located at Xp11.21, have been identified. All have been single-base substitutions. Most lead to changes in protein structure, causing instability or loss of function. They are found scattered over the seven exons (out of 11) encoding the C-terminal, catalytically active part of the protein. Mutations affecting the promoter have also been shown to cause disease. Function may be rescued to a variable degree by administration of pyridoxal phosphate (the essential cofactor for ALAS2), the best responses occurring when the mutation affects the pyridoxal phosphate-binding domain of the enzyme. The response is better if iron overload is reduced by phlebotomy or chelation. Female carriers of X-linked sideroblastic anaemia may show partial haematological expression, usually with only mild or no anaemia, although rarely a severe dimorphic anaemia occurs. This may depend on variation in the severity of the defect, as well as the degree of lyonization of the affected X chromosome. Late onset in some patients suggests that the degree of lyonization may change with age. Iron loading may also aggravate the defect in haem synthesis in both males and females with sideroblastic anaemia. Patients with X-linked sideroblastic anaemia show a hypochromic, often microcytic, anaemia. There may be a few circulating siderocytes, normoblasts and cells with punctate basophilia, but these features become pronounced only if the spleen has been removed. The bone marrow shows erythroid hyperplasia and the erythroblasts tend to be microcytic with a vacuolated cytoplasm. There are more than 15% ringed sideroblasts. The ineffective erythropoiesis is not usually accompanied by bone deformities, but some bossing of the skull and enlargement of the facial bones may result from the erythroid expansion. The spleen may be enlarged. Patients may present with severe iron overload even when the anaemia is relatively mild, but the rate of iron loading is accelerated if red cell transfusions are needed. Iron loading, however, aggravates the anaemia. ABCB7 mutations A rare form of X-linked sideroblastic anaemia is caused by abnormalities in the ATP-binding cassette transporter gene (ABCB7) at Xq13.3. This form is associated with early-onset, non-progressive cerebellar ataxia. A useful diagnostic distinction is the presence within the red cells of increased zinc pro44

toporphyrin, despite adequate iron stores, rather than the low/ normal levels found in patients with abnormalities in ALAS2. The anaemia is mild to moderately severe. Three abnormalities of protein structure have been described, and these lie within 34 amino acids of one another at the C-terminal end of the transmembrane domain. The enzyme deficiency induces disruption in the maturation of cytosolic iron–sulphur clusters. The ataxia may be due to iron damage to mitochondria in neural cells.

Autosomal Mitochondrial myopathy and sideroblastic anaemia This results from a homozygous mutation in the nuclearencoded gene for pseudouridine synthase. As in Pearson syndrome, there are defects of the mitochondrial electron transport chain affecting reduced access to ferrochelatase. It has been proposed that deficient pseudouridylation of mitochondrial tRNAs underlies this condition. Abnormalities of a high-affinity transporter of thiamine A gene (SLC19A2) encoding a putative thiamine transporter (THTR-1) that is widely and variably expressed has now been mapped to the long arm of chromosome 1 (1q23.3). Abnormalities in this gene are responsible for thiamine-responsive megaloblastic anaemia (Roger syndrome). This syndrome is inherited in an autosomal recessive manner and combines diabetes insipidus, diabetes mellitus, optic atrophy and deafness (DIDMOAD), which respond in varying degrees to pharmacological doses of thiamine (vitamin B1). Ring sideroblasts in varying numbers are typically present and onset is usually in childhood, although some symptoms may be present in infancy. A direct link between the presence of the mutation and mitochondrial iron loading has yet to be demonstrated. There are still a substantial number of cases of inherited sideroblastic anaemias in which the exact underlying genetic defect remains obscure. These cases may or may not show sexlinked inheritance and often show a macrocytic or dimorphic picture. Mutations of glutaredoxin-5 (GLRX5) This enzyme participates in iron–sulphur cluster formation. A single patient has been described with autosomal recessive hypochromic microcytic anaemia with ring sideroblasts with inherited mutations of the enzyme. SLC25A38 mutations This protein is a transporter that may be involved in glycine transfer to the mitochondria, an essential step in synthesis of ALA. Mutation in congenital sideroblastic anaemia is described.

Mitochondrial DNA mutations Deletions of mitochondrial DNA, sometimes associated with duplications, are known to be the cause of the Pearson marrow–

Iron metabolism, iron deficiency and disorders of haem synthesis

pancreas syndrome, typically consisting of sideroblastic anaemia, pancreatic exocrine dysfunction and lactic acidosis. This is a severe disorder of early onset, presenting usually with failure to thrive, persistent diarrhoea and lactic acidosis. All haemopoietic cell lineages can be affected, and the anaemia is typically macrocytic with prominent vacuoles in cells of both myeloid and erythroid lineages. Mitochondrial DNA has its own genetic code and encodes mitochondrial tRNA and ribosomal RNA as well as several mitochondrial proteins. In Pearson syndrome, deletions may encompass tRNA as well as mitochondrial genes and therefore have an effect on the function of all mitochondrion-encoded proteins, causing considerable loss of mitochondrial function. The presence of many different mitochondria within nucleated cells enable the coexistence of normal and abnormal species, the proportion of which is likely to vary within different tissues, a phenomenon known as heteroplasmy. The extent to which different tissues are affected depends to some extent on this proportion and detection often requires the study of different tissues. Inheritance is difficult to determine for the same reason and most cases are described as of ‘sporadic’ occurrence.

thesis and in some patients causes ring sideroblast formation, presumably as a result of impaired haem formation in the mitochondria. Lead inhibits several enzymes involved in haem synthesis and may damage structural mitochondrial proteins. In some cases, ring sideroblasts are visible in the marrow. Ring sideroblasts may occur in erythropoietic porphyrias (see p. 41). Treatment

Pyridoxine Some patients with X-linked sideroblastic anaemia respond to pyridoxine, given initially in doses of 100–200 mg daily. The response is usually partial. Patients may require only small doses (less than 10 mg daily) to maintain a higher haemoglobin concentration. Pyridoxine therapy is almost always ineffective in refractory anaemia with ring sideroblasts. However, some secondary sideroblastic anaemias may be completely reversed by pyridoxine therapy. This has been described in alcoholism, haemolytic anaemia and gluten-induced enteropathy, as well as in patients receiving antituberculous chemotherapy, in whom the drugs have been stopped and pyridoxine administered.

Acquired sideroblastic anaemias (see also Chapter 28)

Refractory anaemia with ring sideroblasts This is a form of myelodysplasia and arises as a clonal disorder of haemopoiesis. The anaemia is often macrocytic with raised red cell protoporphyrin concentrations, in contrast to X-linked sideroblastic anaemia. Marked erythroid hyperplasia may be present, together with increased iron stores. In these patients, abnormalities in the white cell or platelet precursors are usually absent and the risk of transformation to acute myeloid leukaemia appears less than in other myelodysplastic disorders. Smaller numbers (< 15%) of ring sideroblasts may be present in patients with any of the other myelodysplastic and myeloproliferative diseases. Recent data suggest that acquired defects of mitochondrial DNA may underlie iron transport abnormalities in refractory anaemia with ring sideroblasts. Secondary sideroblastic anaemias Sideroblastic anaemia associated with pyridoxine deficiency has been described, although not completely documented, in a few patients with gluten-induced enteropathy, in pregnancy, and with haemolytic anaemias, such as sickle cell disease and mechanical or autoimmune haemolytic anaemia. Sideroblastic anaemia may be found as a complication of antituberculous chemotherapy, particularly with isoniazid and cycloserine (pyridoxine antagonists). Sideroblastic anaemia occurs in alcoholism if there is associated malnutrition and folate deficiency. Suggested mechanisms include interference with haem formation and pyridoxine metabolism. The anaemia rapidly reverses with abstinence from alcohol, a normal diet and pyridoxine therapy. Chloramphenicol inhibits mitochondrial protein syn-

Other forms of treatment Folic acid may benefit patients with secondary anaemia. For refractory patients, the anaemia may remain stable and, if the patient is transfusion independent, no treatment is needed. Patients requiring regular red cell transfusions require iron chelation therapy. Iron loading may aggravate the anaemia and, in some patients, improvement in the anaemia has followed iron removal by phlebotomy or iron chelation therapy. Splenectomy should usually be avoided, as it does not benefit the anaemia and leads to persistently high platelet counts postoperatively, with a high incidence of thromboembolism.

Selected bibliography Anderson GJ, Frazer DM (2006) Iron metabolism meets signal transduction. Nature Genetics 38: 503–4. Andrews NC (2008) Forging a field: the golden age of iron biology Blood 112: 219–30. Andriopoulos B Jr (2009) BMP6 is a key endogenous regulator of hepcidin expression and iron metabolism. Nature Genetics 41: 482–7. Anker SD, Colet JC, Filippatos G et al. (2009) Ferric carboxymaltose in patients with heart failure and iron deficiency. New England Journal of Medicine 361: 2436–48. Babitt JL, Huang FW, Xia Y et al. (2007) Modulation of bone morphogenetic protein signalling in vivo regulates systemic iron balance. Journal of Clinical Investigation 117: 1933–9. Barrett TG, Bundey S, Macleod AF (1995) Neurodegeneration and diabetes: UK nationwide study of Weifrom (DIDMOAD) syndrome. Lancet 346: 1458–63. 45

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Beguin Y (2003) Soluble transferrin receptor for the evaluation of erythropoiesis and iron status. Clinica Chimica Acta 329: 9–22. Bottomley, S (2006) Congenital sideroblastic anemias. Current Hematology Reports 5: 41–49 Camaschella, C (2008) Recent advances in the understanding of inherited sideroblastic anaemia. British Journal of Haematology 143: 27–38. Cazzola M (2002) Hereditary hyperferritinaemia/cataract syndrome. Best Practice and Research. Clinical Haematology 15: 385–98. De Domenico I, McVey Ward D, Kaplan J (2008) Regulation of iron acquisition and storage: consequences for iron-linked disorders. Nature Reviews. Molecular Cell Biology 9: 72–81. Drysdale J, Arosio P, Invernizzi R et al. (2002) Mitochondrial ferritin: a new player in iron metabolism. Blood Cells, Molecules and Diseases 29: 376–83. Du X, She E, Gelbart T (2008) The serine protease TMPRSS6 is required to sense iron deficiency. Science 320: 1088–92. Finberg KE, Heeney MM, Campagna DR et al. (2008) Mutations in TMPRSS6 cause iron-refractory iron deficiency anaemia (IRIDA). Nature Genetics 40: 569–71. Goswami T, Andrews NC (2006) Hereditary hemochromatosis protein, HFE, interaction with transferrin receptor 2 suggests a molecular mechanism for mammalian iron sensing. Journal of Biological Chemistry 281: 28494–8. Hershko C, Hoffbrand AV, Keret D (2005) Role of autoimmune gastritis, Helicobacter pylori and coeliac disease in refractory or unexplained iron deficiency anaemia. Haematologica 90: 585–95. Hoffbrand AV (ed.) (2009) Recent advances in the understanding of iron metabolism and iron-related diseases. Acta Haematologica 122: 75–184, 12 articles covering all aspects of iron metabolism and related diseases. Iolascon A, De Falco L, Beaumont C (2009) Molecular basis of inherited microcytic anaemia due to defects in iron acquisitions or haem synthesis. Haematologica 94: 395–408. Lakhal S, Talbot NP, Crosby A et al. (2009) Regulation of growth differentiation factor 15 expression by intracellular iron. Blood 113: 1555–63. Lebay V, Ras T, Baran D et al. (1999) Mutations in SCL19AZ cause thiamine-responsive megaloblastic anaemia associated with diabetes mellitus and deafness. Nature Genetics 22: 300–4.

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Locatelli F, Aljama P, Bárány P et al. (2004) Revised European best practice guidelines for the management of anaemia in patients with chronic renal failure. Nephrology, Dialysis, Transplantation 19 (Suppl. 2): 1–47. Lozoff B, De Andraca I, Castillo M et al. (2003) Behavioural and developmental effects of preventing iron-deficiency anaemia in healthy full-term infants. Pediatrics 112: 846–54. Mehta A, Beck M, Eyskens F et al. (2010) Fabry disease: a review of current management strategies. Quarterly Journal of Medicine, epub ahead of print. Meynard D, Kautz L, Darnaud V et al. (2009) Lack of the bone morphogenetic protein BMP6 induces massive iron overload. Nature Genetics 41: 478–81. Online Mendelian Inheritance in Man (OMIM) Available at www. ncbi.nlm.nih.gov/OMIM/. Knowledge base of human genes and genetic disorders. Peyssonnaux C, Zinkernagel AS, Schuepbach RA et al. (2007) Regulation of iron homeostasis by the hypoxia-inducible transcription factors (HIFs). Journal of Clinical Investigation 117: 1926–32. Ramsay AJ, Hooper JD, Folgueras AR et al. (2009) Matriptase-2 (TMPRSS6): a proteolytic regulator of iron homeostasis. Haematologica 94: 840–9. Silvestri L, Pagani A, Nai A et al. (2008) The serine protease matriptase-2 (TMPRSS6) inhibits hepcidin activation by cleaving membrane hemojuvelin. Cell Metabolism 6: 502–11. Srai SK, Bomford A, McArdle HJ (2002) Iron transport across cell membranes: molecular understanding of duodenal and placental iron transport. Best Practice and Research. Clinical Haematology 15: 243–59. Tanno T, Bhanu NV, Oneal PA et al. (2007) High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin. Nature Medicine 13: 1096–101. WHO (2001) Iron Deficiency Anaemia. Assessment, Prevention and Control. A Guide for Programme Managers. World Health Organization, Geneva. Wrighting DM, Andrews NC (2006) Interleukin-6 induces hepcidin expression through STAT3. Blood 108: 3204–9. Zhang AS, Enns CA (2009) Iron homeostasis: recently identified proteins provide insight into novel control mechanisms. Journal of Biological Chemistry 284: 711–15.

CHAPTER 4

4

Iron overload Clara Camaschella1 and A Victor Hoffbrand2 1

Vita-Salute University, Milan, Italy Royal Free and University College Medical School, Royal Free Hospital, London, UK

2

Introduction, 47 Genetic haemochromatosis, 47 Classification, 47 Type 1 haemochromatosis, 47 Type 2 (juvenile) haemochromatosis, 52 Type 3 haemochromatosis, 52 Type 4 haemochromatosis, 52

Neonatal haemochromatosis, 52 Increased iron intake, 52 Other causes of iron overload, 53 Iron and neurodegeneration, 53 Hereditory hyperferritinaemia–cataract syndrome, 53

Introduction Excessive iron accumulation may eventually lead to tissue damage. Iron overload of the parenchymal cells of the liver commonly arises when there is excessive iron absorption, whereas iron administered parenterally (e.g. as multiple transfusions) is first taken up in senescent red cells by macrophages. However, there is no absolute distinction between the two sources of iron loading, as iron in macrophages is slowly released to transferrin, from which it can be taken up by parenchymal cells. Causes of iron overload are shown in Table 4.1. Severe iron overload, arbitrarily defined as an excess of more than 5 g, is confined to the genetic haemochromatoses, together with the iron-loading anaemias and sub-Saharan African dietary iron overload.

Genetic haemochromatosis Classification Genetic haemochromatosis is now classified according to the genetic defect causing iron overload (Table 4.2). The vast majority of cases are of type 1, involving the HFE gene (see Chapter 3). In populations of northern European origin, about 90% of patients with haemochromatosis are homozygous for the HFE Cys282Tyr mutation (C282Y). In southern Europe, homozygosity for C282Y is found in only about 60% of patients

Postgraduate Haematology: 6th edition. Edited by A. Victor Hoffbrand, Daniel Catovsky, Edward G.D. Tuddenham, Anthony R. Green © 2011 Blackwell Publishing Ltd.

Iron loading anaemias, 53 Tests of body iron burden, 54 Tests of organ function, 56 Iron chelation therapy, 56 Thalassaemia intermedia, 59 Acute iron poisoning, 59 Selected bibliography, 59

with haemochromatosis. Type 2, the severe juvenile haemochromatosis caused by mutations of the hemojuvelin or hepcidin genes, type 3, due to mutations of transferrin receptor 2, and type 4, due to mutations of ferroportin, are all very rare disorders reported mainly in European countries but also identified in some Asian patients with novel mutations. Types 1, 2 and 3 are autosomal recessive diseases and share common features due to hepcidin deficiency, including high transferrin saturation and hepatocyte iron accumulation. Type 4 haemochromatosis, inherited as a dominant condition, is a heterogeneous disease with variable clinical phenotype.

Type 1 haemochromatosis This is one of the most common genetic conditions found in populations of northern European origin but it also occurs in African and Asian populations at very low gene frequency. In the UK, about one in eight people are carriers of the C282Y mutation of the HFE gene, and about 1 in 200 are homozygous for this mutation. Homozygosity is strongly associated with haemochromatosis, with about 90% of patients with genetic iron overload having this genotype. In homozygotes, there is a gradual accumulation of iron, leading to tissue damage, which may present as cirrhosis of the liver, diabetes, hypogonadism, cardiomyopathy, arthritis and a slate-grey skin pigmentation. Hepatocellular carcinoma develops in 25% of established cases with cirrhosis. Most patients present between the ages of 40 and 60 years, but the clinical penetrance is low (see p. 50). Full phenotypic expression of the disorder is dependent on other factors, including dietary iron intake, blood donations or blood loss and other genetic factors modifying the genotype. Menstrual losses and pregnancies account for a generally delayed onset in women. 47

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Nature of the defect A defect in the regulation of intestinal iron absorption leading to increased mucosal iron transfer to plasma is due to deficient hepcidin production. The responsible gene, HFE, is an atypical MHC class I-like gene that maps to the short arm of chromosome 6. The reported association with HLA-A3 and, to a lesser extent, B7 suggested a founder mutation in a chromosome carrying the A3, B7 haplotype. In over 80% of patients there is homozygosity for a G→A substitution at nucleotide 845 of the HFE gene that results in a cysteine to tyrosine substitution at Table 4.1 Causes of iron overload. Severe iron overload (> 5 g excess) Excess iron absorption Hereditary haemochromatosis Massive ineffective erythropoiesis (e.g. β-thalassaemia intermedia, sideroblastic anaemia, congenital dyserythropoietic anaemia) Increased iron intake Sub-Saharan dietary iron overload (in combination with a genetic determinant of increased absorption) Excess parenteral iron therapy Repeated red cell transfusions Congenital anaemias (e.g. β-thalassaemia major, sickle cell anaemia, red cell aplasia) Acquired refractory anaemias (e.g. myelodysplasia, aplastic anaemia) Modest iron overload (< 5 g excess) Chronic liver disease (e.g. alcoholic cirrhosis) Porphyria cutanea tarda Rare genetic disorders of iron metabolism (e.g. atransferrinaemia, acaeruloplasminaemia, DMT1 mutations) Focal iron overload* Pulmonary haemorrhage, idiopathic pulmonary haemosiderosis Chronic haemoglobinuria (e.g. paroxysmal nocturnal haemoglobinuria) *May occur in association with general body iron deficiency.

amino acid 282 (C282Y). A second variant (187C→G) results in a histidine to aspartic acid substitution at amino acid 63 (His63Asp or H63D). This is carried by about 20% of the general population. In the UK, about 90% of patients presenting with haemochromatosis are homozygous for HFE C282Y, and another 4% are compound heterozygotes for the two mutations. That HFE was the haemochromatosis gene was confirmed by the demonstration that HFE knockout mice and mice homozygous for the C282Y mutation develop iron overload. Hepcidin synthesis requires expression of HFE and in mice lacking HFE or expressing the C282Y protein hepcidin is low or at least inappropriate to the degree of iron overload (see Chapter 3). In addition, tissue-specific inactivation of HFE protein in hepatocytes but not in duodenal cells or macrophages leads to iron overload, indicating a specific function of HFE in the liver. Hepcidin is a negative regulator of iron absorption by binding ferroportin and causing its internalization and lysosomal degradation. Lack of hepcidin upregulates ferroportin expression in duodenal mucosa, thus increasing mucosal iron transfer to transferrin and leaving the duodenal cells iron deficient. Hepcidin also controls iron release from macrophages. Low hepcidin explains the findings in the early stages of haemochromatosis: increased iron absorption, a raised serum iron and a paucity of iron in macrophages. Most recent data suggest that all types of haemochromatosis (except Type 4; Table 4.2) are characterized by low hepcidin secretion, the defect being the most severe in juvenile haemochromatosis. The more recent model of the pathophysiology of the disease suggests that HFE competes with diferric transferrin for TFR1 binding and that when diferric transferrin binds TFR1, HFE is free to bind TFR2, thus forming a complex that results in hepcidin synthesis (see Chapter 3). Lack of HFE on the hepatic cell plasma membrane in the case of C282Y homozygosity will lead to decreased hepcidin synthesis. HFE mutation frequencies worldwide HFE genotypes have been reported from large population studies throughout the world. In a study of 100 000 multiethnic participants from primary care practices, the estimated prevalence of the C282Y homozygous genotype was higher in white

Table 4.2 Classification of genetic haemochromatosis. Type

Gene

Inheritance and phenotype

Severity

Incidence

1 2 (juvenile)

HFE Hemojuvelin (HFE2) Hepcidin (HAMP) TFR2 Ferroportin 1 (SLC11A3)

AR, parenchymal iron overload AR, parenchymal iron overload AR, parenchymal iron overload AR, parenchymal iron overload AD, RE iron or parenchymal iron

Highly variable Severe Severe Variable Variable

Common Rare Rare Rare Rare

3 4

AD, autosomal dominant; AR, autosomal recessive; RE, reticuloendothelial. For gene symbols see Table 3.1. 48

Iron overload

(1 in 227, 0.44%) than in black or Asian people, in whom it was extremely rare. The frequency is higher in northern Europe and lower in southern Italy and Greece. The H63D mutation is found throughout the world but is most common in Europe, where allele frequencies vary from 10 to 20%, with a mean of 15%. Other HFE gene mutations associated with iron accumulation have been described mostly in individual families. HFE mutations and iron status The haemochromatosis gene may have increased in frequency because of a selective advantage for heterozygotes, namely protection against iron deficiency anaemia. Homozygotes would be unlikely to suffer the effects of iron overload before reproducing. Transferrin saturation and serum ferritin are significantly higher in C282Y/C282Y subjects than in other genotypes. Increased levels of iron parameters are present in 75–100% of males and 40–60% of females in different studies. In population surveys slight but significantly higher values for serum iron and transferrin saturation have been found in heterozygotes for either C282Y or H63D compared with subjects lacking these mutations. The differences in ferritin levels are smaller and not significant. In compound heterozygotes, and those homozygous for H63D, there are larger increases in transferrin saturation and serum ferritin, although significant iron accumulation is rare. In heterozygotes for C282Y or H63D, haemoglobin levels are slightly higher than in subjects lacking these mutations, but it has not been clearly demonstrated that this leads to a lower prevalence of anaemia among women carrying either mutation. HFE mutations and morbidity Although advanced haemochromatosis is characterized by diabetes, arthritis and cirrhosis, there is no evidence that possession of HFE mutations is a risk factor for these conditions except through iron overload. The frequency of homozygosity or heterozygosity for C282Y or H63D mutations is not generally increased in patients with arthritis, diabetes and heart disease. Homozygosity for C282Y is more frequent in patients with cirrhosis and hepatoma than in the general population. Alcohol is a definite risk factor for the development of cirrhosis in patients homozygous for C282Y. The significance of other genetic modifiers remains uncertain. Diagnosis: clinical The variety of clinical presentations and their lack of specificity for haemochromatosis means that a high degree of clinical suspicion is needed. Fatigue, diabetes mellitus, signs and symptoms of gonadal failure and arthritis may be present for several years before the diagnosis is made. Arthritis particularly affects the second and third metacarpophalangeal joints (Figure 4.1), and destructive arthropathy of hip and knee joints occurs in 10% of patients. There is chondrocalcinosis with pyrophosphate deposition in the joints. Abdominal pain may result from

Figure 4.1 Radiograph of hand: patient with haemochromatosis showing loss of joint space and erosion of cartilage at the metacarpophalangeal joints.

hepatic enlargement or hepatocellular carcinoma. Grey skin pigmentation results from excess melanin deposition. Diagnosis: iron status

Transferrin saturation and serum ferritin In asymptomatic subjects, iron accumulation is indicated by a raised transferrin saturation (> 45%). Most men and about 50% of women who are homozygous for HFE C282Y will have a raised transferrin saturation. As iron accumulates, the serum ferritin concentration rises, and values in excess of 200 µg/L (women) and 300 µg/L (men) suggest iron overload. Serum ferritin concentrations largely reflect iron turnover in phagocytic cells and do not provide an early indication of iron accumulation in liver parenchymal cells. Thus, measurement of transferrin saturation is essential for early detection of iron loading. However, in patients with infection, inflammation or malignancy or in those undergoing surgery, transferrin 49

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saturation may be depressed and the serum ferritin concentration elevated. In most cases, HFE genotyping will confirm the diagnosis of haemochromatosis.

Liver biopsy Since the advent of genetic testing, confirmation of iron overload by liver biopsy is not usually performed and is not necessary in the absence of liver damage. In patients homozygous for C282Y with evidence of liver disease and serum ferritin concentration above 1000 µg/L, liver biopsy is essential to assess tissue damage. In patients with an unexplained raised transferrin saturation and serum ferritin, who are not homozygous for C282Y, a liver biopsy may be required to confirm iron overload and to address the subsequent diagnostic process (Figure 4.2). The availability of non-invasive measurement of liver iron concentration has restricted the diagnostic use of liver biopsy.

(a)

(b)

Figure 4.2 Liver histology (Perls stain). (a) Liver biopsy from a patient with type 1 haemochromatosis, showing staining predominantly in parenchymal cells. (b) Liver biopsy from a patient with type 4 haemochromatosis, showing iron staining predominantly in Kupffer cells. 50

Liver iron concentration can be measured on a dried fragment of liver biopsy. Values in excess of 80 µmol/g dry weight (4.5 mg/g dry weight) indicate iron overload. Since in haemochromatosis there is a progressive increase in liver iron concentration with age in some cases, it is useful to express the result as the ‘hepatic iron index’ (µmol iron/g dry weight divided by age in years).

Non-invasive methods Techniques using magnetic resonance imaging (MRI) are being increasingly used as an indirect non-invasive measure of both liver and heart iron (see p. 55). They require special analytical skill. Although most experience is with secondary iron overload, MRI is also increasingly used in genetic haemochromatosis. The superconducting quantum interface device (SQUID) biosusceptometry technique is sensitive, accurate and reproducible. It depends on the paramagnetic properties of haemosiderin and ferritin. Unlike MRI, it does not distinguish parenchymal from reticuloendothelial iron, but the result closely correlates with chemical estimation of liver iron, except when fibrosis is present. Machines are expensive to build and run and at present there are only four worldwide (none in the UK). Mobilization of iron by phlebotomy to calculate iron stores The amount of iron removed to reach iron depletion can be calculated (see Treatment) and provides a good estimate of total body iron. Since the advent of genetic testing, confirmation of iron overload by liver biopsy is not necessary in the absence of liver damage and quantitative phlebotomy provides the only practical way of confirming the presence of iron overload. The amount of iron removed at each venesection is calculated by weighing the blood bag before and after venesection (density of blood is 1.05 g/mL) and assuming that 450 mL of blood (haemoglobin concentration 13.5 g/dL) contains 200 mg of iron. Iron absorption should be allowed for at the rate of 3 mg daily (20 mg/week). With these assumptions, 25 weekly venesections will remove 4.5 g of iron. The amount of storage iron measured by the technique in normal adults has been shown to be about 750 mg in men and 250 mg in women. Clinical penetrance Before the discovery of the HFE gene, it was assumed that every family member who was homozygous for haemochromatosis would eventually accumulate sufficient iron to cause tissue damage. Recent studies, in which subjects homozygous for HFE C282Y have been compared with ‘wild-type’ subjects, have shown that the frequencies of lethargy, arthralgia and diabetes are the same. There is, however, a small but significant increase in the percentage of subjects with either raised serum transaminase activity or fibrosis/cirrhosis in the C282Y homozygous

Iron overload

group in several studies. Population surveys have shown that less than 5% of subjects homozygous for C282Y ever receive a diagnosis of haemochromatosis. Despite much debate about ascertainment bias in family and population surveys, it is becoming clear that most men who are homozygous for C282Y will have a raised transferrin saturation before the age of 30 years; a proportion will have an elevated serum ferritin concentration, but only a minority will eventually develop fibrosis and cirrhosis of the liver. Studies in Australia and Norway found that 5% of homozygote males but no females showed cirrhosis. There is evidence that ferritin concentration above 1000 µg/L is a risk factor for liver fibrosis in different studies. These patients should undergo liver biopsy and complete cardiac and endocrinological evaluation. Only about 50% of homozygous women have a raised transferrin saturation, and progression through iron accumulation and tissue damage is usually, but not always, slower. In one population study in the USA, based on reported clinical complications, the clinical penetrance was estimated to be as low as 1%. In a more recent study, which evaluated for 12 years a cohort of C282Y homozygous patients of middle age (40–69 years) using strict criteria to define liver disease, this clinical complication was estimated to be present in about 30% of males and 1% of females. However, the iron accumulation rate of C282Y patients is not constant and may vary greatly from patient to patient. Many homozygotes followed for 10 years or more have shown no change in serum ferritin. Family testing Physicians should discuss with the patient the desirability of testing all first-degree relatives over the age of consent in order to identify those at risk. Transferrin saturation and serum ferritin concentration should be measured along with HFE genotyping. Genetic testing may identify other family members homozygous for HFE C282Y. If the serum ferritin concentration is normal and there is no evidence of liver disease, transferrin saturation and serum ferritin should be measured at yearly intervals and treatment instituted if necessary. Compound heterozygotes are at lesser risk of iron overload but should also be tested by measuring transferrin saturation and serum ferritin, perhaps at 3-yearly intervals. Iron status should be determined in heterozygotes and, if normal, reassessed after 5 years to ensure that no other iron-loading genes are present.

Associations with other conditions The HFE C282Y mutation is relatively common; heterozygosity, and even homozygosity, may occur with other haematological conditions, including inherited sideroblastic anaemia. The occasional presence of iron overload in patients with haematological disorders such as congenital spherocytosis, in whom it is otherwise uncommon, may be due to a combined effect on iron absorption of increased eythropoiesis and coincidental inheritance of the heterozygous state for haemochromatosis.

Porphyria cutanea tarda, which may be associated with iron overload, an important trigger of disease manifestations, is discussed in Chapter 3. Population screening Widespread population screening by iron status or genetic testing is considered unwarranted as the level of risk for a C282Y homozygote developing iron overload appears to be low. Once the factors that convey a high risk of developing significant iron overload and tissue damage have been identified, it may be appropriate to reconsider this question but at present screening should be limited to at-risk populations. Treatment Removal of excess iron by regular phlebotomy greatly reduces the mortality from cardiac and hepatic failure, although hepatocellular carcinoma accounts for a substantial proportion of deaths in those with established disease. Early diagnosis is therefore a priority, as patients identified and treated before the onset of cirrhosis of the liver have a normal life expectancy. Since no test is available to identify those patients who will progress to fibrosis and those who will not, the present recommendation is to phlebotomize all patients with evidence of iron overload. Phlebotomy should be at a rate of 450 mL of blood each week and should be performed until iron depletion is reached (serum ferritin < 20 µg/L and transferrin saturation < 16%). Haemoglobin levels should be measured weekly and the rate of venesection reduced if anaemia develops. Serum ferritin should be monitored monthly. The transferrin saturation should be measured weekly when the ferritin concentration drops below 50 µg/L. Weekly phlebotomy will need to be continued for at least 6 months to remove total iron excess, which is usually greater than 5 g in established symptomatic disease but may be more than 20 g. When iron stores are exhausted, the frequency of phlebotomy should be reduced to two to four units each year, to continue indefinitely. The aim is to maintain a normal transferrin saturation (< 50%) and a serum ferritin in the lownormal range (< 50 µg/L). Fatigue and transaminase elevation usually reverse on venesection. In some patients, diabetes mellitus, hypogonadism and arthralgia improve, but cirrhosis and arthritis are not reversible. Early cardiac disease may respond to phlebotomy but severe cardiomyopathy does not and requires iron chelation. Iron chelation with subcutaneous desferrioxamine (DFX) may be used in patients who do not tolerate phlebotomy or have concomitant anaemia. DFX given as a continuous intravenous infusion with or without an oral iron chelator (see below) may have a role in the short-term management of patients with lifethreatening cardiac failure. Well-tolerated oral iron chelators are likely to become an option for treatment of selected patients in the future. 51

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Type 2 (juvenile) haemochromatosis Juvenile haemochromatosis is a rare autosomal recessive disease, with clinical symptoms related to iron overload appearing in the second and third decades of life. The disease is genetically heterogeneous since it can be due either to hemojuvelin mutations or, more rarely, to mutations in the hepcidin (HAMP) gene (see Chapter 3). More than 30 hemojuvelin mutations have been reported in European and Asian families with G320V being the most frequent. The age of onset has been as low as 5 years but the disease usually presents in teenagers or those in their early twenties. Hemojuvelin modulates hepcidin expression by acting as a coreceptor of bone morphogenetic proteins (see Chapter 3). Inactivation of hemojuvelin strongly decreases hepcidin production. This explains why the clinical phenotypes of HAMP or hemojuvelin (HFE2) mutations are indistinguishable. In both cases iron absorption is greater than in type 1 haemochromatosis and iron deposition occurs not only in the hepatocytes but also in cardiac myocytes, pancreas and pituitary; liver disease is present but symptoms related to cardiomyopathy, diabetes and hypogonadism are more prominent. Digenic disease has been rarely described in patients with severe disease expression who are heterozygous for HFE C282Y and have a mutation in the HAMP (hepcidin) gene.

Type 3 haemochromatosis Haemochromatosis type 3 is a rare autosomal recessive disease reported in those of European and Asian ancestry. It is phenotypically similar to type 1 haemochromatosis, although it may have an earlier age of onset. It is due to mutations in the gene for transferrin receptor 2 (TFR2) (see Table 4.2). TFR2 shows moderate homology to TFR1, may bind transferrin but is not iron regulated. Hepcidin levels are very low in type 3 haemochromatosis, disproportionate to the degree of iron overload (see Table 3.1). TFR2 binds HFE when transferrin saturation is high and this complex activates hepcidin (see Chapter 3). Thus mutations of either HFE or TFR2 cause a similar disorder.

Type 4 haemochromatosis (ferroportin disease) Haemochromatosis type 4 has peculiar genetic and clinical features compared with the other forms. First, it is inherited as an autosomal dominant trait. Second, all patients have increased serum ferritin levels, but some have a normal transferrin saturation. Third, at liver biopsy iron is usually increased in the reticuloendothelial cells, as well as in hepatocytes, features that suggest a different pathophysiology of the disease. Haemochromatosis type 4 is due to heterozygous missense mutations in the gene for the iron exporter ferroportin, located 52

on chromosome 2q32. In the typical form (type 4a), so-called ferroportin disease, the phenotype differs from haemochromatosis and is similar to that found in the anaemia of chronic disease (see Figure 4.2b), with iron accumulation predominantly in the macrophages and normal or low transferrin saturation. These cases are due to loss-of-function ferroportins that are unable to target correctly to the cell surface and so export iron from macrophages. It seems that reticuloendothelial iron is less toxic than parenchymal iron. For this reason and because some of these patients develop mild anaemia, tolerance to phlebotomy should be carefully monitored and intensive regimens are not indicated. In the rare type 4b with mutations occurring in the binding site of ferroportin for hepcidin, mutant ferroportins reach the cell surface but are resistant to the effect of hepcidin, causing increased iron export to plasma, saturation of transferrin and iron deposition in hepatocytes similar to HFE haemochromatosis. In people of African origin, the Gln248His mutation in ferroportin is a common variant that may be associated with a tendency to iron loading and mild anaemia.

Neonatal haemochromatosis This is a condition that is recognized at birth but may occur in utero. It is characterized by heavy parenchymal iron deposition in several organs and irreversible liver failure. The only therapeutic option used to be liver transplantation. No mutations in the known haemochromatosis genes have been reported and a genetic cause has been more recently doubted in favour of an immunological pathogenesis, but heterogeneous causes cannot be ruled out. Neonatal haemochromatosis has been linked in some cases to the presence of a maternal factor, for example an antiribonuclear factor antibody. Infusions of gammaglobulin in pregnancy appear to reduce the severity of the condition and it has been proposed that the disease is due to an alloantibody (as rhesus incompatibility) but the target antigen is unknown.

Increased iron intake African iron overload (Bantu siderosis) results from the combination of a dietary component (a traditional beer that contains iron) and an unknown susceptibility gene. Mutations in the HFE gene have been excluded but mutations of the ferroportin gene may play a role (see above). Iron deposition, as in type 4 haemochromatosis, occurs in both hepatocytes and reticuloendothelial cells. Serum ferritin is usually elevated, but transferrin saturation may be normal. The condition occurs in sub-Saharan Africa. It is a cause of hepatic fibrosis and cirrhosis, and associations with diabetes mellitus, peritonitis, scurvy and osteoporosis have been described. The iron overload is associated with a poor outcome of tuberculosis, an infection that is highly prevalent in sub-Saharan Africa.

Iron overload

Other causes of iron overload Atransferrinaemia This is a rare recessive genetic disorder associated with a severe hypochromic anaemia with, in some cases, excessive deposition of non-transferrin-bound iron (NTBI) in the parenchymal cells. In all cases tested, some iron-transferrin has been detected by iron-binding ability or immunologically defining a type of hypotransferrinaemia. Complete absence of transferrin would presumably lead to fetal death.

Iron and neurodegeneration Acaeruloplasminaemia This is also a rare recessive disorder in which there is a deficiency of ferroxidase activity as a consequence of mutations in the caeruloplasmin gene. Clinically, the condition presents in middle age, with progressive degeneration of the retina and basal ganglia and with diabetes mellitus. Iron accumulates in the liver, pancreas and brain with smaller amounts in the heart, kidneys, thyroid, spleen and retina. The serum iron is low and mild anaemia may be present. The total iron-binding capacity of transferrin (TIBC) is normal and ferritin is normal or raised. Unfortunately, no effective treatment is available to reduce neurodegeneration but deferiprone, which penetrates the blood–brain barrier, may be an option. Hallervorden–Spatz syndrome This is an autosomal recessive neurodegenerative disorder associated with iron accumulation in the brain. Clinical features include extrapyramidal dysfunction, onset in childhood and a relentlessly progressive course. Histological study reveals iron deposits in the basal ganglia. Hallervorden–Spatz syndrome is caused by a defect in a novel pantothenate kinase gene that causes accumulation of cysteine. Iron binding by cysteine may cause iron accumulation and oxidative stress, which is a likely explanation for the pathophysiology of the disease. Neuroferritinopathy A rare, dominantly inherited, late-onset basal ganglia disease that variably presents with extrapyramidal features similar to those of Huntington disease or parkinsonism also shows iron accumulation in the forebrain and cerebellum. The responsible gene (FTL) codes for ferritin light-chain polypeptide. An adenine insertion at position 460–461 was found that was predicted to alter carboxy-terminal residues of the gene product. Abnormal aggregates of ferritin and iron in the brain contrasted with low serum ferritin levels. These diseases may serve as a model for complex neurodegenerative diseases such as Parkinson disease, Alzheimer disease and Huntington disease, in which accumulation of iron in the brain is also observed. Possession of the C282Y mutation of the HFE gene does not appear to be a risk factor for these conditions.

Friedreich ataxia This is a neurodegenerative disease characterized by loss of sensory neurones in the spinal cord and dorsal root ganglia. There is mitochondrial iron overload and loss of activity of iron–sulphur cluster-containing enzymes. Patients frequently die from cardiomyopathy. The majority of cases of Friedreich ataxia result from the expansion of triple nucleotide repeats within an intron of the FXN gene, leading to reduced expression of frataxin mRNA and protein. Point mutations have also been identified in a small number of cases. Frataxin is found in the mitochondria where, in Friedreich ataxia, there is increased oxidative stress and decreased activity of iron–sulphur proteins. Oxidative damage following iron accumulation is thought to precipitate the neurone loss. This is confirmed by experiments in yeast that show that iron is redistributed to the mitochondria of Yfh (yeast frataxin homologue)-deficient yeast and that this iron accumulation precedes oxidative damage. Frataxin shows structural similarity to ferritin, suggesting that frataxin may regulate mitochondrial iron homeostasis by storing excess iron. A preliminary trial of deferiprone therapy has shown some improvement in neuropathy and gait in the youngest patients and larger-scale trials are now in progress.

Hereditary hyperferritinaemia–cataract syndrome This syndrome is characterized by elevated serum ferritin levels, early-onset bilateral cataracts and normal or low serum iron and transferrin saturation. It is usually due to heterozygous point mutations in the l-ferritin iron-response element so that a monoclonal ferritin is synthesized due to impaired negative feedback of ferritin synthesis. There is no general tissue iron overload and serum transferrin saturation is normal, but ferritin accumulates in the lens causing cataracts. In a few cases, the mutation is in the coding regions of the gene.

Iron-loading anaemias Removal of iron is essential in patients with transfusiondependent anaemias, such as thalassaemia major, to prevent death from iron overload, usually caused by cardiac failure or arrhythmia. Red blood cell requirements are about 160 mL/kg annually in non-splenectomized and 120 mL/kg annually in splenectomized patients with thalassaemia major. The iron content of each transfusion is determined by volume (mL) × haematocrit × 1.16 mg. Intake of iron ranges from 0.32 to 0.64 mg/kg body weight daily. Patients with anaemias associated with increased iron absorption (e.g. thalassaemia intermedia), who are too anaemic to be venesected to remove iron, may also require iron chelation therapy, although the rate of iron loading is considerably lower at about 0.1 mg/kg daily. The iron-chelating drug widely available is desferrioxamine (DFX). 53

Postgraduate Haematology

Table 4.3 Characteristics of desferrioxamine, deferiprone and deferasirox.

Structure Molecular weight Iron–chelator complex Plasma clearance (t½) Absorption Iron excretion Therapeutic daily dose Route Clinical experience Side-effects

Desferrioxamine

Deferiprone

Deferasirox

Hexadentate 560 1:1 20 min Negligible Urine + faecal 40 mg/kg Parenteral >40 years Ototoxicity, retinal toxicity, growth defects, cartilage and bone abnormalities

Bidentate 139 1:3 53–166 min Peak 45 min Urine 75–100 mg/kg Oral >20 years Agranulocytosis, arthropathy, gastrointestinal disturbance, transient transaminitis, zinc deficiency

Tridentate 373 1:2 1–16 hours Peak 1–2.9 hours Faecal 20–30 mg/kg Oral >8 years Skin rashes, gastrointestinal disturbance, rising serum creatinine

This is orally inactive and given by slow subcutaneous or intravenous infusion. Deferiprone, an orally active iron chelator first used clinically in 1987, is now licensed in 61 countries. A third drug, deferasirox, is orally active and was introduced into clinical practice in 2002 and is now widely prescribed (Table 4.3 and see Figure 4.5). Iron chelation therapy is monitored by: 1 tests of body iron burden; 2 tests of function of the organs sensitive to iron overload (Table 4.4); 3 tests to detect potential side-effects of the particular chelating drug being used. Tests (1) and (2) are discussed first. The results and sideeffects with the individual iron-chelating drugs are then described.

Tests of body iron burden Serum ferritin Serum ferritin is useful in monitoring changes in body iron, although the absolute level is an imprecise measure of total body iron. There is a wide range of liver iron at any given serum ferritin level. This is partly because serum ferritin mainly reflects reticuloendothelial iron and partly because inflammation, (e.g. hepatitis C infection) raises the level, whereas vitamin C deficiency, frequent in iron overload, lowers it. The Thalassaemia International Federation (TIF) guidelines recommend maintaining the level below 1000 µg/L in thalassaemia major. Liver iron Liver iron may be measured chemically after liver biopsy, by MRI or, in a few specialized units, by SQUID (see p. 50). Chemical estimation is the gold standard but can be inaccurate 54

Table 4.4 Monitoring for iron-induced organ damage. Cardiac function ECG ± exercise 24-hour monitoring Echocardiography, MUGA ± stress test Doppler echography, MRI Liver structure and function Liver function tests Liver histology Bone Osteoporosis: bone density (Dexa scan) Endocrine system Diabetes: urine glucose, HbA1c, glucose tolerance test, IGF-1 Growth and sexual development: sitting and standing height, Tanner staging, radiography for bone age, testosterone, estradiol, LH, FSH, SHBG, pulsatile GnRH release, sperm tests Thyroid: T4, TSH Parathyroid: calcium, phosphate, PTH ECG, electrocardiogram; FSH, follicle-stimulating hormone; GnRH, gonadotrophin-releasing hormone; HbA1c, glycated haemoglobin; IGF, insulin-like growth factor; LH, luteinizing hormone; MRI, magnetic resonance imaging; MUGA, multigated acquisition scan; PTH, parathyroid hormone; SHBG, sex hormone-binding globulin; TSH, thyroid-stimulating hormone.

if fibrosis is present. Levels greater than 15 mg/g dry weight have been associated in DFX-treated patients with a high risk of cardiac disease, liver fibrosis and cirrhosis. Levels between 7 and 15 mg/g dry weight are associated with liver damage only if

Iron overload

(b)

Figure 4.3 Magnetic resonance imaging T2* technique. Tissue appearances of liver and spleen: (a) normal; (b) tissue iron overload; (c) severe liver iron overload with normal cardiac iron;

there is also hepatitis C infection and have been considered indicative of relative safety from cardiac disease but are associated with damage to the endocrine organs. Levels less than 7 mg/g dry weight are found in carriers of haemochromatosis and are considered safe. MRI techniques are being increasingly used as indirect measures of liver and cardiac iron (Figure 4.3). They have the advantage of being non-invasive and are more widely available than SQUID (which is suitable for liver but not cardiac iron). MRI is also the only practical method of performing sequential studies of iron in the heart, pituitary or other endocrine organs. Different MRI techniques have been used. They all rely on a shortening of relaxation time and thus reduction in signal intensity with iron overload. Gradient-echo imaging with the calculation of the T2* has a short total imaging time, reducing movement artefacts. It is also extremely sensitive and reproducible. The spin-echo technique is less sensitive to iron and requires longer imaging time, making it less valuable for testing cardiac iron. Cardiac iron As cardiac failure or arrhythmia is the usual cause of death in transfusional iron overload, it is essential to monitor cardiac iron. Iron is deposited in myocytes and interstitial fibrosis develops. Direct measurement of cardiac iron by endomyocardial biopsy is inappropriate as the technique is highly invasive and inaccurate as iron localizes mainly in the ventricular myocardium and epicardium. T2* cardiovascular magnetic resonance offers a reproducible (around 5% coefficient of variation between different observers or between two studies of the same patient), sensitive, albeit indirect measure: the lower the T2* value, the greater the cardiac iron (Figure 4.3). The majority of patients with T2* greater than 20 ms have normal left ventricular function. A T2* below 20 ms correlates with the presence of cardiac dysfunction detected by echocardiography (left and right ventricles) or by 24-hour rhythm monitoring or the need for cardiac therapy (Figure 4.4). The great majority of patients

(c)

(d)

(d) severe cardiac iron deposition with minimal liver iron deposition. (From Anderson et al. 2001 with permission.)

100 Left ventricular ejection fraction (%)

(a)

80 60 40 20 0 0

20

40 60 Heart T2* (ms)

80

100

Figure 4.4 Relationships of myocardial T2* and left ventricular ejection fraction in patients with thalassaemia major and iron overload. (From Anderson et al. 2001 with permission.)

who develop cardiac failure have T2* less than 10 ms. Poor correlation has been found between myocardial iron and liver iron (MRI derived) or serum ferritin in patients receiving DFX, so serum ferritin and liver iron cannot be used as surrogate measures of cardiac iron. Urine iron excretion Iron excretion after a single infusion of a standard dose of DFX or oral dose of deferiprone is related to body iron. Urine iron is derived from the labile iron pool chelated mainly extracellularly with DFX and probably intracellularly with deferiprone. With DFX (but not deferiprone) urine iron excretion is increased by ascorbate and is proportionately higher if the haemoglobin is lower. The test is useful when commencing therapy with DFX or deferiprone, with which iron excretion is highly dose related, and for monitoring therapy, in some studies correlating closely with liver and cardiac iron. However, several estimations must be performed at any given dose in view of the variability found. 55

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Non-transferrin-bound iron This is present in plasma in patients with gross iron overload and 100% saturation of transferrin. It is highly toxic, promoting the formation of free radicals that cause peroxidation of membrane lipids. Part of the early improvement in liver and cardiac function with chelation therapy may be due to removal of this fraction, even before iron burden is substantially lowered. Its clearance by DFX is short-lived as it reappears in plasma within hours of stopping an infusion. Deferasirox, with its long clearance time, provides 24-hour removal of NTBI after a single oral dose. Deferiprone removes NTBI for about 6 hours after a single dose. NTBI is absent from plasma of well-chelated patients.

(a) HOOC

(b)

O OH CH3

N N N

CH3

N OH HO

(c)

HO

O

H N

N O

CH3

Tests of organ function The tests that are usually needed are listed in Table 4.4. Heart function is best tested by measurement of left ventricular ejection fraction and by tests for rhythm disturbance. Liver function assessment requires routine liver function tests as well as liver biopsy to assess liver structure and liver iron burden (histologically and chemically). The endocrine system is also damaged by iron and appropriate tests are listed in Table 4.4. The anterior pituitary is particularly sensitive, with damage resulting in reduced growth and impaired sexual maturation. Direct damage to the ovaries or testes may also occur but is usually less important. Hypogonadic hypogonadism, defects of growth hormone secretion and in its receptor, and deficiency of insulin-like growth factor mainly account for growth failure; DFX may also cause this. Diabetes mellitus and pre-diabetes, due to iron deposition in the pancreatic islets, are frequent, especially in patients with genetic susceptibility. Hypothyroidism and hypoparathyroidism are also common in poorly chelated patients. Osteoporosis is well recognized in iron-overloaded thalassaemia patients; it is due to multiple factors and is detected by bone density studies.

Iron chelation therapy Desferrioxamine

Pharmacokinetics Desferrioxamine mesylate is licensed in all countries. It is not absorbed orally, and after parenteral injection is rapidly cleared from the plasma, being excreted in the urine, taken up by hepatocytes or metabolized in the tissues (Table 4.3). This accounts for the much greater mobilization of iron by continuous intravenous or slow subcutaneous infusions, which allow more prolonged exposure of the drug to the chelatable iron than with intramuscular injection. DFX is a trihydroxamic acid (hexadendate) (Figure 4.5), one molecule binding covalently to all six oxygen sites on one ferric ion to form the red chelate, ferrioxamine. This is excreted in 56

N

O H2N+

HN

O

HO

O

N OH

Figure 4.5 Chemical structures of three iron chelators: (a) deferasirox; (b) deferiprone; (c) desferrioxamine.

urine and bile. Faecal iron is derived from hepatocytes. Urine iron also derives, at least partly, from hepatocytes, although other body sources, especially iron released from macrophages, contribute. Urinary iron excretion tends to level off at higher doses, but this does not occur with bile excretion so bile iron may therefore predominate at high doses, and this is also the major route of excretion when total body iron has been reduced to relatively low levels. Increased erythropoiesis, as in haemolytic anaemias, is associated with an increase in urine iron excretion in relation to body iron stores.

Clinical studies Most studies have involved thalassaemia major, but patients with other inherited anaemias (e.g. Diamond–Blackfan syndrome, Fanconi anaemia, sickle cell anaemia, sideroblastic anaemia) or acquired disorders, especially myelodysplasia, myelofibrosis, red cell aplasia or aplastic anaemia, may require iron chelation therapy. In these conditions, as well as in elderly patients with acquired, transfusion-dependent, refractory anaemias and otherwise good prognosis, deferasirox is widely used if iron overload is likely to cause significant morbidity or mortality. In children, tissue damage from iron may be present from very early life; regular iron chelation should begin in thalassaemia major after transfusion of about 12 units of blood or when serum ferritin exceeds 1000 µg/L. In young children, treatment with DFX should be started at 20 mg/kg to prevent tissue damage due to iron without causing toxicity due to excess DFX. A local anaesthetic cream (e.g. EMLA) reduces pain from the needle insertion. Oral chelation may be preferred to DFX.

Iron overload

The standard adult dose of DFX is 40 mg/kg s.c., given as an 8–12 hour infusion on at least 5 days each week. Repletion of ascorbic acid deficiency, which sometimes accompanies iron overload, or ascorbate therapy even in those with normal tissue levels of ascorbate, increases urinary iron excretion with DFX. Vitamin C supplements should be given at a dose of 100–200 mg per day. For those with iron-induced cardiomyopathy, continuous intravenous DFX may be given via an indwelling catheter (e.g. Hickman) or Port-a-Cath chamber. Removal of liver iron is more rapid than removal of cardiac iron with this intensive chelation regimen. Combined therapy with deferiprone may also be used. Studies with deferasirox are in progress. Body iron stores can be restricted to 5–10 times normal in well-chelated, regularly transfused patients. There is improved cardiac function and survival in patients who comply with DFX therapy. Growth and pubertal development are improved in many, but not all, patients; diabetes and other endocrine abnormalities still occur frequently. Serum ferritin levels in wellchelated with DFX thalassaemia major patients usually plateau between 1500 and 2500 µg/L. Unfortunately, through lack of compliance with DFX, premature deaths usually from ironinduced cardiac damage may occur in a proportion of thalassaemia major patients. Combination therapy or orally active drugs alone should be used in these patients.

Side-effects These include rare generalized sensitivity reactions, local soreness related to the site of injection (usually due to the needle being inserted too superficially) and exacerbation of some infections, notably of the urinary tract and precipitation of Yersinia enterocolitis. Auditory (high-tone sensorineural hearing loss) and visual neurotoxicity (night blindness, visual field loss, retinal pigmentation and changes on electrical tests) are relatively frequent. Growth and bone defects may also occur. The spine may be affected, with sitting height reduced; rickets-like bone lesions, genu valgum and metaphyseal changes are described, especially in children (Figure 4.6). Auditory, visual and growth side-effects of DFX occur mainly if the body iron burden is low and doses of DFX high, particularly in children. A therapeutic index can be calculated as follows: mean daily dose (mg/kg)/current serum ferritin (µg/L). If this is below 0.025 at all times, these side-effects of DFX do not occur. Deferiprone

Pharmacokinetics Deferiprone (1,2-dimethyl-3-hydroxypyrid-4-one) is rapidly absorbed, appearing in plasma within 15 min of ingestion (see Table 4.3). The chelator–iron complex is excreted with the free drug and glucuronide derivative in urine. Its iron chelation site

Figure 4.6 Bone and cartilage defects due to desferrioxamine.

is inactivated by glucuronidation, the speed of which varies from patient to patient. This explains much of the individual variation in response. It is available both as tablets (500 mg) and as a liquid formulation containing 100 mg/mL. Deferiprone mobilizes iron from parenchymal and reticuloendothelial pools and from transferrin, ferritin and haemosiderin. The enhanced ability of deferiprone to cross cell membranes may underlie its superior ability compared with DFX to protect the heart from iron and also the ‘shuttle’ effect for iron when the two drugs are given simultaneously (see p. 59 and Figure 4.7). Deferiprone crosses the blood–brain barrier to treat neurological conditions with iron loading in the brain. Few balance studies have been performed. These suggest that, on average, deferiprone 75 mg/kg is about as effective as DFX 40 mg/kg. Wide individual variations occur, especially with deferiprone. Moreover, it is easier for patients to comply with deferiprone than DFX on all 7 days each week.

Clinical studies The usual dose used has been 75–100 mg/kg daily. Serum ferritin levels tend to plateau around 2000–2500 µg/L. MRI studies suggest that liver iron may generally be higher in patients treated with deferiprone (75 mg/kg 7 days per week) compared with those treated with DFX (40 mg/kg 5 days per week). In the liver, DFX has the advantage of facilitated transport into cells by an active mechanism. Deferiprone, on the other hand, may have greater penetration of myocardial cells because of its lower molecular weight and because it is lipophilic. Retrospective and prospective studies have shown, on the basis of T2* MRI measurement of cardiac iron, echocardiography, clinical incidence of cardiac disease, need for cardiac therapy and survival, that 57

Postgraduate Haematology

Intracellular compartment

Extracellular compartment

Protein–Fe

Protein

3DFP+Fe

(DFP)3–Fe

DFP

(DFP)3–Fe

Excretion: Urine

DFO–Fe

Excretion: Urine, bile

deferiprone 75 mg/kg is more effective than DFX at protecting the heart from iron-induced cardiomyopathy in routine clinical practice.

Side-effects The most severe is agranulocytosis (neutrophils < 0.5 × 109/L on two consecutive counts), with an estimated incidence of around 0.5–1.0% (0.2–0.3 episodes per 100 patient-years). It is most frequent in the first year of therapy. Lesser degrees of neutropenia (0.5–1.5 × 109/L) are more frequent, around 3.5–8.5%, occurring more frequently in non-splenectomized patients. Agranulocytosis and neutropenia spontaneously recover when the drug is discontinued, usually within 4–28 days (median duration 9 days) but occasionally are more prolonged. Granulocyte colony-stimulating factor produces a faster recovery. The mechanism appears to be idiosyncratic, more common in females, with no definite evidence of an immune mechanism established. Patients should be monitored by blood counts every week for at least the first 8–12 weeks of therapy and every 2 weeks thereafter for 2 years. Agranulocytosis may be more frequent in patients with stem cell or progenitor cell defects, for example aplastic anaemia, Diamond–Blackfan syndrome or myelodysplasia. Deferasirox may be safer for these conditions. Painful joints, especially the knees, occur in around 5–10% in most large series. The incidence has been highest in Indian patients. Some but not all studies show that this complication is most frequent in the most iron-loaded patients and with higher doses of deferiprone. It usually, but not invariably, resolves with withdrawal of the drug and it is often possible to reintroduce the drug, commencing with lower doses. About 2.0% of patients permanently discontinue therapy because of joint symptoms. Gastrointestinal side-effects (e.g. nausea and abdominal pain) occur in about 30% of patients in the first year but decrease to 3% in subsequent years. In most the drug can be 58

Parenteral administration NTBI

NTBI DFP

Oral administration

DFO

Figure 4.7 The concept of combination therapy: DFO, desferrioxamine; DFP, deferiprone; NTBI, non-transferrin-bound iron.

reintroduced long term, initially at a lower dose. The liquid preparation appears to produce fewer gastrointestinal symptoms. Zinc deficiency has been described in diabetic and prediabetic patients. Rarely, it can lead to clinical features, such as skin rashes and hair loss. It is easily treated by oral zinc therapy. Liver fibrosis was suggested as a complication of deferiprone therapy in one study, but larger studies show that liver fibrosis is not a consequence of deferiprone. Nevertheless, transient increases in liver enzymes have been associated with deferiprone therapy in about 7% of patients and about 1% of patients have been withdrawn from therapy because of a persistent rise in liver enzyme levels. There have been no reports of renal, cardiac or neurological side-effects. Embryo toxicity and teratogenicity have been reported in non-iron-loaded animals treated with deferiprone. Women of childbearing age should be counselled to avoid the drug or use contraception but a few uneventful pregnancies with healthy newborns have been reported. Combination therapy Urine iron excretion when DFX and deferiprone are given simultaneously is equivalent to the sum of the excretion when the drugs are given on separate days. There is evidence for a ‘shuttle’ effect in which deferiprone enters cells, chelates iron and then returns to plasma, where the iron is transferred to DFX for excretion in urine or bile (Figure 4.7). All studies of combination therapy, for example deferiprone on 7 days a week and DFX on 2 days, have shown a significant fall in serum ferritin and improvement in cardiac and liver iron over 6–18 months. It has been associated with improved survival and reversal of endocrinological complications including diabetes, hypothyroidism and hypogonadism. The combination has also proved successful in reversing severe myocardial siderosis. Alternating therapy has also been studied, for example 4 or 5 days of deferiprone and 2 or 3 days of DFX each week, with improved compliance and improved iron status in previously poorly compliant (with DFX) children or adults.

Iron overload

Deferasirox

Pharmacokinetics Deferasirox, 4-[3,5-bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl] benzoic acid (Figure 4.5), is a tridentate chelator forming a 2 : 1 chelator–iron complex and increases predominantly faecal iron excretion. After a single oral dose, only 6% of iron excretion occurs in the urine (see Table 4.3). It is highly selective for iron. Peak plasma concentration after a single oral dose occurs at about 2 hours, and the drug is still detectable in plasma in almost all patients at 24 hours, with a mean elimination half-life of between 11 and 19 hours after multiple-dose administration. The single daily dose ranges from 20 to 40 mg/kg. Clinical studies Deferasirox has been shown to be effective at eliminating NTBI in plasma and reducing serum ferritin and liver iron in heavily iron-loaded patients. The effect is dependent on dose and on transfusion requirements of the patient. The drug has been shown to be safe and effective in children as young as 2 years. The starting dose in children is 20 mg/kg daily with subsequent dose adjustments. In adults 30–40 mg/kg daily may be required according to iron stores. Trials lasting for up to 5 years on children and adults have not shown any progressive renal, hepatic or bone marrow dysfunction and there are no reports of deferasirox having negative impact on growth or sexual development. Emerging data suggest that deferasirox is effective at removing cardiac iron and preventing cardiac siderosis in thalassaemia major. It has also been shown to maintain or reduce iron overload in transfusion-dependent patients with myelodysplasia, Diamond–Blackfan anaemia and aplastic anaemia and in iron-loaded sickle cell anaemia patients. Adverse effects in these groups appear similar to those with thalassaemia major. The use of deferasirox is also being explored in hereditary haemochromatosis, chronic hepatitis C infection, porphyria cutanea tarda and mucormycosis. The most common adverse effects have been abdominal pain, nausea, diarrhoea, vomiting and skin rashes. These decrease in frequency annually. They usually respond to dose adjustments, taking the drug in the evening or adding products such as Lactaid to the diet. Non-progressive increases in serum creatinine (defined as a rise above the mean pretreatment measurement by more than 33% on two consecutive tests) occur in about one-third of patients. These increases are dose dependent and resolve spontaneously. Serum creatinine should be measured in duplicate before therapy and then monthly, with significantly increased levels managed by dose reduction or interruptions.

Thalassaemia intermedia For these patients, and other severely anaemic patients who are not transfusion dependent or only need a few transfusions each

year, iron loading occurs mainly through increased iron absorption. When anaemia is too severe for venesection, DFX has been used but oral iron chelation with deferasirox or deferiprone, more easily managed and with better patient compliance, has been shown effective in ‘de-ironing’ such patients, potentially reducing serum ferritin and liver iron to normal. A rise in haemoglobin level may occur. This may be due to removal of iron from the renal oxygen sensor, augmenting the effect of hypoxia and increasing erythropoietin secretion from the kidney. The rise in haemoglobin may also be a result of deferiprone directly removing iron from erythroblasts and mature red cells, reducing ineffective erythropoiesis and haemolysis. Improved haemopoiesis has also been described in myelodysplasia after chelation with DFX.

Acute iron poisoning Acute oral iron poisoning produces a severe necrotizing gastritis and enteritis, followed by metabolic acidosis and, after a day or two, cardiovascular collapse and evidence of liver damage. DFX should be given both orally and parenterally. The instillation of 5 g into the stomach after a 1% sodium bicarbonate gastric lavage (to reduce further absorption) and an injection of 1–2 g i.m. may be tried. If a large number of tablets have been taken, an intravenous DFX infusion up to a maximum dose of 80 mg/ kg in 24 hours should be used. Deferiprone and deferasirox have not yet been used in this setting.

Selected bibliography Adams PC, Barton JC (2007) Haemochromatosis. Lancet 370: 1855–60. Anderson LJ, Holden S, Davis B et al. (2001) Cardiovascular T2star (T2*) magnetic resonance for the early diagnosis of myocardial iron overload. European Heart Journal 22: 2171–9. Anderson LJ, Wonke B, Prescott E et al. (2002) Improved myocardial iron levels and ventricular function with oral deferiprone compared with subcutaneous desferrioxamine in thalassaemia. Lancet 360: 516–20. Angelucci E, Barosi G, Camaschella C et al. (2008) Italian Society of Haematology practice guidelines for the management of iron overload in thalassemia major and related disorders. Haematologica 93: 741–52. Beutler E, Felitti VJ, Koziol JA et al. (2002) Penetrance of 845G→A (C282Y) HFE hereditary haemochromatosis mutation in the USA. Lancet 359: 211–18. Boddaert N, Le Quan Sang KH, Rötig A et al. (2007) Selective iron chelation in Friedreich ataxia: biologic and clinical implications. Blood 110: 401–8. Camaschella C, Poggiali E (2009) Towards explaining ‘unexplained hyperferritinemia’. Haematologica 94: 307–9. Camaschella C, Poggiali E (2009) Rare types of genetic hemochromatosis. Acta Haematologica 122: 140–5. 59

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Capellini MD, Cohen A, Piga A et al. (2006) A phase 3 study of deferasirox (ICL 670), a once-daily oral iron chelator, in patients with β-thalassemia. Blood 107: 3455–62. Cappellini MD, Porter JB, El-Beshlawy A et al. (2010) Tailoring iron chelation by iron intake and serum ferritin: prospective EPIC study of deferasirox in 1744 patients with transfusiondependent anemias. Haematologica, in press. Farmaki K, Tzoumari I, Pappa C et al. (2009) Normalisation of total body iron with very intensive combined therapy reverses cardiac and endocrine complications of thalassaemia major. British Journal of Haematology 148: 466–75. Galanello R, Piga A, Alberti D et al. (2003) Safety, tolerability and pharmokinetics of ICL 670, a new, orally active iron chelating agent in patients with transfusion-dependent iron overload due to beta-thalassemia. Journal of Clinical Pharmacology 43: 565–72. Gordeuk VR, Caleffi A, Corradini E (2003) Iron overload in Africans and African-Americans and a common mutation in the SCL40A1 (ferroportin 1) gene. Blood Cells, Molecules and Diseases 31: 299–304. Gurrin LC, Osbourne NJ, Constantine CC et al (2008) The natural history of serum iron in HFE C282Y homozygosity associated with hereditary hemochromatosis. Gastroenterology 135: 1945–52. Hoffbrand AV (ed.) (2009) Recent advances in the understanding of iron metabolism and iron-related diseases. Acta Haematologica 122: 75–7. 12 articles covering all aspects of this topic. Hoffbrand AV, Cohen A, Hershko C (2003) Role of deferiprone in chelation therapy for transfusional iron overload. Blood 102: 17–24. Ke Y, Qian ZM (2003) Iron misregulation in the brain: a primary cause of neurodegenerative disorders. Lancet Neurology 2: 246–53. Kirk P, Roughton M, Porter JB et al. (2009) Cardiac T2* magnetic resonance for prediction of cardiac complications in thalassemia major. Circulation 109: 1961–8. Lee PL, Beutler E (2009) Regulation of hepcidin and iron-overload disease. Annual Review of Pathology 4: 489–515. Lok CY, Merryweather-Clarke AT, Viprakasit V et al. (2009) Iron overload in the Asian community. Blood 114: 20–5.

60

McLaren GD, McLaren CE, Adams PC et al. (2008) Clinical manifestations of hemochromatosis in HFFE homozygotes detected by screening. Canadian Journal of Gastroenterology 22: 923– 30. Modell B, Khan M, Darlison M et al. (2008) Improved survival of thalassaemia major in the UK and relation to T2* cardiovascular magnetic resonance. Journal of Cardiovascular Magnetic Resonance 10: 42. Pennell DJ, Berdoukas V, Karagiorga M et al. (2006) Randomized controlled trial of deferiprone or deferoxamine in betathalassemia major patients with asymptomatic myocardial siderosis. Blood 107: 3738–44. Pennell DJ, Porter JB, Cappellini DM et al. (2010) Efficacy of deferasirox in reducing and preventing cardiac iron overload in beta-thalassemia. Blood 115: 2364–71. Pootrakul PS, Sirankapracha P, Sankote J et al. (2003) Clinical trial of deferiprone iron chelation therapy on β-thalassaemia/haemoglobin E patients in Thailand. British Journal of Haematology 122: 305–10. Porter JB, Galanello L, Saglio G et al. (2008) Relative response of patients with myelodysplastic syndromes and other transfusiondependent anaemias to deferasirox (ICL670): a 1-yr prospective study. European Journal of Haematology 80: 168–76. Taher A, Cappellini MD, Vichinsky E et al. (2009) Efficacy and safety of deferasirox doses >30 mg/kg per day in patients with transfusion-dependent anaemia and iron overload. British Journal of Haematology 147: 752–9. Tanner MA, Galanello R, Dessi C et al. (2007) A randomized, placebo-controlled double blind trial of the effect of continued therapy with deferoxamine and deferiprone on myocardial iron in thalassemia major using cardiovascular magnetic resonance. Circulation 115: 1876–84. Tanner MA, Galanello R, Dessi C et al. (2008) Combination chelation therapy in thalassemia major for the treatment of severe myocardial siderosis with left ventricular dysfunction. Journal of Cardiocasicular Magnetic Resonance 10: 12. Voskaridou E, Plata E, Douskou M et al. (2010) Treatment with deferasirox (Exjade) effectively decreases iron burden in patients with thalassaemia intermedia: results of a pilot study. British Journal of Haematology 148: 332–4.

CHAPTER 5

5

Megaloblastic anaemia A Victor Hoffbrand University College Medical School, Royal Free Hospital, London, UK

Introduction, 61 Underlying basic science, 61 Biochemical basis of megaloblastic anaemia, 61 Cobalamin–folate relationship, 62 Clinical features, 62 General tissue effects of cobalamin and folate deficiencies, 64 Haematological findings, 66 Peripheral blood, 66 Bone marrow, 67 Chromosomes, 67 Ineffective haemopoiesis, 67 Cobalamin, 68 Dietary sources and requirements, 68 Absorption, 68 Enterohepatic circulation, 69 Transport, 69

Cobalamin analogues, 70 Causes of cobalamin deficiency, 70 Diagnosis of cobalamin deficiency, 74 Tests for the cause of cobalamin deficiency, 75 Folate, 76 Dietary folate, 76 Body stores and requirements, 76 Absorption, 76 Enterohepatic circulation, 77 Transport, 77 Biochemical functions, 77 Causes of folate deficiency, 77 Diagnosis of folate deficiency, 79 General management of megaloblastic anaemia, 79 Treatment of cobalamin deficiency, 80 Treatment of folate deficiency, 80

Introduction The megaloblastic anaemias are a group of disorders characterized by the presence of distinctive morphological appearances of the developing red cells in the bone marrow. The cause is usually deficiency of either cobalamin (vitamin B12) or folate, but megaloblastic anaemia may arise because of inherited or acquired abnormalities affecting the metabolism of these vitamins or because of defects in DNA synthesis not related to cobalamin or folate (Table 5.1).

Underlying basic science Biochemical basis of megaloblastic anaemia The common feature of all megaloblastic anaemias is a defect in DNA synthesis that affects rapidly dividing cells in the bone marrow and other tissues. All conditions that give rise to megaloblastic changes share in common a disparity in the rate of synthesis or polymerization of the four immediate precursors

Postgraduate Haematology: 6th edition. Edited by A. Victor Hoffbrand, Daniel Catovsky, Edward G.D. Tuddenham, Anthony R. Green © 2011 Blackwell Publishing Ltd.

Folinic acid (5-formyl-THF), 80 Prophylactic folic acid, 80 Pregnancy, 80 Prematurity, 80 Haemolytic anaemia and dialysis, 80 Megaloblastic anaemia not due to cobalamin or folate deficiency or altered metabolism, 80 Other nutritional anaemias, 80 Protein deficiency, 80 Scurvy, 80 Other deficiencies, 80 Selected bibliography, 80

of DNA: the deoxyribonucleoside triphosphates (Figure 5.1). In deficiencies of either folate or cobalamin there is a failure to convert deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP). The coenzyme 5,10-methylene tetrahydrofolate polyglutamate is needed for this reaction and the availability of this coenzyme is reduced in either cobalamin deficiency or folate deficiency (see below). The reduced supply of deoxythymidine triphosphate (dTTP) in megaloblastic anaemia owing to folate or cobalamin deficiency slows elongation of newly originated replicating segments from multiple sites of origin. Thus small fragments accumulate, single-stranded areas become points of weakness where mechanical or enzymatic breakage may occur, and the failure to form bulk DNA impairs contraction of newly replicated lengths of DNA, leaving the chromosomes elongated, despirillated and with random breaks. Late-replicating DNA is particularly affected and some cells become arrested and die at this stage by apoptosis, which can be prevented in vitro by preformed thymidine. Surprisingly, measurements of dTTP concentration in megaloblasts have not shown a deficiency. This may be because the overall cell concentration masks a localized deficiency at the multienzyme complex directly concerned with DNA replication. An alternative hypothesis for megaloblastic anaemia in cobalamin or folate deficiency is the misincorporation of uracil into DNA because of a build-up of deoxyuridine triphosphate 61

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Table 5.1 Causes of megaloblastic anaemia. Cobalamin deficiency or abnormalities of cobalamin metabolism (Table 5.4) Folate deficiency or abnormalities of folate metabolism (Table 5.7) Therapy with antifolate drugs (e.g. methotrexate) Independent of either cobalamin or folate deficiency and refractory to cobalamin and folate therapy: Some cases of acute myeloid leukaemia, myelodysplasia* Therapy with drugs interfering with DNA synthesis (e.g. cytarabine, hydroxycarbamide, 6-mercaptopurine, azidothymidine) Orotic aciduria (responds to uridine) Lesch–Nyhan syndrome (? responds to adenine)

(dUTP) at the replication fork as a consequence of the block in conversion of dUMP to dTMP (Figure 5.1). There is a mechanism for recognition of this aberrant material for excision and repair, but with dTTP in short supply this may not be possible. Repeated cycles of futile excision and misrepair may occur, with disruption of the normal programme of DNA synthesis leading to apoptotic cell death. Data on this hypothesis are conflicting. It does not explain megaloblastic anaemia due to defects of DNA synthesis at sites other than thymidylate synthesis, for example with drugs such as hydroxycarbamide (hydroxyurea), cytarabine or 6-mercaptopurine, or with enzyme deficiencies such as orotic aciduria or thiamine-responsive megaloblastic anaemia (see below).

Cobalamin–folate relationship *Folate deficiency also occurs frequently in these diseases.

DNA

dATP

dGTP

dCTP

dTTP

dTDP

Methotrexate

THF polyglutamate

DHF polyglutamate

dTMP

5,10-methylene-THF polyglutamate

dUMP

THF

B12

Methionine Homocysteine

Methyl-THF Cell membrane Methyl-THF

Plasma Small intestine

Folate is required for many other reactions in mammalian tissues, including two in purine synthesis (Table 5.2), but impairment of these is far less important clinically. Only two reactions in the body are known to require cobalamin (Figure 5.2). Methylmalonyl-CoA isomerization, which requires deoxyadenosyl(ado)-cobalamin, is discussed later. The methylation of homocysteine to methionine requires both 5-methyltetrahydrofolate (methyl-THF) as methyl donor and methylcobalamin as coenzyme (Figure 5.3). This reaction, which is almost completely irreversible, is the first step in the pathway by which methyl-THF, which enters bone marrow and other cells from plasma, is converted into all the intracellular folate coenzymes (Figure 5.3). The coenzymes are all polyglutamated (the larger size aiding retention in the cell), but the enzyme folate polyglutamate synthase requires THF and not methyl-THF as substrate. In cobalamin deficiency, methylTHF accumulates in the plasma, while intracellular folate concentrations fall due to failure of formation of intracellular folate polyglutamates because of ‘THF starvation’ or ‘methylfolate trapping’. This theory explains the abnormalities of folate metabolism that occur in cobalamin deficiency (high serum folate, low cell folate, reduced thymidylate synthesis, positive purine precursor AICAR excretion; Table 5.2) and also why the anaemia that occurs in cobalamin deficiency will respond to folic acid in large doses. The explanation of why serum cobalamin falls in folate deficiency may also be related to impairment of the homocysteine–methionine reaction, with reduced formation of methylcobalamin, the main form of cobalamin in plasma, but other mechanisms may be responsible.

Dietary folates

Figure 5.1 Role of folate (as 5,10-methylene-THF polyglutamate and methylcobalamin) in DNA synthesis. THF, tetrahydrofolate; MP, monophosphate; TP, triphosphate; d, deoxyribose; A, adenine; T, thymine; C, cytosine; G, guanine. 62

Clinical features Many symptomless patients are detected through the finding of a raised mean corpuscular volume (MCV) on a routine blood

Megaloblastic anaemia

Table 5.2 Biochemical reactions of folate coenzymes. Reaction

Coenzyme form of folate involved

Single-carbon unit transferred

Importance

Formate activation

THF

–CHO

Generation of 10-formyl-THF

Purine synthesis Formation of glycinamide ribonucleotide

5,10-Methenyl-THF

–CHO

Formation of purines needed for DNA, RNA synthesis, but reactions probably not rate limiting

10-Formyl-THF

–CHO

5,10-Methenyl-THF

–CH3

Rate limiting in DNA synthesis Oxidizes THF to DHF Some breakdown of folate at the C-9–N-10 bond

THF 5-Methyl-THF

=CH2 –CH3

THF

–HN–CH=

Entry of single-carbon units into active pool Demethylation of 5-methyl-THF to THF; also requires cobalamin, flavine adenine dinucleotide, ATP and adenosylmethionine Basis of the Figlu test (now obsolete)

Formylation of amino-imidazolecarboxamide-ribotide (AICAR) Pyrimidine synthesis Methylation of deoxyuridine monophosphate (dUMP) to thymidine monophosphate (dTMP) Amino acid interconversion Serine–glycine interconversion Homocysteine to methionine

Forminoglutamic acid to glutamic acid in histidine catabolism

DHF, dihydrofolate; THF, tetrahydrofolate.

Figure 5.2 Intracellular cobalamin metabolism. Cbl1+, Cbl2+ and Cbl3+ refer to the oxidation state of the central cobalt atom of cobalamin. A–G refer to the sites of blocks that have been identified by complementation analysis in infants with metabolic defects. AdoCbl, adenosylcobalamin; MeCbl, methylcobalamin; TC, transcobalamin. The mitochondrial, lysosomal and cytoplasmic compartments are indicated. (From Lilleyman JS, Hann IM, Blanchette VS (eds) (1999) Paediatric Haematology, 2nd edn. Churchill Livingstone, Edinburgh with permission.)

TC OHCbl

TC OHCbl

TC OHCbl

Homocysteine Methionine MS–MeCbl E,G

F OHCbl C,D Cbl2+ (Cbl3+)

Methionine synthase-Cbl+ (MS–Cbl+)

Cbl2+ A

Cbl+ B AdoCbl L-Methylmalonyl CoA Succinyl CoA Mutase

63

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Methylated product (e.g. methylated lipids, myelin basic protein, DOPA, DNA)

Pyruvate

Methyltransferases

Substrate

GSH S-Adenosylhomocysteine (SAH)

Cysteine

Cystathionine Cystathionine synthase vitamin B6

THE METHYLATION CYCLE

Homocysteine

Cell

ATP Methionine Polyglutamate synthase + glutamates

Methionine synthase methylcobalamin

Plasma 5-Methyl tetrahydrofolate 5-Methyl tetrahydrofolate (monoglutamate)

S-Adenosylmethionine (SAM)

5,10-Methylenetetrahydrofolate reductase

Tetrahydrofolate Serine Glycine

DHF reductase

Formate Purines

Dihydrofolate 5,10-Methylene tetrahydrofolate Deoxyuridine monophosphate Folic acid

DNA CYCLE (CELL REPLICATION)

10-Formyl tetrahydrofolate Deoxythymidine monophosphate Folic acid

Figure 5.3 The role of folates in DNA synthesis and in formation of S-adenosylmethionine (SAM), which is involved in numerous methylation reactions. Enzymes are shown in yellow boxes. (Figure prepared in conjunction with Professor John Scott.)

count. The main clinical features in more severe cases are those of anaemia. Anorexia is usually marked and there may be weight loss, diarrhoea or constipation. Other particular features include glossitis, angular cheilosis, a mild fever in the more severely anaemic patients, jaundice (unconjugated) and reversible melanin skin hyperpigmentation, which may occur with either deficiency. Thrombocytopenia sometimes leads to bruising (and this may be aggravated by vitamin C deficiency in malnourished patients. The (anaemia and) low leucocyte count may predispose to infections, particularly of the respiratory or urinary tracts. Cobalamin deficiency has also been associated with impaired bactericidal function of phagocytes.

General tissue effects of cobalamin and folate deficiencies Epithelial surfaces These deficiencies, when severe, affect all rapidly growing (DNA-synthesizing) tissues. After the marrow, the next most affected tissues are the epithelial cell surfaces of the mouth, stomach, small intestine and respiratory, urinary and female genital tracts. The cells show macrocytosis, with increased 64

numbers of multinucleate and dying cells. The deficiencies may cause cervical smear abnormalities. Complications of pregnancy The gonads are also affected and infertility is common in both men and women with either deficiency if severe. Maternal folate deficiency has been implicated as a cause of prematurity and both folate and cobalamin deficiency have been implicated in recurrent fetal loss. Neural tube defects Folic acid supplements at the time of conception and in the first 12 weeks of pregnancy reduce by about 70% the incidence of neural tube defects (NTDs) – anencephaly, meningomyelocele, encephalocele and spina bifida – in the fetus. Most of this protective effect can be achieved by taking folic acid 0.4 mg daily. The incidence of cleft palate and harelip can also be reduced by prophylactic folic acid. There is no clear simple relationship between maternal folate status and these fetal abnormalities, although the lower the maternal folate, the greater the risk to the fetus. NTDs can also be caused by antifolate and antiepileptic drugs.

Megaloblastic anaemia

An underlying maternal folate metabolic abnormality has also been postulated. One abnormality has been identified: reduced activity of the enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) (Figure 5.3) caused by a common 677C→T polymorphism in the MTHFR gene. In one study, the prevalence of this polymorphism was found to be higher in the parents of NTD fetuses and in the fetuses themselves: homozygosity for the TT mutation was found in 13% compared with 5% in control subjects. The polymorphism codes for a thermolabile form of MTHFR. The homozygous state results in lower mean serum and red cell folate compared with control subjects, as well as significantly higher serum homocysteine levels. Tests for mutations in other enzymes possibly associated with NTDs, for example methionine synthase or serine–glycine hydroxymethylase, have been negative. Autoantibodies to folate receptors (see p. 77) had been suggested to cause NTDs but this has not been confirmed in a recent large study. Cardiovascular disease Children with severe homocystinuria (blood levels of 100 µmol/L or more) due to deficiency of one of three enzymes, methionine synthase, MTHFR or cystathionine synthase (Figure 5.3), suffer from vascular disease (e.g. ischaemic heart disease, cerebrovascular disease or pulmonary embolus) as teenagers or in young adulthood. Meta-analysis shows a significant association between lesser degrees of raised serum homocysteine (normal range 5–15 µmol/L) and of homozygosity for mutated MTHFR with ischaemic heart disease, stroke, deep vein thrombosis and pulmonary embolism. The odds ratios for a 5-µmol/L increase in serum homocysteine were 1.42 in 72 genetic (MTHFR) studies and 1.32 in 20 prospective studies of serum homocysteine, 1.60 for deep vein thrombosis with or without pulmonary embolism in genetic studies, and for stroke 1.65 in genetic studies and 1.59 in prospective studies. As the genetic and prospective studies do not share the same potential sources of error but both yield highly significant results, the authors considered the results strong evidence of a causal association between homocysteine and cardiovascular disease. Heterozygosity for the C677T mutation has also been shown to be associated with an increase in the risk of thrombosis in subjects heterozygous for factor V Leiden. It remains possible that homocysteine levels may be high as a consequence of the vascular damage or may merely be a marker for some other underlying factor that is responsible for both the vascular damage and the raised homocysteine. Folate deficiency, for example, may be such a factor. Folate levels have been found in various studies to be lower in patients with myocardial infarct and carotid artery disease than control subjects. There are some reports of prevention of arterial disease recurrence or progression by prophylactic folic acid or cobalamin but the results are conflicting. Meta-analysis of data from large multicentre prospective trials of folic acid in prevention of coronary vascular disease do not show a positive effect. Meta-analysis

does suggest that folic acid supplementation reduces the risk of stroke by 18% but even this is not certain, depending on which data are included in the analysis. Extremely high levels of homocysteine (>50 µmol/L) are toxic to endothelia. When homocysteine levels are only mildly (15–25 µmol/L) or moderately (25–50 µmol/L) elevated, then another mechanism needs to be invoked to explain vascular damage or increased risk of thrombosis. Several mechanisms have been proposed, including oxidant damage through the generation of peroxide produced during thiol oxidation to form disulphides and interaction of free reduced homocysteine with cysteine residues on coagulation factors, platelets, adhesion molecules or endothelial cells. Promotion of vascular wall inflammation through the generation of proinflammatory cytokines and interference with key methylation reactions are also possible mechanisms. Malignancy Prophylactic folic acid in pregnancy has been found in some but not all studies to reduce the subsequent incidence of acute lymphoblastic leukaemia (ALL) in childhood. A significant negative association has been found with the MTHFR 677C→T and 1298A→C polymorphisms and the incidence of both paediatric and adult ALL. There are various positive and negative associations between polymorphisms in other folatedependent enzymes and the incidence of paediatric and adult ALL. Other tumours that have been associated with folate polymorphisms or status include follicular lymphoma, breast cancer and gastric cancer. The C677T polymorphism is thought to lead to increased thymidine pools and ‘better quality’ of DNA synthesis by shunting one-carbon groups towards thymidine and purine synthesis. This may also explain its reported association with a lower risk for colorectal and gastric cancer. The incidence of colon cancer was also lower in subjects taking vitamin supplements containing folic acid and in those with higher folate intake compared with control subjects in the Nurses Health Study, and increased folate intake may mask the protective effect of the C677T and A1298C polymorphisms. There is no correlation between pre-diagnostic plasma folate and risk of death in patients with colorectal cancer. One study showed no overall difference in incidence of colonic adenoma in subjects taking folic acid or controls but a higher incidence of multiple (>3) adenomas in those taking folic acid. However, the statistics have been questioned and other studies have shown a lower incidence of colonic adenoma with higher folate intake. Folate deficiency is postulated to predispose to malignancy by reducing the ratio of S-adenosylmethionine (SAM) to Sadenosylhomocysteine (SAH), causing DNA hypomethylation and, by resulting in uracil misincorporation into DNA, possibly leading to double-strand breaks. Most recent data from large prospective trials suggest no significant effect of folic acid on the incidence or progress of any cancer. 65

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Other tissues Folate deficiency causes reduced regeneration of cirrhotic liver. Patients with gluten-induced enteropathy and those with sickle cell anaemia have also been reported to show stunted growth, which has been improved coincidentally with commencement of folic acid therapy, but it is not certain how much the growth improvement in these children was due to folic acid and how much to other, simultaneously administered vitamins. In the fragile X syndrome, sister chromatid exchange and DNA breaks are increased in vitro in a folate-deficient medium, apparently at the Xq28 site. No in vivo abnormality of folate metabolism can be detected. Neurological manifestations Cobalamin deficiency may cause bilateral peripheral neuropathy or degeneration (demyelination) of the posterior and pyramidal tracts of the spinal cord and, less frequently, optic atrophy or cerebral symptoms. The patient classically presents with paraesthesiae, muscle weakness or difficulty in walking and sometimes dementia, psychotic disturbances or visual impairment. Long-term nutritional cobalamin deficiency in infancy leads to poor brain development and impaired intellectual development. Folate deficiency may cause mental changes such as depression and slowness and has been suggested to cause organic nervous disease, but this is uncertain. Autoantibodies to the folate receptor involved in transport of folate into cerebrospinal fluid have been postulated to be associated with autismassociated neurological abnormalities. Methotrexate injected into the cerebrospinal fluid may cause brain or spinal cord damage. Neural tube defects in the fetus are discussed above. The biochemical basis for cobalamin neuropathy remains obscure. Its occurrence in the absence of methylmalonic aciduria in transcobalamin deficiency, and in monkeys given nitrous oxide (N2O), suggests that the neuropathy is related to the defect in conversion of homocysteine to methionine. Accumulation of SAH in the brain, resulting in inhibition of transmethylation reactions due to an altered SAM to SAH ratio, has been suggested. SAM is needed in methylation of biogenic amines (e.g. dopamine), as well as of proteins, phospholipids and neurotransmitters in the brain (see Figure 5.3). A reduced ratio of SAM to SAH is postulated to result in reduced methylation. However, measurements of methylation of arginine in myelin basic protein in fruit bats with cobalamin neuropathy, or in rats exposed to N2O, showed no defect of methylation. Psychiatric disturbance is common in both folate and cobalamin deficiencies. Loss of cognitive function in the elderly has been associated with high plasma homocysteine and low vitamin B12 and folate levels. This, like the neuropathy, has been attributed to a failure of the synthesis of SAM. Plasma homocysteine is a risk factor for dementia and Alzheimer disease, shown in a median follow-up period of 8 years in one study of 1092 subjects. Studies showing an association between lower serum levels of folate or cobalamin and 66

higher homocysteine levels and Alzheimer disease, loss of cognitive function or brain volume loss have been reported. However, trials of supplementation with folic acid, vitamin B12 and vitamin B6 have not shown a benefit in preventing progression of the dementia compared with a control group, or in improving cognitive function.

Haematological findings Peripheral blood Oval macrocytes, usually with considerable anisocytosis and poikilocytosis, are the main feature (Figure 5.4a). The MCV is usually more than 100 fL unless a cause of microcytosis (e.g. iron deficiency or thalassaemia trait) is present, when there is a raised red cell distribution width (RDW) and the film is dimorphic. In other cases, the MCV may be normal owing to excess fragmentation of red cells. Some of the neutrophils are hypersegmented (more than five nuclear lobes). Both macrocytosis and hypersegmented neutrophils may also occur in other situations (Table 5.3). Together, however, they strongly suggest megaloblastic haemopoiesis. There may be leucopenia due to a reduction in granulocytes and lymphocytes; the platelet count may be moderately reduced, rarely to less than 40 × 109/L. Occasionally, a leucoerythroblastic blood picture is seen. In the non-anaemic patient, the presence of a few macrocytes and Table 5.3 Conditions in which macrocytosis or hypersegmented neutrophils may occur in the absence of megaloblastic anaemia. Macrocytosis Alcohol Liver disease (especially alcoholic) Reticulocytosis (haemolysis or haemorrhage) Aplastic anaemia or red cell aplasia Hypothyroidism Myelodysplasia Myeloma and macroglobulinaemia Leucoerythroblastic anaemia Myeloproliferative disease Pregnancy Newborn Congenital dyserythropoietic anaemia (type II) ? Chronic respiratory failure Hypersegmented neutrophils Renal failure Congenital (familial) ? Iron deficiency Note: Falsely high MCV recorded when cold agglutinins, paraproteins or marked leucocytosis are present.

Megaloblastic anaemia

(a)

(b)

Figure 5.4 Severe megaloblastic anaemia: (a) peripheral blood; (b) bone marrow.

hypersegmented neutrophils in the peripheral blood may be the only abnormalities.

Bone marrow In the severely anaemic patient, the marrow is hypercellular with accumulation of primitive cells due to selective death of more mature forms. The most characteristic finding is dissociation between nuclear and cytoplasmic development in the erythroblasts, with the nucleus maintaining a primitive appearance despite maturation and haemoglobinization of the cytoplasm; fully haemoglobinized (orthochromatic) erythroblasts, which retain nuclei, may be seen. The nucleus of the megaloblast has an open, fine, lacy appearance; the cells are larger than normoblasts and an increased number of cells with eccentric lobulated nuclei or nuclear fragments may be present (Figure 5.4b). Mitoses and dying cells are more frequent than normal. Giant and abnormally shaped metamyelocytes and enlarged hyperpolyploid megakaryocytes are characteristic. Severe florid megaloblastic changes may be confused with acute erythroid leukaemia. Rarely, the marrow may be hypocellular or red cell precursors are lost almost completely from the marrow and a mistaken diagnosis of myeloid leukaemia may be made. Iron staining shows increase in both reticuloendothelial stores and in the developing megaloblasts. In less anaemic patients, the changes in the marrow may be difficult to recognize. The terms ‘intermediate’, ‘mild’ and ‘early’ have been used. The changes may be mild and difficult to recognize, even in a severely anaemic patient, if the anaemia is largely due to other factors (e.g. iron deficiency, infection,

malignant disease, haemolysis) and the megaloblastosis is an incidental phenomenon. The term ‘megaloblastoid’ has several different connotations including the dysplastic changes seen in the myelodysplastic syndromes and is best avoided.

Chromosomes Bone marrow cells, transformed lymphocytes and other proliferating cells in the body show a variety of changes including random breaks, reduced contraction, spreading of the centromere, and exaggeration of secondary chromosomal constrictions and overprominent satellites. Similar abnormalities may be produced by antimetabolite drugs (e.g. cytarabine, hydroxycarbamide and methotrexate) that interfere with either DNA replication or folate metabolism and which also cause megaloblastic appearances.

Ineffective haemopoiesis There is accumulation of unconjugated bilirubin in plasma due to the death of nucleated red cells in the marrow (ineffective erythropoiesis). Other evidence for this includes raised urine urobilinogen, reduced haptoglobins and positive urine haemosiderin, raised serum lactate dehydrogenase to values between 1000 and 10 000 IU/dL, and raised serum iron, non-transferrinbound iron and ferritin levels. Carbon monoxide production is also increased. Serum lysozyme may also be raised, suggesting ineffective granulopoiesis. In rare patients, ineffective haemopoiesis is associated with features of disseminated intravascular coagulation, with raised 67

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serum fibrin degradation products. Thrombocytopenia, when it occurs, is usually caused by ineffective megakaryopoiesis. A weakly positive direct antiglobulin test due to complement can lead to a false diagnosis of autoimmune haemolytic anaemia.

Cobalamin Cobalamin (vitamin B12) exists in a number of different chemical forms. The molecule consists of two halves: a planar group and a nucleotide set at right angles to it (Figure 5.5). The planar group is a corrin ring and the nucleotide consists of a base, 5,6-dimethylbenzimidazole, and a phosphorylated sugar, ribose-5-phosphate. In nature, the vitamin is mainly in the 5′-deoxyadenosyl (ado) form. This is the main form in human tissues and is located in the mitochondria. It serves as the cofactor for methylmalonyl-CoA mutase. The other major natural cobalamin is methylcobalamin, the main form in human plasma and cell cytoplasm. It serves as the cofactor for methionine synthase. There are also minor amounts of hydroxocobalamin, the form to which methyl- and ado-cobalamin are rapidly converted by exposure to light, hydroxocobalamin having its cobalt atom in the fully oxidized Cbl3+ state, whereas the cobalt exists as reduced Cbl1+ in the methyl- and ado-cobalamin forms (see Figure 5.2). A glutathionyl cobalamin form has also been identified.

Dietary sources and requirements Cobalamin is synthesized solely by microorganisms. Ruminants obtain cobalamin from the foregut but the only source for N

C C

N

+

N

CH2 CH2

C

D

N

Co

N

A

N

C

B

C

H3

C

N

CO CH 3

O

CH2 CH

OH O-

O

NH

O

P O

CH2.OH

CH3

Figure 5.5 The structure of vitamin B12 (cyanocobalamin). 68

humans is food of animal origin. The highest amounts are found in liver and kidney (up to 100 µg per 100 g), but it is also present in shellfish, organ and muscle meats, fish, chicken and dairy products (eggs, cheese and milk) in small amounts (6 µg/L). Vegetables, fruits and all other foods of non-animal origin are free from cobalamin unless they are contaminated by bacteria. Cooking does not usually destroy cobalamin. A normal Western diet contains 5–30 µg of cobalamin daily. Adult daily losses (mainly in the urine and faeces) are about 1–2 µg (about 0.1% of body stores) and because the body does not have the ability to degrade cobalamin, daily requirements are also about 1 µg. Body stores are of the order of 2–3 mg and are sufficient for 3–4 years if supplies are completely cut off.

Absorption Two mechanisms exist for cobalamin absorption. One is passive, occurring equally through the duodenum and the ileum; it is rapid but extremely inefficient as less than 1% of an oral dose can be absorbed by this process. Passive absorption of cobalamin can also occur through other mucous membranes such as the sublingual and nasal mucosae. The other mechanism is active; it occurs through the ileum in humans and is efficient for small (a few micrograms) oral doses of cobalamin. This is the normal mechanism by which the body acquires cobalamin and is mediated by gastric intrinsic factor (IF). Dietary cobalamin is released from protein complexes by enzymes in the stomach, duodenum and jejunum; it combines rapidly with a salivary glycoprotein (R binder) related to plasma transcobalamin I (TCI). These belong to the family of cobalamin-binding proteins known as haptocorrins (HCs), which differ only in glycosylation. They are products of a single gene (TCN1), and they occur in saliva, gastric juice, bile, milk and other body fluids. Subsequently, HC is digested by pancreatic trypsin and the cobalamin transferred to IF. Binding of cobalamin to IF is favoured by an alkaline pH; it binds one molecule for one molecule. All forms of cobalamin are absorbed by the same IF mechanism (Figure 5.6). Pseudo-cobalamin compounds, in which the 5,6-dimethylbenzimidazole nucleotide is replaced by other nucleotides that may attach to HC, do not attach to IF and therefore remain unabsorbed. Intrinsic factor is a glycoprotein (molecular weight 45 000) encoded by a gene on chromosome 11q13. It is produced in gastric parietal cells in the fundus and body of the stomach. The IF–cobalamin complex, in contrast with free IF, is resistant to enzyme digestion, having a more closed structure. The IF– cobalamin complex passes to the ileum, where IF attaches to a specific receptor (cubilin, molecular weight 460 000) on the microvillus membrane of the brush border surface of the ileal absorptive cells. Cubilin (gene located on chromosome 10p12.1) is also present in yolk sac and renal proximal tubular epithelium. The attachment of the IF–cobalamin complex requires calcium ions and a pH around neutral. It is probably a physical

Megaloblastic anaemia

(a)

(b)

CN (or

CH3 or

OH or deoxyadenosyl)

C0

Figure 5.6 (a) Intrinsic factor; (b) intrinsic factor–cobalamin complex. Intrinsic factor has been estimated to have a molecular radius of 3.6 nm, vitamin B12 0.8 nm and the complex 3.2 nm.

process, not requiring energy. Cubulin traffics by means of the protein amnionless (AMN, molecular weight 480 000). Cubulin and AMN are subunits of a novel complex in which AMN binds tightly to cubulin and directs sublocalization and endocytosis of cubulin with its ligand (IF–cobalamin complex). Defects in cubulin and AMN are implicated in autosomal recessive megaloblastic anaemia, characterized by intestinal malabsorption of cobalamin (see p. 73). A third protein, megalin (LRP2), has been suggested to play a role in stabilizing the cubilin–AMN complex. Cobalamin then enters the ileal cell, but the exact fate of IF is unknown. IF does not enter the bloodstream as such, as after a delay of about 6 hours absorbed cobalamin appears in portal blood attached to transcobalamin (TCII), which is probably synthesized in the ileum, either by mucosal cells or by venous endothelial cells in the submucosa. The ileum has a restricted capacity to absorb cobalamin because of limited receptor sites. Although 50% or more of a single dose of 1 µg of cobalamin may be absorbed, with doses above 2 µg the proportion absorbed falls rapidly. Moreover, after one dose of IF–cobalamin complex has been presented, the ileal cells become refractory to further doses for about 6 hours.

Enterohepatic circulation Between 0.5 and 5.0 µg of cobalamin enter the bile each day. This binds to IF and a portion of biliary cobalamin is normally reabsorbed together with cobalamin derived from sloughed intestinal cells. Bile may enhance cobalamin absorption. Cobalamin deficiency develops more rapidly in individuals who malabsorb cobalamin than it does in vegans, who ingest no cobalamin but in whom reabsorption of biliary cobalamin is intact.

Transport Two main cobalamin-binding proteins exist in human plasma; they both bind cobalamin one molecule for one molecule

Mol wt = 56 000–58 000 Source: granulocytes ? other tissues TCl + III (HCs) 800 ng/L 450 ng/L

30–40 ng/L

TCII (TC) 1000 ng/L Mol wt = 38 000 Source: liver ileum macrophages

Figure 5.7 The serum cobalamin-binding proteins (TCs, transcobalamins). Dark blue rectangles indicate endogenous serum cobalamin; light blue rectangles indicate unsaturated cobalamin-binding protein; HCs, haptocorrins.

(Figure 5.7). One HC (also known as TCI) is a glycoprotein. TCIII was a name used to describe a minor isoprotein of TCI in plasma. These HCs are derived primarily from the specific granules in neutrophils and are normally about two-thirds saturated with cobalamin, which they bind tightly. They do not enhance cobalamin entry into tissues. Glycoprotein receptors on liver cells are concerned in the removal of HCs from plasma, and HC may have a role in the transport of cobalamin analogues to the liver for excretion in bile. The gene, TCN1, is located on chromosome 11q11–q12.3, has nine exons and codes a protein of 433 amino acids. Heterozygous, homozygous or compound heterozygous mutations in the gene may cause mild or severe reductions in serum vitamin B12 respectively with no known clinical consequences. 69

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The other major cobalamin transport protein in plasma is transcobalamin (TC, also known as TCII). TC is a β-globulin (molecular weight 38 000) synthesized by liver, and by other tissues including macrophages, endothelial and possibly ileal cells. It normally carries only 20–60 ng of cobalamin per litre of plasma and readily gives up cobalamin to marrow, placenta and other tissues, which it enters by receptor-mediated endocytosis via clathrin-coated pits. TC is not reutilized. The gene is on chromosome 22q11–q13.1 and, as for IF and HC, there are nine exons. The three proteins are likely to have a common ancestral origin. TC has 20% amino acid homology and more than 50% nucleotide homology with human HC and with rat IF. The regions of homology of HC, TC and IF are involved in cobalamin binding. Five different inherited isoproteins of TC, separated by polyacrylamide gel electrophoresis, have been described; all are functionally active. TC occurs in cerebrospinal fluid and binds cobalamin (approximately 10 ng/L) there. Alterations may occur in TC and HC levels in a variety of disease states (Table 5.4). In general, an increase in HC causes an increase in serum cobalamin, whereas an increase in TC does not. The TC receptor belongs to the low-density lipoprotein receptor family. It is composed of 282 amino acids and is heavily Table 5.4 Alterations in plasma cobalamin-binding proteins in disease. Haptocorrin Increased (usually with elevated serum cobalamin) Myeloproliferative diseases, especially chronic myeloid leukaemia, myelosclerosis, polycythaemia vera Hepatoma Increased granulocyte production (e.g. inflammatory bowel disease, liver abscess) Eosinophilia due to hypereosinophilic syndrome Congenital absence Low total serum cobalamin No clear clinical abnormality Transcobalamin (II) Increased (sometimes with no elevation of serum cobalamin) Liver disease Gaucher disease Autoimmune disease Long-continued hydroxocobalamin therapy Congenital absence Normal or decreased total serum cobalamin Megaloblastic anaemia or pancytopenia within a few weeks of birth Impaired cobalamin absorption, may be associated with defective cellular and humoral immunity

70

glycosylated; the gene is located at chromosome 19p15.2. It is more heavily expressed in dividing than quiescent cells, and is recycled to the cell surface. Megalin (LRP-2) is also involved in the endocytosis of TC–cobalamin.

Cobalamin analogues Cobalamin analogues are corrinoids, which exist as cobamides (containing substitutions in the place of ribose, e.g. adenosyl) or as cobinamides (which have no nucleotide whatever). HC may carry analogues to the liver for excretion in the bile. It is unclear whether they are inert or inhibit cobalamin-dependent reactions. The proportion of analogues derived from diet, gut bacteria or endogenous breakdown of cobalamins is unknown. They are present in fetal blood and tissues.

Causes of cobalamin deficiency Cobalamin deficiency is usually due to malabsorption. The only other cause is inadequate dietary intake. Cobalamin deficiency due to excess degradation occurs as a result of exposure to the anaesthetic gas N2O. N2O causes irreversible oxidation of the active Cbl1+ during catalytic shunting of labile methyl groups in the methionine synthase reaction (see Figure 5.2). Inadequate dietary intake

Adults Dietary cobalamin deficiency arises in vegans who omit meat, fish, eggs, cheese and other animal products from their diet. The largest group in the world consists of Hindus, and many millions of Indians are at risk of deficiency on a nutritional basis. However, not all vegans develop cobalamin deficiency of sufficient severity to cause anaemia or neuropathy, even though subnormal cobalamin levels have been found in up to 50% of randomly selected, young, adult Indian vegans. Dietary cobalamin deficiency may also arise rarely in non-vegetarian subjects who exist on grossly inadequate diets because of poverty or psychiatric disturbance. Explanations of why nutritional cobalamin deficiency may not progress to megaloblastic anaemia include the following. 1 The diet of most vegans is probably not totally lacking cobalamin. The serum cobalamin level may not be an accurate measure of their body stores. 2 The enterohepatic circulation of cobalamin is still intact in vegans and thus losses are less (about 1 µg daily) than in conditions of malabsorption (about 2 µg gdaily). 3 Daily losses of cobalamin are thought to be related to body stores; therefore, as the body stores become depleted, daily losses become smaller and the amount of cobalamin needed to maintain the status quo may also become smaller.

Megaloblastic anaemia

Table 5.5 Causes of cobalamin deficiency causing megaloblastic anaemia. Nutritional Vegans Malabsorption Pernicious anaemia Gastric causes Congenital intrinsic factor deficiency or functional abnormality Total or partial gastrectomy Intestinal causes Intestinal stagnant loop syndrome: jejunal diverticulosis, ileocolic fistula, anatomical blind loop, intestinal stricture, etc. Ileal resection and Crohn’s disease Selective malabsorption with proteinuria Tropical sprue Transcobalamin deficiency Fish tapeworm

Infants Cobalamin deficiency has been described in infants born to severely cobalamin-deficient mothers. These infants develop megaloblastic anaemia at about 3–6 months of age since they are born with low stores of cobalamin and are then fed breast milk of low cobalamin content. This occurs most commonly in Indian vegans, but a similar condition has also been described in unrecognized maternal pernicious anaemia and in strict practitioners of veganism living in Western countries whose offspring have shown growth retardation, impaired psychomotor development and other neurological sequelae. Gastric causes of cobalamin malabsorption (Tables 5.5 and 5.6)

Pernicious anaemia Pernicious anaemia (PA) may be defined as a severe lack of IF due to gastric atrophy. It is a common disease in northern Europeans but occurs in all countries and ethnic groups. The overall incidence is about 120 per 100 000 population in the UK, but there is wide variation between one area and the next. The prevalence rate in Western countries may be as high as 2–3%. The ratio of incidence in men and women is approximately 1 : 1.6 and the peak age of onset is 60 years, with only 10% of patients presenting being less than 40 years of age. In some ethnic groups, notably black people and Latin Americans, the age of onset of PA is generally lower. The disease occurs more commonly than by chance in close relatives, in subjects with other organ-specific autoimmune diseases (see below), in those

Table 5.6 Malabsorption of cobalamin may occur in the following conditions but is not usually sufficiently severe and prolonged to cause megaloblastic anaemia. Gastric causes Simple atrophic gastritis (food cobalamin malabsorption) Zollinger–Ellison syndrome Gastric bypass surgery Use of proton pump inhibitors Intestinal causes Gluten-induced enteropathy Severe pancreatitis HIV infection Radiotherapy Graft-versus-host disease Deficiency states Cobalamin, folate, protein, ?riboflavin, ?nicotinic acid Drug therapy Colchicine, p-aminosalicylate, neomycin, slow-release potassium chloride, anticonvulsant drugs, metformin, phenformin, cytotoxic drugs Alcohol

with premature greying, blue eyes and vitiligo, and in persons of blood group A. An association with human leucocyte antigen (HLA)-3 has been reported in some but not all series and in those with endocrine disease, with HLA-B8, -B12 and -BW15. The life expectancy has been estimated as normal in women once regular treatment has begun. Men have a slightly subnormal life expectancy as a result of a higher incidence of carcinoma of the stomach than in control subjects. Diagnosis This is usually suspected from the clinical picture and the findings of megaloblastic anaemia due to cobalamin deficiency. A lack of IF has been demonstrated by cobalamin absorption studies but this test is no longer available in most countries. Tests for circulating gastric autoantibodies are also important. Direct measurements on gastric juice following pentagastrin stimulation are now rarely performed. Hydrochloric acid or pepsin production and IF output were previously measured. The serum gastrin level is usually raised in PA (>200 µg/L), the hormone coming from endocrine cells in the gastric fundus. Raised serum gastrin also occurs in simple atrophic gastritis. Serum pepsinogen I levels are low (< 30 µg/L) in over 90% of those affected and a low ratio of serum pepsinogen I to pepsinogen II correlates with the presence of chronic atrophic gastritis.

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Gastric biopsy This usually shows atrophy of all layers of the body and also fundal atrophy, with loss of glandular elements, an absence of parietal and chief cells and replacement by mucous cells, a mixed inflammatory cell infiltrate and perhaps intestinal metaplasia. The infiltrate of plasma cells and lymphocytes contains an excess of CD4 cells. The antral mucosa is usually well preserved. Helicobacter pylori infection is infrequent in PA, but it has been suggested that H. pylori gastritis may represent an early phase of atrophic gastritis, which is gradually replaced, in some individuals, by an immune process with disappearance of H. pylori infection. Immune phenomena In addition to the appearance of the gastric mucosa, there is a large body of evidence that suggests that immune mechanisms play an important role in the pathogenesis of PA. This aspect of the disease is discussed under four main headings. Antibodies to gastric antigens 1 IF antibodies. Two types of IF antibody may be found in the sera of patients with PA, both being IgG. One, the ‘blocking’ or ‘type I’ antibody, prevents the combination of IF and cobalamin, whereas the other, the ‘binding’, ‘type II’ or ‘precipitating’ antibody, which attaches to IF whether joined to cobalamin or not, prevents attachment of IF to ileal mucosa. The blocking antibody occurs in the serum of about 55% of patients and the binding antibody in 35%. IF antibodies cross the placenta and cause temporary IF deficiency in the newborn infant. Patients with PA also show cell-mediated immunity to IF. An increased CD4/CD8 lymphocyte ratio in blood has been described in PA patients with IF antibodies. IF antibodies are rarely found in conditions other than PA. Type I antibody has been detected rarely in the sera of patients without PA but with thyrotoxicosis, myxoedema, Hashimoto disease or diabetes mellitus, and in relatives of PA patients. IF antibodies have also been detected in gastric juice in about 80% of patients with PA. These antibodies may reduce absorption of dietary cobalamin by combining with small amounts of remaining IF in the gastric juice. Achlorhydria favours the formation of this antigen–antibody complex. 2 Parietal cell and gastrin receptor antibodies. Parietal cell antibody is present in the sera of almost 90% of adult patients with PA, but it is frequently present in other subjects. Thus, it occurs in as many as 16% of randomly selected female subjects aged over 60 years and in a smaller proportion of younger control subjects; it is found more frequently than in control subjects in relatives of PA patients. These antibodies are also found more frequently in patients with simple atrophic gastritis, chronic active hepatitis and thyroid disorders and their relatives, as well as in Addison disease, rheumatoid arthritis and other conditions. The parietal cell antibody is directed against the α- and β-subunits of the gastric proton pump (H+/K+72

ATPase). The sera of PA patients may also contain an autoantibody to the gastrin receptor, although this test is not used clinically. Association with other ‘autoimmune’ diseases There is a clinical association between PA and thyroid diseases, vitiligo, hypoparathyroidism and Addison disease. These diseases are often found in close relatives of patients with overt disease due to one of these conditions. Response to steroid therapy Steroid therapy improves the gastric lesion, at least temporarily, in a proportion of patients with PA. There may be regeneration of the mucosa with a return of secretion of acid and IF, and an improvement in cobalamin absorption. When steroid therapy is withdrawn, there is relapse within a few weeks. These findings suggest that an autoimmune process is continuously damaging the gastric mucosa in PA and preventing regeneration. Hypogammaglobulinaemia PA is found more often than by chance in patients with a deficiency of IgA or with complete hypogammaglobulinaemia. These subjects resemble others with PA, except that they often present relatively early (before the age of 40 years), they have a lower incidence of serum IF and parietal cell antibodies, and they may show intestinal malabsorption. They may also have a history of recurrent infections. The gastric lesion is similar to that in other causes, except that plasma cells are absent from the inflammatory cell infiltrate and the antrum is involved. Serum gastrin levels are normal.

Juvenile pernicious anaemia This usually occurs in older children and resembles PA of adults. Gastric atrophy, achlorhydria and serum IF antibodies are all present, although parietal cell antibodies are usually absent. About half of these patients show an associated endocrinopathy such as autoimmune thyroiditis, Addison disease or hypoparathyroidism; in some, mucocutaneous candidiasis occurs. Congenital intrinsic factor deficiency or functional abnormality The affected child usually shows no demonstrable IF but has a normal gastric mucosa and normal secretion of acid. The inheritance is autosomal recessive. These patients usually present with megaloblastic anaemia in the first, second or third year of life when stores of cobalamin accumulated from the mother in utero are used up; a few have presented as late as the second decade. Parietal cell and IF antibodies are absent. Variants have been described in which the child is born with IF that can be detected immunologically but which is unstable or functionally inactive, being unable either to bind cobalamin or to facilitate its uptake by the ileum.

Megaloblastic anaemia

Gastrectomy Following total gastrectomy, cobalamin deficiency is inevitable and prophylactic cobalamin therapy should be commenced immediately following the operation. After partial gastrectomy, 10–15% of patients also develop this deficiency. The exact incidence and time of onset are most influenced by the size of the resection and the pre-existing size of the cobalamin body store. Simple atrophic gastritis (food cobalamin malabsorption) The normal IF-mediated mechanism of cobalamin absorption requires adequate gastric output of acid and pepsin to ensure the release of food cobalamin. Failure of this mechanism is believed to be responsible for a condition more common in the elderly known as food cobalamin malabsorption, but there is no definitive proof of this. The syndrome has also been described in association with H. pylori infection, long-term use of histamine H2-receptor antagonists and proton-pump inhibitors, chronic alcoholism, pancreatic exocrine failure, Sjögren syndrome and systemic sclerosis. The syndrome is associated with low serum cobalamin levels, with or without evidence of cobalamin deficiency, such as raised serum levels of methylmalonic acid and homocysteine. A minority of patients with food cobalamin malabsorption may go on to develop clinically significant cobalamin deficiency including polyneuropathy, confusion, dementia and subacute combined degeneration of the cord and anaemia, but the frequency of occurrence and reasons for this progression are not clear. Intestinal causes of cobalamin malabsorption Malabsorption of cobalamin occurs in a variety of intestinal lesions in which there is colonization of the upper small intestine by faecal organisms. This may occur in patients with jejunal diverticulosis, enteroanastomosis, intestinal stricture or fistula, or with an anatomical blood loop due to Crohn’s disease, tuberculosis or an operative procedure. Bacterial overgrowth in the small intestine may also cause spurious elevation of serum methylmalonate (see below). Some bacteria produce copious quantities of propionate, the immediate precursor of methylmalonate. Removal of 1.2 m or more of terminal ileum causes malabsorption of cobalamin. In some patients, following ileal resection, particularly if the ileocaecal valve is incompetent, colonic bacteria may contribute further to the onset of cobalamin deficiency. Nearly all patients with acute and subacute tropical sprue show malabsorption of cobalamin; this may persist as the principal abnormality in the chronic form of the disease, when the patient may present with megaloblastic anaemia or neuropathy due to cobalamin deficiency. Absorption of cobalamin usually improves after antibiotic therapy and, in the early stages, after folic acid therapy. Malabsorption of cobalamin occurs in about 30% of untreated patients with gluten-induced enteropathy and correlates with the degree of steatorrhoea. Cobalamin defi-

ciency is not usually severe in these patients and is probably never the cause of megaloblastic anaemia unless another lesion causing malabsorption of cobalamin (e.g. stagnant loop syndrome) is present. The absorption improves when these patients are treated with a gluten-free diet. Selective malabsorption of cobalamin with proteinuria (also known as Imerslünd syndrome, Imerslünd–Grasbeck syndrome, congenital cobalamin malabsorption or autosomal recessive megaloblastic anaemia MGA1) is an autosomal recessive disease and is the most common cause of megaloblastic anaemia due to cobalamin deficiency in infancy in Western countries. More than 200 cases have been reported, with familial clusters in Finland, Norway, the Middle East and North Africa. The patients usually present with megaloblastic anaemia between the ages of 1 and 5 years, and secrete normal amounts of IF and gastric acid. In some cases, such as in Finland, impaired synthesis, processing or ligand binding of cubilin due to inherited mutations, for example 391C→T (named FM1) and a mutation at an intron causing a truncated protein (FM2), have been implicated. In others, for example in Norway, mutation of the gene for AMN has been reported. Other tests of intestinal absorption are normal. Over 90% of these patients show nonspecific proteinuria but renal function is otherwise normal and renal biopsy has not shown any consistent renal defect. A few of these patients have shown aminoaciduria and congenital renal abnormalities, such as duplication of the renal pelvis. The fish tapeworm (Diphyllobothrium latum) lives in the small intestine of humans and accumulates cobalamin from food, rendering this unavailable for absorption. People acquire the worm by eating raw or partly cooked fish. Infestation is common around the lakes of Scandinavia, Germany, Japan, North America and Russia. Megaloblastic anaemia or cobalamin neuropathy occurs only in those with a heavy infestation, with the worm high in the small intestine. Many carriers have no cobalamin deficiency. In severe chronic pancreatitis, lack of trypsin is thought to be the reason why dietary cobalamin attached to gastric non-IF (R) binder is unavailable for absorption. It has also been proposed that in pancreatitis, the concentration of calcium ions in the ileum falls below the level needed to maintain normal cobalamin absorption. Serum cobalamin levels tend to fall in patients with HIV infection and are subnormal in 10–35% of those with AIDS. Increased levels of apoTC, possibly derived from macrophages, are usual. Malabsorption of crystalline cobalamin not corrected by IF has been shown in some, but not all, patients with subnormal serum cobalamin levels. Cobalamin deficiency sufficiently severe to cause megaloblastic anaemia or neuropathy is rare. Malabsorption of cobalamin has been reported in Zollinger– Ellison syndrome. It is thought that there is a failure to release cobalamin from R binding protein due to inactivation of pancreatic trypsin by high acidity, as well as interference with IF binding of cobalamin. 73

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Both total body irradiation and local radiotherapy to the ileum (e.g. as a complication of radiotherapy for carcinoma of the cervix) may cause malabsorption of cobalamin. Graftversus-host disease commonly affects the small intestine: malabsorption of cobalamin due to abnormal gut flora, as well as damage to ileal mucosa, is frequent. Neomycin, colchicine, phenytoin, p-aminosalicylic acid, phenformin, metformin, slow-release potassium chloride and alcohol have all been reported to cause malabsoption of cobalamin; rarely, megaloblastic anaemia due to cobalamin deficiency has been reported with phenformin therapy. The use of histamine H2-blockers for treatment of peptic ulcer disease causes a decrease in cobalamin absorption, and continued use may lead to lowering of the serum cobalamin level. Both severe cobalamin and folate deficiencies affect the function of the small intestine; malabsorption of cobalamin due to ileal dysfunction may be found in patients with either deficiency. It may take several weeks of cobalamin therapy to correct the ileal absorptive defect in patients with PA. Deficiencies of protein, riboflavin and pyridoxine have also been reported to cause malabsorption of cobalamin. Abnormalities of cobalamin metabolism

Congenital transcobalamin deficiency or abnormality Infants with TC deficiency usually present with megaloblastic anaemia within a few weeks of birth. Serum cobalamin and folate levels are normal but the anaemia responds to massive (e.g. 1 mg three times weekly) injections of cobalamin, which cause free cobalamin to enter marrow cells by passive diffusion in the absence of functional TC. Some cases show neurological complications. In some cases, the protein is present in normal amounts but is unable to bind cobalamin or to attach to the cell surface and so is functionally inert. Genetic abnormalities so far found include mutations of an intra-exonic cryptic splice site, extensive or single nucleotide deletion, nonsense mutation and an RNA editing defect. These infants do not show methylmalonic aciduria, but malabsorption of cobalamin occurs in all cases and reduced immunoglobulins in some. Less severe cases present later in childhood. Failure to institute adequate cobalamin therapy or treatment with folic acid may lead to neurological damage. Congenital methylmalonic acidaemia and aciduria Infants with this abnormality are ill from birth, with vomiting, failure to thrive, severe metabolic acidosis, ketosis and mental retardation. Anaemia, if present, is normocytic and normoblastic. The condition may arise as a result of a functional defect in either the mitochondrial methylmalonyl-CoA mutase or its cofactor ado-cobalamin (see Figure 5.2). Mutations in methylmalonyl-CoA mutase are not responsive, or only poorly responsive, to treatment with cobalamin. Two disorders result in 74

cobalamin-responsive methylmalonic acidaemia. In cobalamin (Cbl)A disease, there is failure of reduction of cobalamin III (Cbl3+) or cobalamin II (Cbl2+) to cobalamin I (Cbl1+) in mitochondria; in CblB disease, there is a defect of an adenosyltransferase required for synthesis of ado-cobalamin (see Figure 5.2). A proportion of infants with CblA and CblB disease respond to cobalamin in large doses, whereas others are unresponsive. In those who do not respond to cobalamin, the enzyme methylmalonyl-CoA mutase is lacking (mut0) or defective (mut−). Some children have combined methylmalonic aciduria and homocystinuria due to defective formation of both cobalamin coenzymes. The defects are in the transfer of cobalamin from the endocytic compartment of lysosomes to the cytoplasm (CblF disease) or in the reduction of cobalamin 3+ to cobalamin 2+ after transfer to the cytoplasm (CblC and CblD diseases). Over 100 cases of CblC disease have been described. It usually presents in the first year of life with feeding difficulties, developmental delay, microcephaly, seizures, hypotonia and megaloblastic anaemia. Some patients present with homocystinuria and megaloblastic anaemia, often with neurological defects but without methylmalonic aciduria. There is a selective deficiency of methylcobalamin. These conditions have been termed CblE and CblG disease (lack of association of methylcobalamin with methionine synthase).

Acquired abnormality of cobalamin metabolism: nitrous oxide inhalation N2O irreversibly oxidizes methylcobalamin from its active, fully reduced Cbl1+ state to an inactive Cbl2+ precursor. This has been shown to inactivate methylcobalamin and methionine synthase. This occurs in both humans and experimental animals and was of importance in the megaloblastic anaemia that occurred in patients undergoing prolonged N2O anaesthesia (e.g. in intensive care units). A neuropathy resembling cobalamin neuropathy has been described in dentists and anaesthetists who are repeatedly exposed to N2O and in monkeys exposed to the gas for many months. In patients with low cobalamin stores, megaloblastic anaemia or cobalamin neuropathy may be precipitated after shorter exposure to N2O. Recovery from N2O exposure requires regeneration of methionine synthase, as this protein is damaged by active oxygen derived from the N2O–cobalamin reaction. Methylmalonic aciduria does not occur at first as ado-cobalamin is not inactivated by N2O. Later, however, after generalized depletion of cobalamin, methylmalonate levels in serum, urine and cerebrospinal fluid rise.

Diagnosis of cobalamin deficiency The diagnosis of cobalamin or folate deficiency has traditionally depended on the recognition of the relevant abnormalities in the peripheral blood and/or bone marrow and subsequent

Megaloblastic anaemia

analysis of the blood levels of the vitamins. Other causes of macrocytosis and hypersegmented neutrophils are listed in Table 5.3. However, assays of serum methylmalonic acid and homocysteine (see below) have shown these to be raised in some subjects without haematological abnormalities, including a proportion with normal levels of serum cobalamin and folate in whom, nevertheless, the levels of the metabolites fall to normal with cobalamin and/or folate therapy. The significance of these biochemical changes remains controversial. They may imply functional cobalamin or folate deficiency, not reflected by subnormal levels of the vitamins or by disturbed haemopoiesis. If so, it would imply that the accepted normal serum and red cell levels of the vitamins reflect body stores which are sufficiently high to prevent haematological changes but which in some subjects may not be optimal for prevention of other complications of the deficiencies including vascular disease and NTDs in the fetus. Measurement of serum cobalamin Serum cobalamin is usually measured by one of a number of enzyme-linked immunosorbent assays. These are frequently automated. Normal serum cobalamin levels range from 160– 200 ng/L to about 1000 ng/L (ng × 0.738 = pmol, so 200 ng/L = 148 pmol/L). In patients with megaloblastic anaemia due to cobalamin deficiency, the level is usually less than 100 ng/L. In general, the more severe the deficiency, the lower the serum cobalamin level. In patients with spinal cord damage due to the deficiency, levels are very low even in the absence of anaemia. Values of between 100 and 200 ng/L are regarded as borderline. They may occur, for instance, in pregnancy, in patients with megaloblastic anaemia due to folate deficiency, and in patients with heterozygous, homozygous or compound heterozygous mutations of the TCN1 gene that codes for HC (TCI). The relative concentrations of HC and TC also influence the total serum cobalamin level. Raised serum cobalamin levels (if not due to recent therapy) are usually due to a rise in HC (Table 5.4), or to liver or renal disease with increased saturation of HC and TC. Serum holotranscobalamin (holoTCII, holoTC) Since TC is the plasma cobalamin transport protein that is responsible for cellular uptake and delivery of cobalamin, the notion was put forward that measurement of circulating cobalamin that was bound to TC (holoTC) would provide a more meaningful measure of cobalamin status than total serum cobalamin. However, measurement of holoTC is not available or used diagnostically except in research studies. Serum methylmalonate and homocysteine levels In patients with cobalamin deficiency sufficient to cause anaemia or neuropathy, the serum methylmalonate (MMA) and homocysteine levels are raised. Sensitive methods for measuring MMA and homocysteine in serum have been introduced and recommended for the early diagnosis of cobalamin defi-

ciency, even in the absence of haematological abnormalities or subnormal levels of serum cobalamin or folate. However, serum MMA fluctuates in patients with renal failure. Mildly elevated serum MMA and/or homocysteine levels occur in up to 30% of apparently healthy volunteers, with serum cobalamin levels up to 350 ng/L and normal serum folate levels; 15% of elderly subjects, even with cobalamin levels above 350 ng/L, have this pattern of raised metabolite levels. These findings bring into question the exact cut-off points for normal MMA and homocysteine levels. It is also unclear at present whether these mildly raised metabolite levels have clinical consequences and how many of the subjects will progress to clinically overt cobalamin deficiency. When cobalamin supplies to the cell are suboptimal, there may be preferential use of methylcobalamin for methionine synthesis compared with ado-cobalamin for MMA metabolism. Urinary MMA excretion may also be used to screen for cobalamin deficiency but this is also increased in aminoaciduria (e.g. Fanconi syndrome). Homocysteine exists in plasma as single molecules, as two molecules linked together (homocystine) and as mixed homocysteine–cysteine disulphides. Serum homocysteine levels are raised in both early cobalamin and folate deficiency, but they may be raised in other conditions, for example chronic renal disease, alcoholism, smoking, pyridoxine deficiency, hypothyroidism, therapy with steroids, ciclosporin and other drugs. Levels are also higher in serum than in plasma, in men than in premenopausal women, in women taking hormone replacement therapy or oral contraceptive users and in elderly subjects and patients with several inborn errors of metabolism affecting enzymes in trans-sulphuration pathways of homocysteine metabolism. Thus, homocysteine levels are not widely used for diagnosis of cobalamin or folate deficiency. However, homocysteine levels are used in thrombophilia screening and in assessing for cardiovascular risk factors (see Chapter 46).

Tests for the cause of cobalamin deficiency Studies of cobalamin absorption were used but because of the unavailability of radioactive cobalamin have become obsolete. The urinary excretion (Schilling) test is therefore only briefly described here. Serum tests for gastrin and antibodies to parietal cells and intrinsic factor aid in the diagnosis of PA. Upper GI endoscopy including gastric biopsy helps to confirm the diagnosis and exclude gastric neoplasms. Cobalamin absorption The urinary secretion (Schilling) test was carried out with an oral trace dose of crystalline radioactive cyanocobalamin with or without oral IF and a ‘flushing’ intramuscular dose of hydroxocobalamin or cyanocobalamin so that any labelled cobalamin absorbed would appear in a 24-hour urine. Radioactive cyanocobalamin is no longer available and the test 75

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is obsolete, as are similar tests of food absorption using radioactively labelled cobalamin.

Folate Dietary folate Folic acid (pteroylglutamic acid) is a yellow, crystalline, watersoluble substance (molecular weight 441). It is the parent compound of a large family of folate compounds. Pteroylglutamic acid consists of three parts: pteridine, p-aminobenzoate and l-glutamic acid (Figure 5.8). It is only a minor component of normal food folates (probably less than 1%), which differ from it in three respects (Figure 5.8): (i) they are partly or completely reduced at positions 4, 5, 7 and 8 in the pteridine portion to dihydrofolate or THF derivatives; (ii) they usually contain a single carbon unit of varying degrees of reduction, such as a methyl group at N-5 or N-10; and (iii) 70–90% of natural folates contain a chain of three or more glutamate residues linked to each other by the unusual γ-peptide bond and are called pteroyl- or folate-polyglutamates. In human cells, four, five and six glutamate residues are usual. Most foods contain some folate. The highest concentrations are found in liver and yeast (>200 µg per 100 g), spinach, other greens and nuts (>100 µg per 100 g). The total folate content of an average Western diet is about 250 µg daily, but the amount varies widely according to the type of food eaten and the method of cooking. Folate is easily destroyed by heating, particularly in large volumes of water; over 90% may be lost.

Body stores and requirements Total body folate in the adult is about 10 mg, the liver containing the largest store. Daily adult requirements are about 100 µg. Up to 13 µg of folate is lost as such in the urine each day, but breakdown products of folate are also lost in urine. Losses of folate also occur in sweat and skin; faecal folate is largely derived from colonic bacteria. Stores are only sufficient for about 4 months in normal adults, so severe folate deficiency may develop rapidly.

H2N

N 2 3

N

1

N 8

4

5

N

N

7 6

CH2 9

H N

Absorption The principal site of folate absorption is the upper small intestine, and there is a steep fall-off in absorptive capacity in the lower jejunum and ileum. The absorption of all forms tested is rapid, a rise in blood level occurring within 15–20 min of ingestion. The small intestine has a tremendous capacity to absorb folate monoglutamates: about 90% of a single dose is absorbed regardless of whether this is small (100 µg) or large (15 mg). A proton-coupled high-affinity folate transporter with a low pH optimum, termed PCFT/HCP1, is located at the apical brush border of the duodenal, and to a lesser extent jejunal mucosa and in other cells, including the blood–brain barrier. It accounts for the bulk of folate absorption including of folic acid itself, and loss of function in hereditary folate malabsorption is not compensated by other folate transporters expressed on intestinal cells. The absorption of folate polyglutamates with higher numbers of glutamate residues is less. This may be due to the limited capacity of the small intestine to hydrolyse these compounds or to their limited transfer in the mucosal cell. On average, about 50% of food folates is absorbed. Polyglutamate forms are hydrolysed by pteroylpolyglutamate hydrolase (PPH, also known as folylpoly-γ-glutamate carboxypeptidase) to the monoglutamate derivatives, either in the lumen of the intestine or within the mucosa; they do not enter portal blood intact. Monoglutamate or polyglutamate forms of dietary folate, which are already partly or completely reduced, are converted to 5-methyl-THF within the small intestinal mucosa before entering the portal plasma. The monoglutamates are actively transported across the enterocyte by a carriermediated mechanism. Pteroylglutamic acid at doses greater than 400 µg is absorbed largely unchanged and converted to natural folates in the liver. Lower doses are converted to 5-methyl-THF during absorption through the intestine.

Enterohepatic circulation About 60–90 µg of folate enters the bile each day and is excreted into the small intestine. Loss of this folate, together with the folate of sloughed intestinal cells, accelerates the speed with which folate deficiency develops in malabsorption conditions.

O

H

COOH (α)

C

N

CH

10

CH2 CH2 COOH (γ)

76

Figure 5.8 The structure of folic acid (pteroylglutamic acid).

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Transport

Table 5.7 Causes of folate deficiency.

Folate is transported in plasma, about one-third loosely bound to albumin and two-thirds unbound. In all body fluids (plasma, cerebrospinal fluid, milk, bile) folate is largely, if not entirely, 5-methyl-THF in the monoglutamate form. A carrier-mediated active process is involved in the entry of folate into cells, the rate of uptake being linked to the rate of folate polyglutamate synthesis in the cell, which in replicating cells is related to the rate of DNA synthesis. Reduced folates are more rapidly taken up than oxidized folates. In most cells, folates are retained with tight binding to folate-binding proteins, three of which are enzymes involved in methyl group metabolism (sarcosine dehydrogenase, dimethylglycine dehydrogenase and glycine Nmethyltransferase), until the cell dies. Intact liver cells can release folate. Two types of folate-binding protein are involved in entry of methyl-THF into cells. The reduced folate carrier SLC19A1 is a facilitative transporter with a pH optimum of 7.4 and the characteristics of an anion exchanger. Two glycosylphosphatidylinositol (GPI)-linked folate receptors mediate cellular folate uptake by an endocytic mechanism, with internalization in a vesicle (caveola) which is then acidified, releasing folate into the vesicle lumen. Folate is then carried by the membrane folate transporter PCFT/HCPI into the cytoplasm; the caveola recycles to the cell surface, where its high-affinity receptors are reutilized. The GPI-linked transporters may be involved in transport of oxidized folates and folate breakdown products to the liver for excretion in bile.

Dietary Particularly in old age, infancy, poverty, alcoholism, chronic invalids and the psychiatrically disturbed; may be associated with scurvy or kwashiorkor

Biochemical functions Folates (as the intracellular polyglutamate derivatives) act as coenzymes in the transfer of single-carbon units from one compound to another (see Figure 5.3 and Table 5.2). Two of these reactions are involved in purine and one in pyrimidine synthesis necessary for DNA and RNA replication. Folate is coenzyme in another reaction, methionine synthesis, in which cobalamin is also involved and THF is regenerated. THF is the acceptor of single-carbon units newly entering the active pool via conversion of serine to glycine. Methionine, the other product of the methionine synthase reaction, is the precursor for SAM, the universal methyl donor involved in over 100 methyltransferase reactions. During thymidylate synthesis, 5,10-methylene-THF is converted to dihydrofolate (Figure 5.3). The enzyme dihydrofolate reductase converts this to THF. The drugs methotrexate, pyrimethamine and, mainly in bacteria, trimethoprim inhibit dihydrofolate reductase, and this prevents formation of the active folate coenzymes from dihydrofolate. A small fraction of the folate coenzyme is not recycled during thymidylate synthesis but is degraded at the C-9–N-10 bond.

Malabsorption Major causes of deficiency Tropical sprue, gluten-induced enteropathy in children and adults, and in association with dermatitis herpetiformis, specific malabsorption of folate, intestinal megaloblastosis caused by severe cobalamin or folate deficiency Minor causes of deficiency Extensive jejunal resection, Crohn’s disease, partial gastrectomy, congestive heart failure, Whipple disease, scleroderma, amyloid, diabetic enteropathy, systemic bacterial infection, lymphoma, sulfasalazine Excess utilization or loss Physiological Pregnancy and lactation, prematurity Pathological Haematological diseases: chronic haemolytic anaemias, sickle cell anaemia, thalassaemia major, myelofibrosis Malignant diseases: carcinoma, lymphoma, leukaemia, myeloma Inflammatory diseases: tuberculosis, Crohn’s disease, psoriasis, exfoliative dermatitis, malaria Metabolic disease: homocystinuria Excess urinary loss: congestive heart failure, active liver disease Haemodialysis, peritoneal dialysis Antifolate drugs Anticonvulsant drugs (phenytoin, primidone, barbiturates), sulfasalazine Nitrofurantoin, tetracycline, anti-tuberculosis (less well documented) Mixed causes Liver diseases, alcoholism, intensive care units Note: In severely folate-deficient patients with causes other than those listed under Dietary, poor dietary intake is often present.

Causes of folate deficiency (Table 5.7) Nutritional Dietary folate deficiency is common. Indeed, in most patients with folate deficiency a nutritional element is present. Certain individuals are particularly likely to have diets containing inadequate amounts of folate, including the old, edentulous, poor, alcoholic and psychiatrically disturbed, and patients after gastric operations. In relation to the size of the total body folate stores,

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which are in the order of 15–25 mg, the daily requirement of 100–200 mg is large. Consequently, with total cessation of intake or absorption, depletion of stores will occur in 3–6 months. In the USA and other countries where fortification of the diet with folic acid has been adopted to reduce the incidence of NTDs, the prevalence of folate deficiency has dropped dramatically and is now almost restricted to high-risk groups with increased folate needs. Nutritional folate deficiency occurs in kwashiorkor and scurvy, and in infants with repeated infections or who are fed solely on goats’ milk, which has a low folate content (6 µg/L) compared with human or cows’ milk (50 µg/L) as well as high concentrations of a high-affinity folate-binding protein.

patients have a poor diet. The usual presentation of the anaemia is similar to that of other megaloblastic anaemias, but occasionally, when there is an associated infection, acute arrest of haemopoiesis with pancytopenia may occur; this resembles aplastic anaemia, except that the marrow shows obvious megaloblastic changes. A number of consequences of folate deficiency in pregnancy have been described, including antenatal and postpartum haemorrhages, prematurity and congenital malabsorption in the fetus. These have not been fully established, but several studies have shown that prophylactic folic acid therapy reduces the incidence of NTDs (see p. 64).

Malabsorption Malabsorption of dietary folate occurs in tropical sprue and in gluten-induced enteropathy in children and in adults, when it is associated with dermatitis herpetiformis. In the rare recessive congenital syndrome of selective malabsorption of folate, there is an associated defect of folate transport into the cerebrospinal fluid, and these patients show megaloblastic anaemia from the age of a few months, responding to physiological doses of folic acid given parenterally but not orally or large oral doses of 5-formyl-THF. These patients also show mental retardation, convulsions and other central nervous system abnormalities. Loss-of-function mutations, usually homozygous in the gene coding for the low pH transporter PCFT/HCPI, underlie the disease. Minor degrees of malabsorption may also occur following jejunal resection or partial gastrectomy, in Crohn’s disease and in systemic infections but, in these conditions, if severe deficiency occurs, it is usually largely due to poor nutrition. Malabsorption of folate has been described in patients receiving sulfasalazine, cholestyramine and triamterene. It has also been associated with anticonvulsant drug therapy, alcohol abuse and folate deficiency, but these relationships are less well established. In the intestinal stagnant loop syndrome, the predominant effect of the small intestinal bacteria is to cause a rise in serum, red cell and urinary folate by synthesizing folate, which is then absorbed.

Prematurity The newborn infant, whether full term or premature, has higher serum and red cell folate concentrations than the adult, but the newborn infant’s demand for folate has been estimated to be up to 10 times that of adults on a weight basis and the neonatal folate level falls rapidly to the lowest values at about 6 weeks of age. The falls are steepest and liable to reach subnormal levels in premature babies, a number of whom develop megaloblastic anaemia responsive to folic acid at about 4–6 weeks of age. This occurs particularly in the smallest babies (90 µmol/L) suggests coexisting liver disease or biliary tract obstruction due to pigment gallstones or biliary sludge. Mild splenomegaly is common, rarely more than 2–3 cm below the costal margin at presentation. Marked splenomegaly suggests the possibility of a lymphoproliferative disease. Reticulocytosis is present, and the peripheral blood film is characterized by polychromasia, spherocytes (Figure 10.1), circulating nucleated red cells and, in some cases, red cell agglutination. Rarely, there may be reticu-

Reticuloendothelial function The severity of cellular immune red cell destruction depends largely on macrophage function. Reticuloendothelial function may be reduced in SLE by the clearance of immune complexes, a process known as reticuloendothelial blockade. In methyldopa-induced AIHA, the drug has been shown to reduce reticuloendothelial clearance of IgG-coated red cells, which might explain why many patients with a strongly positive DAT due to methyldopa have little or no haemolysis. Hypocomplementaemia Partial protection from complement-mediated lysis may occur in patients with chronic CHAD, in which continuous complement activation may lead to relative complement deficiency. Hypocomplementaemia is common in SLE and may also be caused by chronic activation of the complement pathway. In

Figure 10.1 Warm autoimmune haemolytic anaemia. Blood film showing spherocytosis, polychromasia and nucleated red blood cell (×40). 161

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locytopenia associated with a positive DAT. There is an increase in lactate dehydrogenase (LDH) due to lysis of red cells, but other liver function tests are normal unless there is associated liver or biliary tract disease. The DAT is positive, most commonly as a result of the presence of red cells coated with IgG alone (mainly IgG1 and IgG3 subclasses) or IgG and complement (the latter pattern commonly seen in SLE). Autoantibody in the serum may show specificity within the rhesus system (e.g. auto-anti-e), but in most cases is pan-reactive with all red blood cells. The autoantibody reacts at 37°C. In very rare cases the amount of antibody remaining on the red cell surface is insufficient to be detected by the conventional DAT. Idiopathic warm AIHA In approximately 30% of patients with a DAT-positive haemolytic anaemia, no associated disorder is found. Idiopathic AIHA may occur at any age. There is a peak incidence during infancy and early childhood and a second rise during the third decade, with the majority of cases occurring after the fifth decade. There is a preponderance of female patients in both idiopathic and secondary AIHA. A careful drug history should always be taken to exclude drug-induced AIHA, and chemical exposure at work or in the domestic environment must be assessed. In girls, AIHA may precede clinical or immunological evidence of SLE, so that negative serology for SLE does not exclude the disease at a later date. As mentioned above, the presentation may vary from the gradual onset of anaemia to an acute haemolytic process. Systemic symptoms are rare other than those of anaemia. Pallor, jaundice and mild splenomegaly (usually to between one and a half to five times its normal size) are present. Enlargement of the spleen to the umbilicus or below is not a feature of idiopathic AIHA and suggests a secondary cause. Evans syndrome This is defined as the combination of AIHA and immune thrombocytopenia (ITP) in the absence of any underlying disease. The occurrence of thrombocytopenia may coincide with the haemolysis or may arise as separate episodes. The platelet and red cell antibodies are distinct and do not cross-react. Rarely, episodes of immune neutropenia or pancytopenia have also been described in association with a positive DAT. The diagnosis is important because there appears to be a higher incidence of underlying illness such as immunodeficiency or autoimmune lymphoproliferative disease in children and SLE or T-cell lymphoma in adults. Typically, the disease runs a chronic course characterized by relapses and remissions. Management is as for warm AIHA or ITP (see Chapter 49) but patients with Evans syndrome tend to be more resistant to initial therapy with prednisolone. Options for second-line therapy include immunosuppressive drugs such as vincristine, danazol, ciclosporin and mycophenolate mofetil as single agents 162

or in combination regimens. Splenectomy is usually considered but responses are inferior to those seen in uncomplicated ITP. More recently, the anti-CD20 antibody rituximab has produced remission of both cytopenias in a high proportion of children and adults with steroid-refractory Evans syndrome. Stem cell transplantation offers the only hope for long-term cure for severe refractory patients but carries a significant risk of transplant-related morbidity and mortality. Warm AIHA in infancy and childhood AIHA of unknown cause occurs in infancy and in young children. In infancy, the onset is often acute and anaemia may be profound and difficult to control. The majority of cases in children are transient. It is interesting that in this group the sex difference is reversed, with more boys being affected. In childhood, the haemolytic episode is frequently precipitated by infection. IgG antibodies may be transferred from a mother with AIHA across the placenta to produce haemolysis in the newborn. Warm AIHA associated with other autoimmune diseases AIHA is often associated with SLE, especially in young women. Autoantibodies are usually IgG, and both IgG and C3d are found on the red cell surface. Occasionally, the DAT may be positive because of immune complexes adsorbed onto the red cell surface. The spleen is important for clearing such coated cells and splenectomy should be avoided if possible. Otherwise, treatment is as for idiopathic AIHA (see below). This condition is also described with other autoimmune or presumed autoimmune diseases, notably rheumatoid arthritis, Sjögren syndrome and ulcerative colitis; AIHA is also part of the spectrum of autoimmune diseases associated with agammaglobulinaemia. Warm AIHA in lymphoproliferative diseases The most common association is with B-cell chronic lymphocytic leukaemia (CLL), low-grade B-cell non-Hodgkin lymphoma or Hodgkin disease. The AIHA may precede the diagnosis of lymphoma, sometimes by months or years. On other occasions, the presentations may be simultaneous or the AIHA may be delayed. The antibodies are polyclonal and have no distinct pattern of antibody type or specificity. The formation of antibodies in this group is thought to be due to immune dysregulation rather than direct production by the malignant clone. Both fludarabine and alemtuzumab, used in the treatment of CLL, are associated with an increased incidence of often somewhat refractory autoimmune cytopenias, probably the result of their powerful effect on the immune system. Warm AIHA due to drugs Haemolytic anaemia caused by antibodies directed against self antigens has been described with a number of drugs. Mefenamic acid, levodopa and procainamide have been reported to provoke

Acquired haemolytic anaemias

this condition. The mechanism by which AIHA is produced by exposure to drugs is not known. Alteration to the red cell membrane or modulation of the immune response by the drug have both been suggested. Treatment of patients with CLL with fludarabine and other purine analogues may provoke a very severe and life-threatening acute AIHA and, less commonly, other autoimmune cytopenias. The mechanism may be related to a decrease in autoregulatory T cells caused by treatment with fludarabine. Warm AIHA and carcinoma AIHA has been recorded with a number of malignancies, but it is not clear that there is a true association. It may be associated with ovarian cysts, with the cyst fluid containing the agglutinin. It has been suggested that there is also an association with ovarian carcinoma. Warm AIHA and viral infections In children, but rarely in adults, AIHA may follow a viral infection. Haemolysis is usually brisk but self-limiting. It is possible that the virus alters the red cell membrane, which provokes ‘auto’ antibodies against the altered antigens, or that antiviral antibodies cross-react with membrane antigens. A third possibility is that immune complexes form between the virus and specific antibodies and are secondarily adsorbed onto the red cell surface, leading to immune destruction.

Treatment Corticosteroids First-line treatment of warm AIHA is with corticosteroids. The initial dose should be prednisolone 1–2 mg/kg body weight daily. The dose may be given once daily if tolerated and should be continued for 10–14 days, according to response. In patients who respond, the dose should then be reduced steadily, down to half the starting dose over the next 2 weeks and more gradually thereafter. In practice, the reduction in dose is tailored to individual patients and their response. It is important not to stop the steroids too quickly and allow relapse. About 70–80% of patients improve their haemoglobin initially but only 15– 20% maintain this response long term. For patients who do not respond or who require unacceptably high doses of prednisolone (>20 mg/day) to maintain a reasonable haemoglobin, other measures should be tried as they are at risk of serious steroid side-effects such as avascular necrosis, osteoporosis, infections and diabetes mellitus. A proton pump inhibitor or histamine H2 antagonist should be given at the same time as the steroids to reduce the risk of gastric erosions. Folic acid supplements should be given to patients with chronic haemolysis. Cytotoxic immunosuppressive drugs These agents are used for patients who are refractory to steroids or those who relapse following steroid withdrawal. On its own,

azathioprine is largely ineffective and is used for its steroidsparing action at a dose of 1.5–2.0 mg/kg daily. Cyclophosphamide 1.5–2.0 mg/kg daily or ciclosporin, starting at 5 mg/kg daily in two divided doses to achieve trough plasma levels of 100– 200 mg/L, can also be used. There has been recent interest in the use of mycophenolate mofetil as an alternative immunosuppressive agent for severe refractory AIHA. Response to these drugs is not usually seen for 4–6 weeks and they should be continued for at least 3 months before being deemed ineffective. The mechanism of action of these agents is relatively non-selective, resulting in significant systemic toxicity including bone marrow suppression, renal toxicity and the potential long-term risk of malignancy. It should be emphasized that there are no controlled trials to prove the worth of cytotoxic drugs in AIHA but there are small case reports of success with each agent. Intravenous immunoglobulin Intravenous immunoglobulin has been used in AIHA, particularly when IgG is the main component on the red cell surface. The dose used is the same as for ITP, 0.4 mg/kg daily for 5 days. AIHA responds less frequently to intravenous immunoglobulin than does ITP. Side-effects include headache, fever-chill reactions and a small but present risk of viral transmission. Splenectomy The spleen is the primary site of clearance of antibody-coated red cells and splenectomy is considered if there is no response to corticosteroids after 3 months’ trial. Patients with predominantly IgG on the red cell surface respond best, and those with complement often respond poorly. Splenectomy may be performed laparoscopically; of selected patients, about 30% achieve a complete remission and do not require steroids, 30% have a significantly reduced steroid requirement and the remainder show no or only transient response. There is no certain way to determine who will respond to splenectomy. There is an increased risk of overwhelming sepsis after splenectomy and all patients should receive pneumococcal, meningococcal and Haemophilus influenzae B vaccination at least 2 weeks before the procedure, and prophylactic penicillin indefinitely. The complications of splenectomy are dealt with in more detail in Chapter 8. Because of the emergence of monoclonal antibody therapy as an effective alternative treatment for AIHA, there is a trend towards delaying splenectomy as an option for more resistant/refractory cases. Monoclonal antibody therapy Recently, rituximab, a chimeric murine–human monoclonal antibody that binds to the CD20 antigen on B cells and immature plasma cells and largely used in the treatment of CD20positive B-cell non-Hodgkin lymphoma, has been shown to be effective in idiopathic and secondary warm AIHA as well as in cold agglutinin disease. The standard dose and treatment 163

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schedule used in follicular lymphoma (375 mg/m2 i.v. weekly for 4 weeks) is used for autoimmune cytopenias, although more recent studies in ITP suggest that lower doses may be as effective. Response has been shown in patients who are steroidrefractory and who have received multiple treatment modalities, and some patients who relapse after rituximab have been found to respond to retreatment. Overall response rates of 45–75% have been reported, with durable responses of up to 3 years in patients of all ages. Side-effects are minimal and mainly infusion related, such as fever, chills and hypotension. There are concerns about the long-term effects of the profound B-cell depletion that occurs immediately following rituximab therapy. However, naive and memory B cells can be detected by 4–6 months after therapy and the incidence of viral infections remains low in treated patients. In contrast, the results with alemtuzumab (Campath 1H), the anti-CD52 antibody, have been disappointing in AIHA and ITP. Blood transfusion Blood transfusion must be given if the clinical situation demands it, if the haemoglobin continues to fall or heart failure develops, despite the impossibility of achieving a satisfactory cross-match in the presence of a positive DAT. The least incompatible grouped blood should be used and transfused slowly. Some authors recommend the use of blood lacking antigens to which the autoantibodies react, but others point out that specificity is rarely absolute and that there is a risk of provoking an alloantibody response.

Prognosis The prognosis in warm AIHA depends on a number of variables, including age, associated diseases and severity of haemolysis. In all patients, AIHA should be considered a serious and potentially life-threatening disease. Estimates of mortality of idiopathic AIHA in adults vary from 10% at 5 years to 40% at 7 years. The higher figures are mainly in patients aged over 50 years. Most deaths occur in the first 2 years after diagnosis and are related to associated disease. In children, mortality is much

lower, probably about 5%, with the majority of patients recovering completely. AIHA may carry considerable morbidity, often from prolonged high-dose steroid therapy. Cold-type autoimmune haemolytic anaemias The clinical features of the cold haemagglutinin syndromes vary with the pathogenesis of the disorder. Serological tests are useful in identifying the cause and in determining treatment. The serological characteristics of the antibodies found in these syndromes are shown in Table 10.2.

Clinical features Idiopathic cold haemagglutinin disease Primary CHAD, a relatively uncommon disorder accounting for only 15% of AIHA, is mainly seen in older people and runs a chronic course. Although the condition is mostly benign, the clinical features may be very distressing and disabling. Purplish skin discoloration, maximal over the extremities (acrocyanosis), may be present in cold weather. Acrocyanosis is due to stasis in the peripheral circulation secondary to red cell agglutination. On warming the skin, the colour returns to normal or there is transient erythema. This sequence distinguishes acrocyanosis from Raynaud syndrome. Haemolysis resulting in anaemia is usually present and the patient may be mildly icteric. Occasionally, haemolysis dominates the clinical picture, depending on the ability of the antibody to activate complement on the red cell surface. The cold agglutinins are monoclonal IgM κ, with heavy chain variable regions encoded by the IGHV4–34 gene segment but serum electrophoresis may not reveal a monoclonal band because the concentration of the protein is too low. CHAD may be thought of as a premalignant B-cell disorder, which only presents clinically because of the specificity of the antibody for red cell surface antigens. This is highlighted by the fact that although, traditionally, CHAD has been defined by the absence of an underlying disorder, recent studies using sensitive flow cytometric and immunohistochemical assessments have demonstrated a monoclonal CD20positive κ-positive B-lymphocyte population in the bone

Table 10.2 Serological characteristics of cold-acting antibodies in the cold agglutinin syndromes. Disorder

Specificity Anti-I

Idiopathic (CHAD)

Monoclonal IgM κ

Secondary to Lymphoproliferative disease Mycoplasma pneumoniae Infectious mononucleosis

Monoclonal IgM κ/λ (IgG) Polyclonal Polyclonal

Paroxysmal cold haemoglobinuria

Donath–Landsteier (biphasic)

CHAD, cold haemagglutinin disease; mono. 164

Anti-i

Anti-P, -Pr Monoclonal (rare)

Monoclonal (rare) Polyclonal Polyclonal (anti-P)

Acquired haemolytic anaemias

Treatment General Management of cold haemagglutinin syndromes is difficult. All patients should avoid exposure to cold, and electrically heated gloves and socks are available for use in winter. Wintering in a warm climate is a pleasant alternative. Folic acid supplements should be given to patients with chronic haemolysis.

Figure 10.2 Cold haemagglutinin disease. Blood film showing gross haemagglutination (×20).

marrow of 90% of patients and lymphoplasmacytic lymphoma is a frequent finding. In the laboratory, spontaneous agglutination of red cells is frequently observed, both macroscopically and on the peripheral blood film if made at room temperature (Figure 10.2). Automated blood cell counters detect the agglutinates and record erroneously high mean corpuscular volume and low haemoglobin values, unless the sample is tested at 37°C. The DAT shows only C3d on the red cell surface; IgM cold agglutinins are not detected because they elute from the cell surface in vitro. They are usually anti-I, although anti-Pr, anti-P and other rarer specificities have been described. Cold agglutinin syndromes and lymphoproliferative disorders Occasionally, the cold agglutinin syndrome accompanies or precedes a B-cell lymphoma or CLL. In these cases, the antibody is monoclonal and is a product of the malignant clone. The serological specificity is either anti-I or anti-i. Haemolysis is often more troublesome than symptoms of agglutination. The prognosis is usually that of the underlying lymphoproliferative disease. Cold agglutinin syndromes and infections Haemolysis due to cold agglutinins may follow infections, almost always due to M. pneumoniae, or infectious mononucleosis. Rare cases following Listeria or Toxoplasma infections have been reported. The antibodies are mostly polyclonal IgM in type but occasional IgG cold antibodies are found. The antibodies develop in response to the infecting organism and cross-react with the red cell antigens. Haemolysis appears 2–3 weeks after the infection and is usually mild and self-limiting. Occasionally, very severe and even fatal acute intravascular haemolysis develops after M. pneumoniae infection. Blood transfusion through a blood warmer may be urgently required.

Alkylating agents Chlorambucil may be effective in reducing antibody production when there is an underlying B-cell neoplasm such as in CLL. Intermittent regimens such as 10 mg/day for 14 days every 4 weeks or continuous treatment of 2–4 mg/day are both effective. Long-term treatment carries the risk of bone marrow suppression and the development of myelodysplasia and acute myeloid leukaemia. Alkylating agents including chlorambucil, purine analogues such as cladribine and interferon alfa are usually ineffective in idiopathic CHAD. Corticosteroids Corticosteroids are rarely of use. They should be used only in exceptional circumstances when the antibodies are present in low titres and have a high thermal range. Their use should be avoided in other cases. Splenectomy Removal of the spleen is rarely of any use. The cells are coated with C3b and destruction occurs mainly in the liver. Blood transfusion Blood transfusions should be given with due regard to the difficulty in cross-matching in the presence of cold haemagglutinins. Blood should be given through an in-line blood warmer. The patient should be nursed in a warm environment, preferably at 37°C. Special precautions are needed for surgical procedures to keep the patient warm. Plasma exchange The titre of cold agglutinin may be lowered temporarily by plasma exchange. The procedure may be useful in the control of severe symptoms but has no effect on the underlying disease. Monoclonal antibody therapy The anti-CD20 monoclonal antibody rituximab has been shown to be an effective therapeutic option for both idiopathic CHAD and cold agglutinin syndromes with associated B-lymphoproliferative disorders. Response rates of up to 50% have been reported in Phase II studies but with relatively short response duration of only 11 months. Paroxysmal cold haemoglobinuria This rare syndrome usually occurs in children following acute viral infections. The original cases were described by Donath, 165

Postgraduate Haematology

Landsteiner and Ehrlich in congenital and tertiary syphilis but such cases are no longer encountered. A history of cold exposure is not always present and presentation is with sudden intravascular haemolysis resulting in pallor, dark urine (haemoglobinuria), abdominal pain and sometimes collapse. The cold antibody is a biphasic polyclonal IgG antibody (Donath– Landsteiner antibody) that reacts with red cells below 20°C in the peripheral circulation, causing lysis by complement activation as the red cells are warmed to 37°C in the central vessels. The antibody has specificity for the P antigen. Treatment depends on keeping the patient warm, preferably at an ambient temperature of 37°C. Haemolysis is usually selflimiting but blood transfusion is often required. The rare pp cells are not usually available and transfusion of ABO- and rhesus-compatible P-positive blood should be given through a blood warmer.

ognized, so that large doses of the drug are continued in the presence of increasing antibody levels, complement fixation and acute intravascular haemolysis may occur. The DAT becomes positive after some weeks of treatment and is due to IgG only on the red cell surface. When the drug is stopped, the DAT rapidly becomes negative and haemolysis stops. Antibody in the patient’s serum or eluate from the red cells will react with normal red cells only in the presence of the drug. The clinical and serological features are shown in Table 10.3.

Drug-induced immune haemolytic anaemia Antibody-induced haemolytic anaemia caused by drugs is rare but in some cases may be acute, severe and even lifethreatening. Four main mechanisms have been proposed for antibody-dependent drug-induced haemolytic anaemias: drug adsorption, immune complex and membrane modification mechanisms that lead to antibodies reacting with novel epitopes, and true autoantibody-induced haemolytic anaemia (see above). The same drug at different doses or repeated usage may activate different mechanisms and there are recent suggestions that membrane modification may underlie most of the mechanisms. The diagnosis of drug-induced immune haemolytic anaemia should be made in three stages: (i) diagnosis of a DATpositive haemolytic anaemia; (ii) careful drug history; and (iii) serological demonstration of drug-specific antibody, which interacts with red cells.

Immune complex mechanism: complement-activated acute intravascular haemolysis Several drugs have been reported to cause immune haemolytic anaemia by this mechanism. Those most frequently reported are rifampicin, phenacetin, quinine, quinidine, hydrochlorothiazide and chlorpropamide and, more recently, intravenous cephalosporins and diclofenac (see below). Hapten–carrier complexes are formed between these drugs and plasma proteins, leading to the production of drug-specific antibodies. Once drug antibodies are present, reintroduction of the drug causes immune complexes to form, which are adsorbed onto the red cell membrane and complement is activated. Classically, haemolysis occurs on the second or subsequent exposure to the drug and may develop within minutes or hours of drug ingestion. Severe intravascular haemolysis may occur with fever, rigors or nausea and, in extreme cases, acute renal failure. Several groups have reported fatal immune haemolysis with the third-generation cephalosporin ceftriaxone, and cefotaxime and ceftazidime have also been reported to cause immune haemolytic anaemia. Second-generation cephalosporins have also been implicated, although there are fewer reports with them than with third-generation antibiotics. Diclofenac can also cause an immune haemolytic anaemia with intravascular haemolysis, and this is thought to be mediated by both immune complex and drug adsorption mechanisms.

Pathogenesis Drug adsorption mechanism: IgG antibodies and extravascular haemolysis Penicillin is the prototype drug, although cephalosporins and other penicillin derivatives have also been implicated. Drugs in this group readily form hapten–carrier complexes with plasma proteins, which enhance drug-specific antibody production. It has been estimated that 90% of individuals receiving penicillin produce clinically insignificant IgM anti-penicillin antibodies. When high-dose intravenous penicillin is administered, the drug is adsorbed onto the red cell surface, where it becomes non-specifically attached to red cell surface proteins. A minority of patients on high-dose intravenous penicillin therapy (>l million units daily) develop high-titre IgG anti-penicillin antibodies that attach to the drug bound to the red cell surface and cause predominantly extravascular haemolysis. The clinical picture is usually of mild to moderate haemolysis but if unrec-

Membrane modification mechanism Cephalosporin, in addition to the drug adsorption mechanism, can cause a positive DAT by modifying red cell membrane components. Cisplatin and carboplatin have also been reported to cause immune haemolytic anaemia by this mechanism. As a result, a variety of plasma proteins, including immunoglobulin and complement, may attach via a non-immune mechanism to the red cell membrane. This may result in the finding of a positive DAT but rarely causes immune haemolytic anaemia. Ribavirin in combination with interferon alfa or peginterferon alfa is the treatment for chronic hepatitis C infection. The main dose-limiting toxicity is haemolytic anaemia. Ribavirin enters cells and is phosphorylated to ribavirin triphosphate. In nucleated cells the triphosphate is hydrolysed back to ribavirin, which leaves the cells as the serum level declines. Red cells lack the hydrolysing enzymes: the triphosphate accumulates and alters the deformability of the membrane and extravascular

Alloimmune haemolytic anaemia

166

Acquired haemolytic anaemias

Table 10.3 Drug-induced immune haemolytic anaemias: clinical and serological features.

Examples

Drug adsorption mechanism

Immune complex mechanism

Autoimmune mechanism

Membrane modification mechanism

Penicillin

IV third-generation cephalosporins Quinidine Diclofenac

Methyldopa

Cephalosporins

Procainamide Mefenamic acid Fludarabine* Cladribine* Therapeutic About 6 weeks

Cisplatin Carboplatin

Cephalosporins

Dose/duration

Large therapeutic doses/prolonged

Haemolysis DAT Serum reaction

Extravascular Subacute IgG ± C′3 To drug-treated cells

Eluate reaction

To drug-treated cells

Very low dose on second or subsequent exposure/short Intravascular Acute C′3 only Only in presence of drug or metabolite Non-reactive

Therapeutic

Extravascular Mild/subacute IgG only To normal cells

Rare IgG To drug-treated cells

To normal cells

To drug-treated cells

*May change T-cell regulation.

haemolysis occurs. With ribavirin monotherapy the haemolysis may be compensated by increased marrow activity but the marrow suppression caused by interferon alfa inhibits the response and severe anaemia may result. Occasionally, autoantibodies have been detected, presumably generated to antigens in the altered membrane. Haemolytic anaemia may require dose reduction (20 000 µg/L) and increases in interferon (IFN)-γ and tumour necrosis factor (TNF)-α with variable changes in other cytokines. The syndrome is associated with abnormal T-cell activation, which triggers the macrophage 169

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response and which may be the consequence of a T-cell lymphoma or may be unmasked by a variety of infections. The two main subdivisions of the syndrome are infection-associated HPS and malignant HPS. Clinically, the distinction may be very difficult because in the lymphomas the proliferation may be trivial, the syndrome being derived from the release of cytokines, and because in malignant HPS superadded infection is common. Likewise, in infection-associated HPS it may be impossible to identify the underlying infection. In children, infectionassociated HPS seems to be more common, whereas in adults the majority of cases are associated with lymphoma.

Fragmentation haemolysis: mechanical haemolytic anaemias The relationship between the vascular endothelium, the cellular elements of the blood and the mechanisms of haemostasis and fibrinolysis is clearly intricate and complex. The integrity of the red blood cell may be destroyed by contact with abnormal endothelial surfaces, although not all abnormalities of vessels cause haemolysis. It may be that some adherence between the red cell and the abnormal vessel wall is necessary for fragmentation of the red cell to occur, and that this usually happens in the context of abnormal flow as well as an altered endothelium. The situations in which fragmentation haemolysis may occur are the presence of prosthetic material and altered flow following cardiovascular surgery, the trapping or adherence of red cells in arteriovenous malformations, and the destruction of red cells in pathologically altered small blood vessels (microangiopathic haemolytic anaemia). The characteristic features of fragmentation haemolysis are the appearance of the blood film (Figure 10.3) and the presence of intravascular haemolysis. Depending on the underlying vascular pathology, there may be a reduction in the platelet count and evidence of DIC. The rate of red cell destruction also varies

according to the pathogenesis, so the signs of intravascular haemolysis vary from absence of haptoglobin, elevated LDH and minimal haemosiderinuria to acute intravascular destruction with haemoglobinaemia and haemoglobinuria. The major causes of fragmentation haemolysis are shown in Table 10.5. Cardiac haemolytic anaemia This syndrome was so called because it mainly occurred after cardiac surgery in which prosthetic valves, patches or grafts were inserted. Haemolysis usually becomes significant only when there is turbulent flow that brings the circulating red cells into intimate contact with the prosthetic material. There are certain situations in which the haemolysis may be of considerable clinical importance.

Figure 10.3 Microangiopathic haemolytic anaemia. Blood film from a patient with carcinoma and bone marrow metastases. Note fragmentation of red cells, leucoerythroblastic changes with nucleated red cell and metamyelocyte and low platelets suggesting possible disseminated intravascular coagulation (×40).

Vascular origin

Pathogenesis

Platelets

Cardiac haemolysis

Prosthetic heart valves Patches, grafts Paraprosthetic or perivalvular leaks Kasabach–Merritt syndrome Malignant haemangioendotheliomas TTP/HUS Malignant disease Vasculitis Pre-eclampsia, HELLP Renal vascular disorders Disseminated intravascular coagulation

Normal

Arteriovenous malformations Microangiopathic

Very low Low Normal/low Normal/low Low Normal/low Low

HELLP, haemolysis with elevated liver function tests and low platelets; HUS, haemolytic– uraemic syndrome; TTP, thrombotic thrombocytopenic purpura. 170

Table 10.5 Classification of mechanical anaemias caused by fragmentation haemolysis.

Acquired haemolytic anaemias

Periprosthetic or perivalvular leaks If after insertion of a prosthesis or repair of a heart valve a leak occurs around the valve or through a suture track, there may be severe intravascular haemolysis without evidence of haemodynamic distress. A difficulty may be that fragmentation of red cells is not always prominent, although spherocytes may be present. However, once autoimmune haemolysis is ruled out, the diagnosis can scarcely be anything other than cardiac haemolysis in a patient who has had cardiac surgery. The haemolysis can be cured only by further surgery. Ambulatory haemolysis A patient who has undergone valve replacement may show only slight evidence of haemolysis while in hospital but experience significant anaemia after discharge. This is thought to occur because the higher cardiac output associated with the greater exercise as an outpatient produces more turbulence and hence greater opportunity for red cell fragmentation. A similar mechanism is thought to operate if the patient becomes iron deficient as a result of chronic intravascular haemolysis. Iron replacement and advice about the level of exercise may prevent or delay the need for further surgery. Cardiopulmonary post-perfusion syndrome Acute intravascular haemolysis may occur in patients who have undergone cardiopulmonary bypass surgery. The haemolysis may be accompanied by neutropenia and pulmonary distress. The syndrome does not strictly belong in this section as the haemolysis seems to be caused by complement activation and binding of the membrane attack complex to the red cell surface. The blood film shows ghost red cells rather than fragmentation. The condition is self-limiting and the patient requires only supportive care. Arteriovenous malformation Fragmentation of red cells may be seen in Kasabach–Merritt syndrome, in which platelets are trapped in the vascular network of giant arteriovenous malformations, sometimes with evidence of a consumption coagulopathy. The bleeding disorder is of greater significance than the haemolysis in these patients. A similar picture, usually with clear evidence of a consumptive coagulopathy with evidence of DIC, may be seen in malignant haemangio-endothelioma, in which the tumour tends to invade and grow along veins. Microangiopathic haemolytic anaemias Microangiopathic haemolytic anaemia (MAHA) is a condition in which intravascular haemolysis with fragmentation of red cells is caused by their destruction in an abnormal microcirculation. Proof of microangiopathy may be lacking in those not subjected to a post-mortem, and MAHA should be considered a clinical syndrome. The three main pathological lesions that give rise to MAHA are deposition of fibrin strands, often associ-

Table 10.6 Causes of microangiopathic haemolytic anaemia. Disease

Microangiopathy

Haemolytic–uraemic syndrome Thrombotic thrombocytopenic purpura Renal cortical necrosis Acute glomerular nephritis Malignant hypertension Pre-eclampsia HELLP Polyarteritis nodosa Wegener granulomatosis Systemic lupus erythematosus Homograft rejection

Endothelial cell swelling, microthrombi in renal vessels Platelet plugs, microaneurysms, small-vessel thrombi

Mitomycin C Ciclosporin Carcinomatosis

Primary pulmonary hypertension Cavernous haemangioma (Kasabach–Merritt)

Necrotizing arteritis

Fibrinoid necrosis Vasculitis

Microthrombi in transplanted organ Uncertain Renal vessel anomalies Abnormal tumour vessels, intravascular coagulation (disseminated or localized) Abnormal vasculature Local vascular changes, thrombosis

HELLP, haemolysis with elevated liver function tests and low platelets.

ated with DIC; platelet adherence and aggregation; and vasculitis. The vessel abnormalities may be generalized or confined to particular sites or organs. In most cases, the haemolysis is of less consequence than the underlying cause of the microangiopathy, but the fragmentation of red cells may be important in pointing to the diagnosis. Some of the disorders producing MAHA are given in Table 10.6. Only well-defined clinical syndromes are described in detail here.

Microangiopathic haemolytic anaemia and malignant disease Fragmentation of red cells with chronic intravascular haemolysis may occur in malignant disease. Clinically significant anaemia may occur, especially when there is invasion of the tumour into a large blood vessel (as in haemangiopericytoma), but more commonly the haemolysis is trivial or well compensated. The fragmentation may simply be noted on the blood film. A blood film that shows evidence of MAHA together with leucoerythroblastic changes is virtually diagnostic of malignant disease with secondary deposits in the bone marrow (Figure 171

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10.3). Mucin-secreting tumours are most likely to produce MAHA. In acute leukaemia, particularly but not exclusively promyelocytic (M3), there may be intense intravascular coagulation that may be accompanied by MAHA. The coagulation changes dominate the clinical picture.

Microangiopathic haemolytic anaemia and infection Infections, particularly septicaemia, may provoke intravascular coagulation and MAHA. Generally, the coagulation changes and septic shock overshadow the mild fragmentation but, occasionally, infections produce a chronic state of partially compensated intravascular haemolysis and marked red cell fragmentation. Haemolytic–uraemic syndrome may be precipitated by infection, particularly Escherichia coli 0157. Haemolytic– uraemic syndrome is discussed in Chapter 44. Thrombotic thrombocytopenic purpura Thrombotic thrombocytopenic purpura is an acute syndrome characterized by fever, neurological signs, haemolytic anaemia with fragmented red cells and thrombocytopenia. The diagnosis is made on the basis of the clinical presentation and evidence for haemolytic anaemia with fragmented red cells and thrombocytopenia. Bilirubin is elevated, as is serum LDH, indicating intravascular haemolysis. LDH is a useful marker for measuring the activity of the microangiopathic process, as is the persistence of red cell fragments in the blood film. It may take up to 1 week for all fragments to be removed from the circulation after the haemolytic process stops. The destruction of red cells occurs at the site of intravascular occlusions; at post-mortem, platelet and fibrin plugs are found in capillaries (Figure 10.4). The condition, including the pathophysiology, is discussed in detail in Chapter 44. March haemoglobinuria Haemoglobinuria following running has been documented for about 100 years. Its origin is mechanical, with destruction of red cells occurring in the feet. It can be cured by wearing soft shoes or running on soft ground. The disorder may arise in joggers and is benign except that it may lead to extensive invasive investigations unless recognized. The blood film does not show any red cell fragmentation or consistent abnormality. Occasionally, haemoglobinuria after running is accompanied by nausea, abdominal cramps and aching legs, and enthusiastic athletes with this condition may exhibit mild splenomegaly and jaundice.

Chemical and physical agents Oxidative haemolysis Oxidative substances may cause haemolysis in people with normal red cell metabolism and normal HbA if the oxidative stimulus is large enough. The major causes of oxidative haemo172

(a)

(b)

Figure 10.4 Thrombotic thrombocytopenic purpura. Microthrombi in capillaries: (a) section from the myocardium; (b) thrombus in a bone marrow capillary (haemotoxylin and eosin, ×100). (Courtesy of Dr Margaret Burke.)

lysis in normal subjects are shown in Table 10.7. The clinical features of this condition are dependent on the main sites of oxidative attack, whether on the membrane of the red cell (intravascular haemolysis), the globin chains (Heinz body formation) or the haem group (methaemoglobin accumulation).

Chronic intravascular haemolysis with Heinz bodies Dapsone and sulfasalazine (Salazopyrin) will cause oxidative intravascular haemolysis in normal subjects if taken in sufficiently high dosage. Red cells show the ‘bite’ abnormality of the chemically damaged cell (Figure 10.5). Heinz bodies may be absent or scanty in patients with an intact spleen. Dapsone may be used in the treatment of G6PD-deficient subjects with leprosy and in the treatment of dermatitis herpetiformis, in which functional hyposplenism occurs; Heinz bodies appear in the latter case, acute intravascular haemolysis in the former. Haemosiderinuria may be detected in patients taking these

Acquired haemolytic anaemias

Table 10.7 Substances causing oxidative haemolysis and/or methaemoglobinaemia in normal people. Substance

Use

Remarks

Dapsone

Leprosy, dermatitis herpetiformis

Maloprim

Antimalarial

Sulfasalazine Phenazopyridine Menadiol Nitrites Nitrates Chlorate Arsine

Ulcerative colitis Analgesic in urinary tract infections Water-soluble vitamin K analogue Fertilizer; present in well water, some vegetable juices Amyl nitrate, butyryl nitrite; abused recreationally Weed killer Gas produced in smelting and other industrial processes

Chronic haemolysis; slow acetylators more susceptible Methaemoglobinaemia in NADH methaemoglobin reductase-depleted subjects Chronic intravascular haemolysis Methaemoglobinaemia Haemolysis/kernicterus in infants Methaemoglobinaemia in infants Methaemoglobinaemia/haemolysis Acute i.v. haemolysis; renal failure; > 30 g fatal Acute i.v. haemolysis; renal failure

Figure 10.5 Oxidative haemolysis caused by drug (phenacetin). Note red cells with contracted haemoglobin.

drugs, and there may be polychromasia and macrocytosis. Haemolysis is usually well compensated and there is no need to stop the treatment unless the anaemia is severe. A dose reduction may sometimes be needed. Methaemoglobinaemia is uncommon unless the patient is partially deficient in NADH methaemoglobin reductase. The gene for this abnormality may not be very uncommon, and it may account for some people becoming cyanosed after taking dapsone-containing antimalarial preparations.

Methaemoglobinaemia with or without haemolysis Nitrites in water or vegetable juices may cause methaemoglobinaemia in infants who have a physiological impairment of the reducing systems. Well-water that comes from land with an excess of nitrites and which is used to reconstitute artificial feeds has produced cyanosis in infants. Cases have also been described following the enthusiastic feeding of juice from carrots grown

on organically fertilized land and of spinach juice (spinach has a high concentration of nitrogen-fixing bacteria on its leaves). Nitrate drugs, for example amyl nitrate, also produce methaemoglobinaemia and have proved fatal when taken in sufficiently high dosage for ‘recreational’ purposes. Water-soluble analogues of vitamin K (menadiol sodium diphosphate) cause haemolysis with or without methaemoglobinaemia in infants and in utero if given to the mother during the third trimester. Fat-soluble vitamin K preparations must be used if required in these situations. Methaemoglobinaemia due to oxidative drugs rarely causes problems with oxygen delivery but severe cases (> 40% methaemoglobin) may be treated with intravenous methylene blue 1–2 mg/kg. Ascorbic acid by mouth may also be used. These measures are ineffective in G6PD-deficient patients and when very strong oxidant substances are implicated. In these circumstances, methylene blue should be avoided because it acts as an oxidant and makes the condition worse.

Acute intravascular haemolysis, methaemoglobinaemia and renal failure These conditions occur following exposure to strong oxidizing substances found mainly in industrial or horticultural settings, for example sodium chlorate is a popular weedkiller and arsine is a gas produced in various industrial situations, including dross removal in smelting procedures and drain clearances where arsenic is a contaminant. Acute intravascular haemolysis and haemoglobinuria develop 1–24 hours after exposure depending on the dose. The serum becomes brown, often very dark, so that blood cells cannot be seen in anticoagulated preparations, due to the presence of methaemalbumin, methaemoglobin and free haemoglobin. Vomiting, abdominal pain and oliguric renal failure usually develop over about 24 hours. The blood film shows microspherocytosis, ghost cells and bizarre forms. Plasma exchange and renal dialysis are the mainstays of treatment, methylene blue being ineffective. Poisoning with 173

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arsine is usually reversible with these measures. Chlorate poisoning is more difficult, 30 g being a generally fatal dose. It is mostly ingested deliberately in suicide attempts. Thermal injury Normal red cells when heated in vitro show no changes when heated to 46°C for 1 hour but show temperature- and durationdependent changes above 47–50°C. Some hereditary membrane defects produce red cells that have increased thermal fragility (see Chapter 8). Haemolysis following burns Severe burns may be accompanied by intravascular haemolysis with haemoglobinuria. The intravascular haemolysis is related to the extent and severity of the burns. The gross haemoglobinuria occurs over the first 24 hours after the burns and ceases thereafter. The blood film shows spherocytosis and schistocytes, the morphological abnormalities reflecting the thermal damage and the amount of lysis. Prolonged anaemia after burning is related to inflammation, occult blood loss and infection rather than haemolysis. Lead poisoning Haemolysis is an important contributor to the anaemia associated with excessive lead exposure. Lead is a potent inhibitor of pyrimidine 5′-nucleotidase 1 (P5N1). This is the prime mechanism of lead-induced haemolysis, producing the same gross basophilic stippling of the red cells as seen in the inherited deficiency of the enzyme (see Chapter 9). P5N1 activity is a good surrogate marker for lead exposure.

Acquired disorders of the red cell membrane The most common acquired disorder is paroxysmal nocturnal haemoglobinuria, caused by somatic mutation of the phosphatidylinositol glycan A (PIGA) gene on the X chromosome, which leads to failure to produce the glycosylphosphatidylinositol (GPI) anchor needed to transport and attach many proteins to the red cell membrane. Intravascular haemolysis occurs through the unchecked action of activated complement. Paroxysmal nocturnal haemoglobinuria is described in detail in Chapter 11. The mature red cell does not have the capacity to repair its membrane. The lipids of the membrane are in equilibrium with the lipids of the plasma and changes in the ratio of free cholesterol to phospholipids in plasma may affect red cell shape and, in some instances, lead to haemolysis. This is most commonly seen in liver disease, but other inherited lipid disorders may affect the red cell secondarily. Liver disease Some degree of shortening of red cell survival occurs in most cases of acute hepatitis, cirrhosis and Gilbert disease, but 174

anaemia is not present and there is only a slight rise in reticulocytes, which may not be detectable. Biliary obstruction is associated with the appearance of target cells and fulminant hepatitis with acanthocytosis, both consequent on changes in the plasma lipid composition. Zieve syndrome is an uncommon disorder seen mainly in alcoholics. It comprises intravascular haemolysis and acute abdominal pain. These patients usually have cirrhosis and jaundice. The cause is unknown but is probably related to lipid changes in the blood. Spherocytes are seen in the peripheral blood. Wilson disease may present as acute intravascular haemolysis. This is probably not a membrane disorder but is consequent on the high levels of copper ions in the blood. The haemolysis may antedate the development of hepatic or neurological features, but Kayser–Fleischer rings are usually present. The blood film may show spherocytosis. The diagnosis is made once the condition is suspected. Apart from caeruloplasmin deficiency, patients have a specific aminoaciduria. Hereditary acanthocytosis (abetalipoproteinaemia) This rare inherited deficiency of low-density lipoproteins is characterized by retinitis pigmentosa, steatorrhoea, ataxia and mental retardation. The haemolysis that occurs is of minor importance to such patients, but the blood film may indicate the diagnosis, with the red cells showing marked acanthocytosis. Vitamin E deficiency Deficiency of vitamin E may occur in infants who are fed a diet rich in polyunsaturated fatty acids. There is haemolysis with contracted cells and a thrombocytosis. Oedema may be present. Vitamin E is an antioxidant, and oxidative damage to the red cell membrane is thought to be the cause of the haemolysis.

Selected bibliography General Dacie JV (1992) The Autoimmune Haemolytic Anaemias. The Haemolytic Anaemias, 3rd edn, Vol. 3. Churchill Livingstone, Edinburgh. Dacie JV (1995) Secondary or Symptomatic Haemolytic Anaemias. The Haemolytic Anaemias, 3rd edn, Vol. 4. Churchill Livingstone, Edinburgh. Petz LD, Garraty G (2004) Acquired Immune Haemolytic Anemias, 2nd edn. Churchill Livingstone, Philadelphia. Autoimmune haemolytic anaemias

Warm type D’Arena G, Califano C, Annunziata M et al. (2007) Rituximab for warm-type idiopathic haemolytic anaemia: a retrospective study of 11 adult patients. European Journal of Haematology 79: 53–8.

Acquired haemolytic anaemias

Garvey B (2008) Rituximab in the treatment of autoimmune haematological disorders. British Journal of Haematology 141: 149–69. Jeffries LC (1994) Transfusion therapy in autoimmune haemolytic anaemia. Hematology/Oncology Clinics of North America 8: 1087–104. Norton A, Roberts I (2006) Management of Evans syndrome. British Journal of Haematology 132: 125–37. Passweg JR, Rabusin M, Musso M et al. (2004) Haematopoietic stem cell transplantation for refractory autoimmune cytopenias. British Journal of Haematology 125: 749–55. Provan D, Butler T, Evangelita ML et al. (2007) Activity and safety profile of low-dose rituximab for the treatment of autoimmune cytopenias in adults. Haematologica 92: 1695–8.

Cold type Berentsen S, Klaus B, Geir ET (2007) Primary chronic cold agglutinin disease: an update on pathogenesis, clinical features and therapy. Haematology 12: 361–70.

Daly JJ, Haeusler MN, Hogan CJ, Wood EM (2006) Massive intravascular haemolysis with T-activation and disseminated intravascular coagulation due to clostridial sepsis. British Journal of Haematology 134: 553. Ekvall H (2003) Malaria and anaemia. Current Opinion in Hematology 10: 108–14. McArthur HL, Dalal BI, Kollmannsberger C (2006) Intravascular hemolysis as a complication of Clostridium perfringens sepsis. Journal of Clinical Oncology 24: 2387–8. Vannier E, Gewurz BE, Krause PJ (2008) Human babesiosis. Infectious Disease Clinics of North America 22: 469–88.

Mechanical haemolytic anaemias Davidson RJL (1969) March or exertional haemoglobinuria. Seminars in Hematology 6: 150. Hall GW (2001) Kasabach–Merritt syndrome: pathogenesis and management. British Journal of Haematology 112: 851–62.

Cardiac haemolysis Drug induced Gaines AR (2005) Disseminated intravascular coagulation associated with acute hemoglobinemia or hemoglobinuria following Rh(0)(D) immune globulin intravenous administration for immune thrombocytopenic purpura. Blood 106: 1532–7. Gaines AR, Lee-Stroka H, Byrne K et al. (2009) Investigation of whether the acute hemolysis associated with Rho(D) immune globulin intravenous (human) administration for treatment of immune thrombocytopenic purpura is consistent with the acute hemolytic transfusion reaction model. Transfusion 49: 1050–8. Marani TM, Trich MB, Armstrong PM (1996) Carboplatin induced immune haemolytic anaemia. Transfusion 36: 1016–18. Myint H, Copplestone JA, Orchard J et al. (1995) Fludarabinerelated autoimmune haemolytic anaemia in patients with chronic lymphocytic leukaemia. British Journal of Haematology 91: 341–4. Salama A (2009) Drug-induced immune haemolytic anaemia. Expert Opinion on Drug Safety 8: 73–9. Salama A, Kroll H, Wittmann G et al. (1996) Diclofenac-induced immune haemolytic anaemia: simultaneous occurrence of red blood cell autoantibodies and drug dependent antibodies. British Journal of Haematology 95: 640–4. Shiffman ML (2004) Side effects of medical therapy for hepatitis C. Annals of Hepatology 3: 5–10. Shiffman ML (2009) What future for ribavirin? Liver International 29 (Suppl. 1): 68–73. Wright MS (1999) Drug induced haemolytic anemias: increasing complications to therapeutic interventions. Clinical Laboratory Science 12: 115–18.

Non-immune haemolytic anaemias

Maraj R, Jacobs LE, Ioli A, Kotler MN (1998) Evaluation of hemolysis in patients with prosthetic heart valves. Clinical Cardiology 21: 387–92. Shapira Y, Vaturi M, Sagie A (2009) Hemolysis associated with prosthetic heart valves. A review. Cardiology in Review 17: 121–4.

Thrombotic thrombocytopenic purpura Aqui NA, Stein SH, Konkle BA et al. (2003) Role of splenectomy in patients with refractory or relapsed thrombotic thrombocytopenic purpura. Journal of Clinical Apheresis 18: 51–4. Fontana S, Kremer Hovinga JA, Studt J-D et al. (2004) Plasma therapy in thrombotic thrombocytopenic purpura: review of the literature and the Bern experience in a subgroup of patients with severe acquired ADAMTS-13 deficiency. Seminars in Hematology 41: 48–59. Kremer Hovinga JA, Meyer S (2008) Current management of thrombotic thrombocytopenic purpura. Current Opinion in Hematology 15: 445–50. Levy GC, Nichols WC, Lian EC et al. (2001) Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature 413: 488–94. Pisoni R, Ruggementi P, Remuzzi G (2001) Drug induced thrombotic microangiopathy. Incidence, prevention and management Drug Safety 24: 491–501. Sadler JE (2009) Von Willebrand factor, ADAMTS13, and thrombotic thrombocytopenic purpura. Blood 112: 11–18. Yarranton H, Machin SJ (2003) An update on the pathogenesis and management of acquired thrombotic thrombocytopenic purpura. Current Opinion in Neurology 16: 367–73.

Infections

Chemical and physical agents

Bruneel F, Gachot B, Wolff M, Regnier B, Danis M, Vachon F (2001) Resurgence of blackwater fever in long-term European expatriates in Africa: report of 21 cases and review. Clinical Infectious Diseases 32: 1133–40.

Kim Y, Yoo CI, Lee CR et al. (2002) Evaluation of activity of erythrocyte pyrimidine 5′-nucleotidase (P5N) in lead exposed workers: with focus on the effect on hemoglobin. Industrial Health 40: 23–7.

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CHAPTER 11

Paroxysmal nocturnal haemoglobinuria Peter Hillmen

11

St James’s University Hospital, Leeds, UK

Introduction, 176 Pathophysiology, 176 Glycosylphosphatidylinositol defect, 176 Association with bone marrow failure, 176 Epidemiology, 176 Clinical features, 176 Clinical triad, 176

Other clinical sequelae, 178 Investigation, 179 Treatment, 179 Supportive care, 179 Thrombosis, 180 Allogeneic bone marrow transplantation, 181 Complement blockade, 181

Introduction Paroxysmal nocturnal haemoglobinuria (PNH) has fascinated haematologists since its first definitive description in 1882 by Paul Strübbing. PNH is unique as an acquired haemolytic disorder in which the defect is intrinsic to the red cell. Patients have a propensity to develop thromboses that are frequently life-threatening. PNH is also associated with bone marrow failure and indeed may provide a unique insight into the pathophysiology of a variety of bone marrow failure syndromes. Recently, the development of the terminal complement inhibitor eculizumab has revolutionized the treatment of PNH but in turn has revealed further insights into the pathophysiology of the disease and into normal physiology. The treatment of PNH with eculizumab is not without problems in that a minority of patients have suboptimal responses and require modifications to maximize the benefits of therapy.

Pathophysiology Glycosylphosphatidylinositol defect PNH results from the expansion of an abnormal haemopoietic clone that arises following an acquired mutation in a gene critical for the biosynthesis of glycosylphosphatidylinositol (GPI) structures. These GPI anchors are highly conserved

Postgraduate Haematology: 6th edition. Edited by A. Victor Hoffbrand, Daniel Catovsky, Edward G.D. Tuddenham, Anthony R. Green © 2011 Blackwell Publishing Ltd.

176

Pregnancy in PNH, 182 Prognosis, 184 Future challenges and developments, 184 Selected bibliography, 184

throughout evolution from yeast to humans, having an identical core structure. GPI anchors are glycolipid structures through which a large number of cell surface antigens are attached to the cell membrane. All patients with PNH have the same biosynthetic defect in one of the early steps of the pathway, namely the transfer of N-acetylglucosamine from UDP-Nacetylglucosamine to phosphatidylinositol. In 1993, Miyata and colleagues reported the cloning of the phosphatidylinositol glycan complementation class A (PIGA) gene, which is part of the enzyme complex involved in this step of the pathway and which has subsequently been shown to be mutated in all cases of acquired PNH reported to date. Mutations of the PIGA gene are different between patients as they are acquired somatic mutations not inherited. It appears that the mutation rate of PIGA is similar to that of other genes and in fact populations of PNH-like GPI-deficient cells can be observed at extremely low levels (1–10 per million cells) in most normal individuals, indicating that occasional haemopoietic stem cells by chance acquire PIGA mutations. Since the PIGA gene is located on the X chromosome (Xp22.1), each somatic cell, male or female, only has a single active copy and therefore a somatic mutation of this active gene leads to deficiency of GPI biosynthesis. GPI-deficient clones only expand if there is an additional factor that encourages their selection. The mechanism of this selective growth advantage of PNH clones is of key importance in understanding the pathophysiology of PNH. This could involve a second genetic event intrinsic to the PNH clone, as described in two separate cases where a chromosomal translocation of the HMGA2 gene has been reported. However, HMGA2 abnormalities have not been discovered in the analysis of a large number of cases of PNH. A much more likely explanation is indicated by the association between PNH and other bone marrow failure syndromes.

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Association with bone marrow failure PNH clones frequently occur at very low levels in normal individuals but very rarely expand to become routinely detectable let alone replace virtually all haemopoiesis, as is frequently observed in haemolytic PNH. In addition, PNH clones are detectable in up to 50% of patients with aplastic anaemia and in a smaller proportion of patients with myelodysplastic syndrome. This suggests that such bone marrow failure syndromes are permissive for the expansion of PNH clones. There is overwhelming evidence that aplastic anaemia is an autoimmune disorder in which there is an aberrant immune attack, probably by CD8 cytotoxic T cells directed against the haemopoietic stem cell. It would therefore appear likely that PNH stem cells, presumably due to GPI deficiency, evade this immune attack. This implies that the immune attack in bone marrow failure syndrome is, at least in part, directed through a GPI-linked structure. Understanding PNH may well provide an insight into the mechanism of a variety of bone marrow failure syndromes and provide a unique therapeutic target for these conditions.

Epidemiology If PNH is defined as the presence of a PNH clone identified during the investigation of bone marrow failure, haemolysis or thrombosis, then the incidence of PNH in a series from the UK was found to be 1.3 newly diagnosed patients per million per year and the prevalence 15.9 patients per million population. However, of these patients, 57% have less than 10% PNH neutrophils, indicating that they are unlikely to have clinically significant haemolysis and only one-third reported macroscopic haemoglobinuria. Thus in the region of 5 patients per million population have haemolytic PNH, and of these only a proportion will be severely affected with symptoms requiring transfusion.

Clinical features Clinical triad The clinical picture in PNH depends on the balance between three components: intravascular haemolysis, bone marrow failure and thrombotic complications. Haemolysis PNH, the disorder with perhaps the most intense chronic intravascular haemolysis, is characterized by episodes of haemoglobinuria, during which the patient’s urine is often black and which, for unknown reasons, is worse in the morning. This episodic haemoglobinuria is frequently associated with disa-

bling symptoms including profound lethargy out of keeping with the patient’s anaemia, abdominal pain sometimes requiring opiates, dysphagia that can be temporarily absolute, and erectile dysfunction. The degree of haemolysis is associated with the size of the PNH clone and particularly the proportion of PNH type III (completely deficient) red cells. Macroscopic haemoglobinuria is rarely seen unless there are at least 10% type III red cells. However, patients with apparently identical PNH clones can have a wide range of disease severity, from very occasional attacks of haemoglobinuria without anaemia to severe recurrent or even continuous haemoglobinuria and transfusion dependence, suggesting that other important factors influence the severity of the symptoms. It is possible that this variation is due to the severity of underlying bone marrow failure, to an inherent variation in complement activity or to some other unidentified factor. Many of the symptoms and complications of PNH result directly from intravascular haemolysis. The presence of extracellular haemoglobin is deleterious as is suggested by the elaborate mechanisms to remove even the smallest quantity. Free plasma haemoglobin is immediately bound to haptoglobin and removed, explaining why haptoglobin is depleted in every patient. Free haemoglobin binds to and removes other gases such as nitric oxide (NO). This depletion of NO in PNH results in smooth muscle dysfunction, vasoconstriction and pulmonary and systemic hypertension. This leads to dysphagia, abdominal discomfort, possibly due to intestinal spasm, erectile dysfunction, severe lethargy, pulmonary hypertension and possibly thrombosis, all classic symptoms of PNH. This syndrome of NO consumption has only become clear since the development of eculizumab (see below), which stops intravascular haemolysis in almost all patients and abrogates the clinical features of PNH. Bone marrow failure The degree of anaemia and other cytopenias is a composite of the activity of intravascular haemolysis and the degree of persisting bone marrow failure. The platelet count in many patients is a suitable surrogate for marrow function but in some patients, for example those with previous intra-abdominal thrombosis and subsequent hypersplenism, there may be other causes for a low platelet count. The presence of a PNH clone in patients with bone marrow failure probably indicates that there is a significant immune component to the marrow failure and may suggest that immune suppression is a reasonable therapeutic option. The degree of bone marrow failure will also impact on the efficacy of eculizumab as this will only treat the component due to complement activity, such as intravascular haemolysis and thrombosis. Thrombosis The precise cause of thrombosis in PNH is not entirely clear and is probably multifactorial. The activation of PNH platelets 177

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Table 11.1 Sites of thrombotic events in haemolytic PNH. Venous thrombosis Deep vein thrombosis Lower extremity Other Mesenteric/splenic vein thrombosis Hepatic/portal vein thrombosis Pulmonary embolus Cerebral/internal jugular thrombosis Superficial vein thrombosis

41 (33.1%) 23 (18.5%) 18 (14.5%) 23 (18.5%) 21 (16.9%) 8 (6.5%) 7 (5.6%) 5 (4.0%)

Arterial thrombosis Cerebrovascular accident/TIA Myocardial infarction/unstable angina

17 (13.7%) 2 (1.6%)

Total

124 (100%)

Source: This research was originally published in Blood. Hillmen P, Muus P, Duhrsen U et al. Effect of the complement inhibitor eculizumab on thromboembolism in patients with paroxysmal nocturnal hemoglobinuria. Blood 2007;110:4123–8. © American Society of Hematology.

by complement is at least part of the cause. Also, intravascular haemolysis and NO consumption may lead to endothelial damage and thrombosis. Recent data indicate that the thrombotic tendency in PNH is in arterial as well as venous sites, with several patients reported as having cerebrovascular accidents and myocardial infarctions at an early age. Thrombosis occurs in 40–50% of patients with haemolytic PNH, with a predilection for certain veins such as the hepatic veins (Budd–Chiari syndrome), other intra-abdominal veins or the cerebral veins (Table 11.1). Approximately 10% of patients present with a thrombosis as their initial clinical manifestation of PNH. After the first thrombosis, patients have a 7.8-fold increased risk of dying compared with those patients who have not had a thrombosis. A classical clinical scenario is that of downward spiralling thrombotic events: after a first thrombosis, patients continue to experience further apparently discrete thromboses despite what would be considered adequate anticoagulation with warfarin and/or heparin until they eventually succumb. Eculizumab has a marked effect on thrombosis in PNH and can interrupt this spiral of thrombosis.

Other clinical sequelae Consequences of haemolysis Even before the development of targeted therapy for PNH, patients usually survived for prolonged periods, with a median survival in the region of 20 years. A minority of patients experienced spontaneous remissions and therefore the majority of patients had decades of life in which they experienced a con178

tinuous high level of intravascular haemolysis. This leads to several complications of the disease. Renal disease All patients with haemolytic PNH develop heavy renal tubular iron loading due to the continuous filtration of haemoglobin. This is evident on both magnetic resonance imaging (MRI) and post-mortem studies. The majority of patients will eventually develop chronic renal disease, with a minority progressing to established chronic renal failure requiring dialysis. In addition, during times of very intense intravascular haemolysis and haemoglobinuria, patients can rarely develop acute renal failure, which is potentially fully reversible even though it may require dialysis. Some patients may develop repeated episodes of acute renal dysfunction. The use of nephrotoxic therapies, such as ciclosporin, adds to the renal insult, as do rare complications such as renal vein thrombosis. Cholelithiasis Patients with PNH have both intravascular and extravascular haemolysis, which increases the risk of developing gallstones. In patients who develop abdominal pain, a common symptom in PNH, and particularly if there is any additional change in liver function tests, then the presence of gallstones should be considered. Leukaemic transformation There have been a number of case reports of myelodysplastic syndrome and acute myeloid leukaemia (AML) in patients with PNH. These have led to the suspicion that PNH may be preleukaemic. However, this is almost certainly not the case for two principal reasons. Firstly, in patients with PNH who develop AML, GPI-linked antigens on myeloid blasts have been reported to be deficient in some but normal in others, indicating that the leukaemia can develop in either the residual normal or the GPIdeficient haemopoietic cells. Secondly, less than 5% of PNH patients in the larger series develop AML. This is similar to the incidence of AML in aplastic anaemia and since it seems the majority, if not all, patients with PNH have an underlying aplasia, then the rate of AML is no higher than would be expected if GPI deficiency has no impact on leukaemogenesis. Therefore it is aplastic anaemia that predisposes to both AML and PNH, and the development of PNH does not increase the development of AML. Spontaneous remission PNH often develops in young adults between 20 and 40 years old. Historically, most patients with PNH have either died as a direct or indirect result of their disease or have suffered from the disease for the remainder of their life. In a series of 80 haemolytic PNH patients reported in 1995, initially diagnosed between 1940 and 1970, median survival was 10 years and 12 of the 35 patients surviving at least 10 years experienced spontaneous remission. Analysis of GPI-linked antigens on blood

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Figure 11.1 Identification of PNH red cells by flow cytometry. (a) GPI anchor protein (e.g. CD59) expression on normal red cells (type I). (b) A total of 46% of red cells are completely GPI anchor-deficient (type III). (c) A mixture of all three types of red

cells are shown: type III, 10%; type II (partial GPI anchor expression), 54%; normal type I cells, 36%. (Figure courtesy of S.J. Richards.)

cells up to 20 years after spontaneous clinical remission demonstrated that the myeloid series was entirely normal with no residual PNH cells, whereas there was a small PNH clone in the lymphoid compartment (presumably these are long-lived memory cells). Early reports suggest that a proportion of patients treated with eculizumab have a progressive decrease in the size of their PNH clone, perhaps suggesting that remission may occur.

deficiency) nor do they provide information about the proportion of PNH leucocytes. The proportion of PNH neutrophils and monocytes probably gives a more accurate assessment of the true size of the PNH clone as they are not influenced by the intensity of intravascular haemolysis or by transfusions. The modern diagnosis of PNH depends on the flow cytometric analysis of at least two cell lineages (e.g. neutrophils and red cells), with a transmembrane marker to positively identify the cell type and at least two separate GPI-linked antigens to clearly separate PNH cells from their normal counterparts (Figures 11.1 and 11.2). In most patients there is a population of red cells with complete deficiency of GPI-linked molecules (PNH type III cells, the most complement-sensitive cells) and in some an additional population with partial GPI deficiency (PNH type II cells, intermediate complement sensitivity). Type II and III are usually not evident in the neutrophils. The size of the PNH clone correlates with the risk of complications, such as thrombosis, and the severity of haemolysis. In addition, the evolution of the clone – either its expansion, leading to more haemolytic disease, or its reduction, ultimately leading to spontaneous remission – can be tracked. Flow cytometry has superseded the complement sensitivity tests in the diagnosis of PNH.

Nitric oxide consumption Intravascular haemolysis leads to the release of cell-free haemoglobin into the plasma. There is now a growing body of evidence in other haemolytic disorders, such as sickle cell anaemia, that many of the symptoms and complications are due to the deleterious effects of free haemoglobin. Free haemoglobin absorbs NO, which is a key mediator in homeostasis and smooth muscle function. Since PNH has extremely high and chronic levels of intravascular haemolysis, it is perhaps not surprising that many of the symptoms of the disease – severe lethargy out of keeping with the level of haemoglobin, dysphagia, abdominal pain, erectile dysfunction, pulmonary hypertension – are most probably due to intravascular haemolysis and NO consumption.

Investigation The diagnosis of PNH was historically made by demonstrating the sensitivity of red cells to lysis by activated complement. The method used to activate complement varied, for example by acidification in the Ham test or by low ionic solutions in the sucrose lysis test. However, these tests only give an indirect assessment of the proportion of PNH red cells and do not assess the type of PNH red cells (complete versus partial GPI

Treatment Supportive care Conventional management in PNH has been supportive. The severity of haemolysis varies greatly between patients, in part dependent on the size of the PNH clone and the degree of underlying bone marrow failure. However, even patients with no evidence of clinically apparent marrow failure and large 179

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PNH clones can have highly variable levels of haemolysis: some patients experience recurrent, even continuous, haemoglobinuria and are transfusion dependent, whereas others experience haemoglobinuria only rarely, or not at all, do not require any transfusions and have a well-compensated haemolytic anaemia. Patients with a significant degree of haemolysis should receive folic acid supplementation. The constant haemosiderinuria means that patients, even those who require regular transfusions, have a tendency to become iron deficient. This iron deficiency can result in failure of the marrow to compensate for the increased red cell destruction and therefore increasing anaemia. Iron supplementation has been reported to precipitate episodes of intravascular haemolysis but usually patients tolerate oral iron supplements well and should be treated where iron deficiency is present. Patients should be transfused according to their symptoms. In most patients the severity of haemoglobinuria lessens immediately after a transfusion but then increases, with a progressive fall in haemoglobin level until the patient requires a further transfusion. Incidental infections lead to an increase in haemolysis, presumably due to activation of the complement system, and frequent sudden drops in the level of haemoglobin. These episodes are often associated with severe symptoms of abdominal pain, dysphagia and debilitating lethargy. Corticosteroids have been widely used in the treatment of haemolysis in PNH. At high doses steroids do appear to have an effect of reducing the activity of complement and some patients report an improvement in symptoms. However, high doses are required to have a clinically useful effect and as the haemolysis in PNH is chronic, the required dose is too high. Steroids are therefore not generally recommended in PNH as the long-term side-effects outweigh the potential benefits.

Thrombosis The major cause of morbidity and mortality in PNH is thrombosis, which usually affects the venous system, particularly 180

Figure 11.2 Identification of PNH granulocytes by multicolour flow cytometry. (a) Granulocytes identified based on CD15 expression and side scatter. (b) Analysis of CD16 and CD24 (both GPI-linked proteins) reveals a small population of PNH granulocytes (10%) that are deficient in both proteins. (Figure courtesy of S.J. Richards.)

the intra-abdominal and cerebral vessels, but also appears to result in arterial thrombosis (Table 11.1). The chance of developing a thrombosis depends on the size of the PNH clone and the severity of haemolysis, two variables that are closely related; half of patients with over 50% PNH neutrophils will develop a thrombosis at some point in their disease course. In most series, approximately one-third of patients will eventually die as a result of thrombosis. In a series reported by Hall and colleagues, 39 patients with PNH who had not previously experienced a thrombosis were treated with warfarin to maintain an International Normalized Ratio (INR) of 2.0–3.0. None of these 39 patients developed a thrombosis compared with a 36.5% chance of thrombosis at 10 years for the 56 patients with large PNH clones who elected not to have warfarin prophylaxis. However, two patients on warfarin experienced intracranial haemorrhage and one of these died as a result. Therefore it appears that primary prophylaxis with warfarin is effective in preventing thrombosis but carries a significant risk in this patient group. Aspirin does not appear to be protective against thrombosis and has no effect on the symptoms of PNH such as abdominal pain (Peter Hillmen, unpublished observation). It seems very likely that primary prophylaxis with warfarin is unnecessary for patients receiving the anti-complement monoclonal antibody eculizumab, as this agent significantly reduces the risk of thrombosis in PNH (see below). The treatment of established thrombosis is similar to the management of thrombosis in patients without PNH. However, in view of the risk of recurrent thrombosis, patients should remain on lifelong anticoagulation after their first episode of thrombosis. Hepatic vein thrombosis (Budd–Chiari syndrome) is one of the more common thromboses seen in PNH and there have been reports of the successful lysis of such thromboses using tissue plasminogen activator; this should be considered even in patients who present with a relatively long history suggesting that their thrombosis occurred days or even weeks before.

Paroxysmal nocturnal haemoglobinuria

Allogeneic bone marrow transplantation The only curative strategy for PNH is allogeneic stem cell transplantation but this carries a considerable risk of mortality. From the reported series and in view of the fact that a proportion of patients will eventually experience a spontaneous remission of PNH and with the advent of potentially effective novel therapies such as eculizumab, transplantation should only be considered in selected cases, such as those with a syngeneic donor or with associated bone marrow failure. In these patients the indications for transplantation are similar to those for aplastic anaemia.

Complement blockade The development of eculizumab (Soliris), a humanized monoclonal antibody that blocks the activation of terminal complement, has dramatically altered the management and almost certainly the prognosis in PNH. The complement cascade can be activated by the classical pathway (antibody dependent), the alternative pathway (directly through microbial membranes) or the lecithin pathway, which all converge at the fifth component of complement (C5). C5 is cleaved and releases C5a (a potent anaphylatoxin) and C5b, to which C6, C7, C8 and numerous C9 molecules bind to form a pore (the membrane attack complex) that punches holes in the membrane of the target. Eculizumab binds to C5 and stops it being cleaved and, as long as trough plasma levels remain above 35 mg/L, prevents any activation of terminal complement. Individuals with inherited terminal complement deficiency are either asymptomatic or present with recurrent Neisseria meningitidis (meningococcus) infections and this highlights the main concern with eculizumab. The clinical features of PNH are due largely to the absence of CD59 from haemopoietic cells and therefore the uncontrolled activity of terminal complement on PNH cells, making eculizumab an ideal candidate for the targeted therapy of PNH. In the initial study, 11 patients with transfusion-dependent haemolytic PNH were treated with eculizumab using a schedule designed to maintain trough levels to ensure that complement was completely blocked. The responses were dramatic, with an immediate resolution of the symptoms of intravascular haemolysis. This study was followed by a randomized placebo-controlled Phase III trial (TRIUMPH) and a non-randomized trial (SHEPHERD). In total, 195 patients were included in these three trials, which led to the licensing of eculizumab in the USA and Europe in 2007. The results of these trials are summarized below.

concentration, which is typically as much as 10–20 times normal in PNH, falls immediately in all patients treated with eculizumab to normal or just above normal. Other markers of haemolysis, such as aspartate aminotransferase, fall to normal and in some patients the haptoglobins become detectable, at least temporarily. In general this results in increasing haemoglobin level which, depending on the degree of coexistent bone marrow failure as well as the extent of extravascular haemolysis (see below), will reach a plateau usually between 9 and 12 g/dL. The most dramatic effect of eculizumab is on the symptoms, with resolution of the abdominal pain and dysphagia and improvement in the severe lethargy and the other features of haemolysis. In the vast majority of patients transfusion requirements improve, with over half of patients becoming transfusion independent. The dramatic improvement in PNH-related symptoms is due to a marked reduction in NO consumption during therapy as a result of the improvement in intravascular haemolysis and thereby the reduction in free haemoglobin. This probably explains many of these benefits of the drug and provides insights into the pathophysiology of the symptoms of PNH. There is a dramatic and clinically significant improvement in the quality of life of patients as measured by validated questionnaires. There is now evidence that the adverse consequences of intravascular haemolysis, such as renal damage and pulmonary hypertension, are ameliorated by eculizumab.

Efficacy of eculizumab

Thrombosis Eculizumab also protects patients from thrombosis. Compared with thrombosis before patients commencing eculizumab (effectively using patients as their own controls), the thrombotic rate reduces by fivefold to tenfold. In patients who have had a previous thrombosis and who are on anticoagulation, there is still a high recurrent thrombosis rate prior to starting eculizumab. However, since eculizumab became available, such recurrent thromboses are extremely uncommon. Patients who commence eculizumab during a ‘spiral’ of thrombotic events stop having further thromboses, indicating that eculizumab specifically targets the mechanism of thrombosis in PNH and that this is a more effective strategy than conventional anticoagulation alone. There is now increasing confidence that the catastrophic thrombotic complications of PNH can be much more successfully managed with a combination of eculizumab and anticoagulation. This observation will have an impact on the decision to use warfarin prophylaxis: in cases where there are concerns over the safety or requirement for warfarin prophylaxis, such as those with low platelets or with borderline PNH clone sizes, it is clearly safer to withhold anticoagulation as long as the use of eculizumab is an option for the patient should a thrombosis occur.

Intravascular haemolysis Eculizumab has a profound and immediate effect on intravascular haemolysis in PNH. The lactate dehydrogenase (LDH)

Renal dysfunction In the 195 patients entering the eculizumab trials, renal dysfunction or damage was observed in 65% of patients before they 181

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were treated with eculizumab. In this series, 27% of patients had developed major clinical kidney disease within 10 years from their initial diagnosis of PNH and 21% of patients developed late-stage chronic kidney disease (stage 3 or 4 as defined by the Kidney Disease Outcomes Quality Initiative) or kidney failure (stage 5). The early analysis of patients treated with eculizumab suggests that many of the patients with early renal dysfunction (stages 1 and 2) will improve, and the deterioration in renal function in patients with advanced renal dysfunction (stages 3–5) is frequently stabilized. Thus eculizumab appears to have a beneficial effect on renal function in PNH and this is presumably due to the marked reduction in intravascular haemolysis and therefore in haemoglobinaemia and haemoglobinuria. Extravascular haemolysis with eculizumab Despite the impressive impact on intravascular haemolysis, most patients remain somewhat anaemic, maintaining their haemoglobin between 9 and 12 g/dL. Virtually all have a persisting reticulocytosis and many continue to have a raised bilirubin. These features are suggestive of ongoing extravascular haemolysis, which has previously been unreported in PNH. On further investigation it transpires that approximately two-thirds of PNH patients on eculizumab develop a positive direct antiglobulin test to complement only. Flow cytometry demonstrates that the PNH red cells are coated by early complement components (C3b and C3d), possibly because PNH red cells, as well as being deficient in the principal controller of terminal complement, namely CD59, do not express the inhibitor of C3 convertase decay-accelerating factor (DAF). It appears that preventing terminal complement activation leads to a build-up of the early complement components, which accumulate on PNH red cells due to their deficiency of DAF. If intense, this extravascular haemolysis results in a poor increase in haemoglobin and a minority of patients continuing to require transfusions. If the transfusions are due to poor marrow reserves and a lack of compensation, particularly in patients with evidence of renal dysfunction, then treatment with erythropoietin can lead to a clinically meaningful increase in haemoglobin. If this fails, then it does not appear that steroids have a major role. When patients become generally unwell, particularly with coexistent infections, there can be an increase in the level of extravascular haemolysis and/or a decrease in marrow compensation, leading to a fall in the level of haemoglobin. When patients recover from infection, they usually goes back to their steady state. Eculizumab administration and dosing Eculizumab is given as a 30-min intravenous infusion. The aim is to rapidly block complement and to maintain complement blockade continuously. The standard dosage schedule for eculizumab comprises a loading dose of 600 mg every week for 4 weeks, followed by 900 mg the next week and then 900 mg every 2 weeks indefinitely. In the majority of patients this is adequate to maintain trough levels of eculizumab above 35 mg/L and 182

therefore to block complement completely. In approximately 5% of patients this dose is inadequate and patients break through complement blockade. Patients appear well with no signs of haemolysis until immediately before a dose of eculizumab but then develop dark urine often with abdominal pain, sometimes with dysphagia, and a sudden deterioration in the laboratory measures of haemolysis, such as LDH and bilirubin, and a fall in the level of haemoglobin. In this situation the maintenance dose of eculizumab is too low and an increase, either by reducing the interval or more conveniently by increasing the dose (from 900 mg every 2 weeks to 1200 mg every 2 weeks is usually effective), will raise trough levels of eculizumab above 35 mg/L and re-establish continuous control of haemolysis (Figures 11.3 and 11.4). Infectious risk with eculizumab Eculizumab is generally very well tolerated with few infusionrelated reactions. However, as noted above, blocking terminal complement activity would be expected to increase the risk of infection with Nesseria meningitidis (meningococcus). All patients commencing eculizumab should be vaccinated with a wide-spectrum meningococcal vaccine (ACWY Vax). Unfortunately, the current vaccines do not cover serotype B, which is common in the UK. Although the risk of meningococcal infection is a concern, the observed risk is less than 0.5 cases of meningococcal infection per 100 patient-years on eculizumab. However, when these infections occur they can be lifethreatening and it is vitally important to impress on patients that they should seek medical help if they suffer any symptoms suggestive of infection, and these are usually of septicaemia rather than meningitis. The role of antibiotic prophylaxis with penicillin is currently being explored. There is no convincing evidence as yet of an increased risk to any other organism except for N. meningitidis.

Pregnancy in PNH It is difficult to estimate the true risk of pregnancy in PNH but there is undoubtedly an increased risk of maternal morbidity and mortality. The reported maternal mortality, mainly from thrombosis, is between 12 and 21%, although this is likely an overestimate due to biased reporting. In addition, an increased fetal loss rate has been reported, although again this is difficult to substantiate and is probably due to maternal factors. There have now been a number of successful pregnancies in women receiving eculizumab either later in pregnancy or throughout the pregnancy from conception to delivery. The reports to date are positive with little or no eculizumab crossing the placenta into the fetus. It does appear that the metabolism of eculizumab may be altered in pregnancy and women seem more likely to break through complement blockade and may need higher doses in the latter part of pregnancy.

Paroxysmal nocturnal haemoglobinuria

Haemoglobinuria Dysphagia

Day:

9

10

11

12

13

0

Early-morning urine Days

0*

1–8

10

2–3

9

10

11

12

13

3

3

3

3

9

0*

1

2

10

3

3

Urine scale

1= Clear; 10 = Black

LDH

160–480 IU/L

2624

–

784

–

–

697

1687

2917

–

–

AST

0–40 IU/L

119

–

38

–

–

31

87

–

–

–

PK

C5 blocked >35 µg/mL

27

–

45

–

–

35

29

26

–

–

PD

No lysis 50 chromosomes) With hypodiploidy (20% of peripheral blood leucocytes or of nucleated bone marrow cells Extramedullary blast proliferation Large foci or clusters of blasts in the bone marrow biopsy Note: In this classification, unlike some other classifications, the acquisition of new cytogenetic abnormalities in addition to the Ph chromosome is not by itself a criterion for ‘promoting’ a chronic-phase patient to accelerated phase.

Advanced phases The haematological picture in acceleration is very variable. It may differ little from chronic phase but blast cell numbers may be increased disproportionately (Table 27.4). There may be anaemia in the presence of a normal leucocyte count. Platelet numbers may be greatly increased (>1000 × 109/L) or reduced ( 95%)

Less than CHR

NA

Less than partial cytogenetic response (Ph+ > 35%) Partial cytogenetic response (Ph+ ≤ 35%)

No cytogenetic response (Ph+ > 95%)

NA

Less than partial cytogenetic response (Ph+ > 35%) Less than complete cytogenetic response Loss of CHR Loss of complete cytogenetic response Mutations‡ Additional chromosome abnormalities in Ph-positive cells§

Less than major molecular response*

12 months

Complete cytogenetic response

18 months

Major molecular response

Any time

Stable or improving major molecular response

Less than major molecular response Loss of major molecular response Mutations†

NA Any rise in transcripts level Other chromosome abnormalities in Ph-negative cells

*Major molecular response denotes BCR–ABL1/ABL1 or other housekeeping genes ≤0.1% on the international scale. BCR–ABL1 kinase domain mutations highly insensitive to imatinib (see Table 27.3). ‡ BCR–ABL1 kinase domain mutations still sensitive to imatinib (see Table 27.3). § Additional chromosome abnormalities in Ph-positive cells are a warning factor at diagnosis, while their occurrence during treatment (clonal progression) is a marker of failure. Two consecutive cytogenetic tests are required, showing the same additional chromosome abnormalities in at least two Ph-positive cells. CHR, complete haematological response; NA, not applicable. †

Resistance to imatinib Resistance to imatinib may be primary or secondary. A very small proportion of patients with newly diagnosed chronicphase CML fail to achieve a haematological response and a small proportion never achieve a complete cytogenetic response. In these cases the resistance seems to be primary. A larger proportion of patients respond initially at the haematological or cytogenetic level and then lose their responses; such secondary resistance occurs most commonly in the first 2 years after starting treatment and appears to be increasingly rare in patients who have taken imatinib for more than 2 years. The cause of primary resistance is essentially unknown but is likely to reflect the underlying heterogeneity of CML at diagnosis. Similarly, the cause of secondary resistance is poorly defined. It has been associated with a variety of diverse mechanisms, including overexpression of the BCR–ABL1 oncoprotein and overexpression of P-glycoprotein, which expedites efflux of the drug from individual cells, and the acquisition of point mutations in the ABL1 kinase domain. Thus far, Ph-positive subclones with at least 50 different point mutations have been identified in leukaemia cells obtained from patients with variable degrees of resistance to imatinib, and some of these, but by no means all, 494

are clearly the cause of the resistance. Each mutation encodes a different amino acid substitution in the ABL1 kinase component of the BCR–ABL1 oncoprotein. Cells with one such substitution, the replacement of threonine by isoleucine at position 315 (referred to as the T315I mutation), seem to be especially resistant to the inhibitory action of imatinib and all other TKIs. Cells with other substitutions are relatively less resistant. It is probable that some of these subclones exist before the administration of imatinib but are allowed to expand when the unmutated oncoprotein molecule is inhibited by imatinib; in other cases the mutation may develop de novo after initiation of imatinib.

Second-generation tyrosine kinase inhibitors The remarkable success of imatinib in chronic-phase CML led rapidly to development of other TKIs, notably dasatinib, nilotinib and bosutinib, all of which are clearly more potent than the original TKI. With each of these drugs the largest experience has been gained in treating patients who have failed imatinib, but all are now being tested as up-front treatment for newly diagnosed individuals with promising early results.

Chronic myeloid leukaemia

Dasatinib Dasatinib (Sprycel) was first used to treat CML in 2003. Somewhat surprisingly it bears little chemical resemblance to imatinib but acts, like imatinib, by occupying the phosphatebinding pocket and blocking access for ATP. Unlike imatinib it is active with the kinase activation loop in both closed and open configurations. It differs also from imatinib in being active against a wide range of tyrosine kinases in addition to ABL1, of which the most notable are SRC and so-called SRC family kinases. It has therefore been termed a dual inhibitor, on the assumption that its value in CML could be based on its relatively wide spectrum of activity. The original dose was 70 mg twice daily but the recommended dose for patients is now 100 mg once daily. For patients who have failed imatinib, dasatinib induces complete cytogenetic responses in about 50% of patients and some of these responses seem very durable. It is definitely more myelosuppressive than imatinib and treatment must occasionally be interrupted or dosage reduced on account of neutropenia or thrombocytopenia Other side-effects observed include nausea, gastrointestinal disturbances, rashes and pleural effusions, which may or may not be asymptomatic. The latter usually resolve on stopping the drug. Nilotinib Nilotinib (Tasigna) was developed by chemical modification of imatinib with the intention of increasing its activity. Nilotinib is about 30 times more active in vitro but the recommended oral dose, 400 mg twice daily, does not reflect the in vitro ratio. Like dasatinib, it induces complete cytogenetic responses in about 50% of patients who fail imatinib. It is usually well tolerated but side-effects have included headaches, nausea, gastrointestinal disturbances, pancreatitis, raised levels of bilirubin, and abnormal liver function tests. Prolongation of the QT interval has been observed in rare instances. Bosutinib The third second-generation TKI in clinical practice is bosutinib, which also targets a relatively wide spectrum of tyrosine kinases. It is also active in patients deemed to have failed imatinib and is being tested in a Phase III study of previously untreated chronic-phase patients. It principal toxic effect is diarrhoea, which may be severe but usually responds to standard antidiarrhoeal agents.

Hydroxycarbamide Hydroxycarbamide (also known as hydroxyurea) is a ribonucleotide reductase inhibitor that targets relatively mature myeloid progenitors in proliferative cycle. Its pharmacological action is rapid and readily reversible. Treatment for patients in chronic phase usually starts with 1.0–2.0 g daily by mouth and continued indefinitely. The leucocyte count starts to fall within days and the spleen reduces in size. It is usually possible to

reverse all features of CML within 4–8 weeks of starting treatment with hydroxycarbamide. The dosage can then be titrated against the leucocyte count, the usual maintenance dose being 1.0–1.5 g daily. In a patient whose leucocyte count is controlled, any reduction in the dose leads to a rapid increase in leucocyte numbers, a phenomenon that disturbs the patient but has no ominous significance. The drug has relatively few side-effects. At high dosage it may cause nausea, diarrhoea or other gastrointestinal disturbance. Some patients get ulcers of the buccal mucosa. Skin rashes are seen. Most patients develop megaloblastic changes in the marrow with macrocytosis in the blood. The drug remains useful today for rapid cytoreduction in the newly diagnosed patient and may also be useful in patients unable to tolerate imatinib, but it rarely causes any degree of reduction in Ph-positive cells in the bone marrow and must therefore be regarded as second- or third-line therapy.

Interferon alfa Interferon alfa, like hydroxycarbamide, can no longer be regarded as first-line therapy for CML, but may still have a role in management of patients who have failed TKIs. It is a member of a large family of glycoproteins of biological origin with antiviral and antiproliferative properties. Studies in the early 1980s using material purified from human cell lines showed that it was active in reducing the leucocyte count and reversing all features of CML in 70–80% of CML patients. Of particular interest at the time was the observation that 5–15% of patients achieved major reduction in the percentage of Ph-positive marrow metaphases with restoration of Ph-negative (putatively normal) haemopoiesis. It raised the important question of whether these ‘cytogenetic responders’ would have their life prolonged by treatment with interferon alfa, and prospectively randomized controlled studies were initiated. Compared with hydroxycarbamide, interferon alfa offered a survival advantage that was maximal for those who achieved complete cytogenetic remissions. These observations meant that interferon alfa replaced hydroxycarbamide and busulfan in the 1980s as primary treatment for CML in chronic phase, and remained so until the introduction of imatinib. Interferon alfa must be administered by subcutaneous injection at daily doses ranging from 3 to 5 million units/m2. There is no good evidence that the higher doses are clinically superior. Toxicity is common in older patients, but is generally mild and reversible. Almost all patients experience fevers, shivers, muscle aches and general flu-like features on starting the drug; these last usually 2–3 weeks but may be alleviated by paracetamol. They recur when dosage is increased. A significant minority of patients cannot tolerate the drug on account of lethargy, malaise, anorexia, weight loss, depression and other affective disorders or alopecia. Autoimmune syndromes, such as thyrotoxicosis, may also occur. A long-acting form of interferon alfa, peginterferon alfa, has been introduced recently, but other than 495

Postgraduate Haematology

ease of administration it seems to have little advantage over conventional formulations.

Busulfan Busulfan (1,4-dimethanesulfonyloxybutane) is a polyfunctional alkylating agent that is now infrequently used (except as conditioning before transplant procedures). It targets a relatively primitive stem cell and the effects of administration are prolonged for some weeks after stopping the drug. It was the mainstay of treatment for CML in the period 1960–80. Treatment was conventionally started with 8 mg daily by mouth and the dosage was reduced as the leucocyte count began to fall. It was essential to reduce the dosage substantially or to stop the drug before the leucocyte count fell below 20 × 109/L because profound and prolonged leucopenia might otherwise be produced. Busulfan could be administered either in finite courses lasting up to 4 weeks or continuously at a maintenance dose between 0.5 and 2.0 mg daily. Occasional patients were hypersensitive to the effects of busulfan and developed severe, sometimes irreversible, pancytopenia with marrow hypoplasia on standard dosage. Overdosage could achieve the same effect in any patient. Gonadal failure (as mentioned above) invariably occurred within a few months of starting treatment and was almost always irreversible. Other toxic effects included cutaneous pigmentation, pulmonary fibrosis and a wasting syndrome resembling hypoadrenalism. These points notwithstanding, the drug may be useful in older patients whose compliance is uncertain as it can also be given orally in the clinic as a single dose of 50 or 100 mg, to be repeated as necessary after 4 weeks or longer.

Homoharringtonine Another drug of interest for CML in chronic phase is homoharringtonine (omecetaxine), a semisynthetic plant alkaloid that enhances apoptosis of CML cells. It produces haematological responses in 60–70% of patients and major cytogenetic responses in 25% in small series of patients in chronic phase. The results appear to improve with the addition of interferon alfa. For the moment, it remains an investigational agent.

Allogeneic stem cell transplantation Younger patients with suitable stem cell donors who fail treatment with TKIs may be offered the option of allogeneic SCT (see Chapter 24). Most specialist centres exclude from consideration patients who are over the age of 50 or 55 years. The major factors influencing survival are patient age, disease phase at time of SCT, disease duration, degree of histocompatibility between donor and recipient, and gender of donor. In general, patients are ‘conditioned’ for transplant with cyclophosphamide at high dosage followed by total body irradiation, or with the combination of busulfan and cyclophosphamide at high dosage. If all goes well, reasonable marrow function is achieved in 3–4 weeks after the infusion of donor haemopoietic stem cells and the patient leaves the hospital. The possible major complications include graft-versus-host disease (GVHD), reactivation of infection with cytomegalovirus or other viruses, idiopathic pneumonitis and veno-occlusive disease of the liver. For patients with CML treated by SCT with marrow from HLA-identical siblings, the overall leukaemia-free survival at 5 years is now 60–80% (Figure 27.9). There is a roughly 20% chance of transplant-related mortality and a 15%

1.0

Probability (%)

0.8

0.6

0.4 HLA-identical sibling, 60 years) treated with CHOP-R, with no increase in treatmentrelated toxicity. The addition of granulocyte colony-stimulating factors can shorten duration of neutropenia, enabling the delivery of chemotherapy at the planned time and dose intensity. Several studies have recently reported encouraging results with autologous SCT in patients older than 60 years (4-year OS in patients with chemosensitive relapsed disease approached 40%), indicating the feasibility of this treatment in selected patients.

Selected bibliography Al-Tourah AJ, Gill KK, Chhanabhai M et al. (2008) Populationbased analysis of incidence and outcome of transformed nonHodgkin lymphoma. Journal of Clinical Oncology 26: 5165–9. Ardeshna KM, Smith P, Norton A et al. (2003) Long-term effect of a watch and wait policy versus immediate systemic treatment for asymptomatic advanced-stage non-Hodgkin lymphoma: a randomized controlled trial. Lancet 362: 516–22. Armitage J, Vose J, Weisenburger D (2008) International peripheral T-cell and natural killer/T-cell lymphoma study: pathology findings and clinical outcomes. Journal of Clinical Oncology 26: 4124–30. Balsalobre P, Diez-Martin JL, Re A et al. (2009) Autologous stemcell transplantation in patients with HIV-related lymphoma. Journal of Clinical Oncology 27: 2192–8. Barr PM, Lazarus HM (2008) Follicular non-Hodgkin lymphoma: long-term results of stem-cell transplantation. Current Opinion in Oncology 20: 502–8. 683

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Bessell EM, Hoang-Xuan K, Ferreri AJ, Reni M (2007) Primary central nervous system lymphoma: biological aspects and controversies in management. European Journal of Cancer 43: 1141–52. Boehme V, Schmitz N, Zeynalova S, Loeffler M, Pfreundschuh M (2009) CNS events in elderly patients with aggressive lymphoma treated with modern chemotherapy (CHOP-14) with or without rituximab: an analysis of patients treated in the RICOVER-60 trial of the German high-grade non-Hodgkin lymphoma study group (DSHNHL). Blood 113: 3896–902. Bohlius J, Herbst C, Reiser M, Schwarzer G, Engert A (2008) Granulopoiesis-stimulating factors to prevent adverse effects in the treatment of malignant lymphoma. Cochrane Database of Systematic Reviews CD003189. Bower M, Powles T, Williams S et al. (2007) Brief communication: rituximab in HIV-associated multicentric Castleman disease. Annals of Internal Medicine 147: 836–9. Cheson BD, Pfistner B, Juweid ME et al. (2007) Revised response criteria for malignant lymphoma. Journal of Clinical Oncology 25: 579–86. Dave SS, Wright G, Tan B et al. (2004) Prediction of survival in follicular lymphoma based on molecular features of tumourinfiltrating immune cells. New England Journal of Medicine 351: 2159–69. Dave SS, Fu K, Wright GW et al. (2006) Molecular diagnosis of Burkitt’s lymphoma. New England Journal of Medicine 354: 2431–42. Diez-Martin JL, Balsalobre P, Re A et al. (2009) Comparable survival between HIV positive and HIV negative non-Hodgkin and Hodgkin lymphoma patients undergoing autologous peripheral blood stem cell transplantation. Blood 113: 6011–14. Dimopoulos MA, Gertz MA, Kastritis E et al. (2009) Update on treatment recommendations from the Fourth International Workshop on Waldenstrom’s Macroglobulinemia. Journal of Clinical Oncology 27: 120–6. Doolittle ND, Abrey LE, Shenkier TN et al. (2008) Brain parenchyma involvement as isolated central nervous system relapse of systemic non-Hodgkin lymphoma: an International Primary CNS Lymphoma Collaborative Group report. Blood 111: 1085–93. Dreyling M, Hiddemann W (2008) Dose-intense treatment of mantle cell lymphoma: can durable remission be achieved? Current Opinion in Oncology 20: 487–94. Ferrucci PF, Zucca E (2007) Primary gastric lymphoma pathogenesis and treatment: what has changed over the past 10 years? British Journal of Haematology 136: 521–38. Feugier P, Van Hoof A, Sebban C et al. (2005) Long-term results of the R-CHOP study in the treatment of elderly patients with diffuse large B-cell lymphoma: a study by the Groupe d’Etude des Lymphomes de l’Adulte. Journal of Clinical Oncology 23: 4117–26. Fisher RI, Gaynor ER, Dahlberg S et al. (1993) Comparison of a standard regimen (CHOP) with three intensive chemotherapy regimens for advanced non-Hodgkin lymphoma. New England Journal of Medicine 328: 1002–6. Forstpointner R, Unterhalt M, Dreyling M et al. (2006) Maintenance therapy with rituximab leads to a significant prolongation of response duration after salvage therapy with a combination of 684

rituximab, fludarabine, cyclophosphamide, and mitoxantrone (R-FCM) in patients with recurring and refractory follicular and mantle cell lymphomas: results of a prospective randomized study of the German Low Grade Lymphoma Study Group (GLSG). Blood 108: 4003–8. Greb A, Bohlius J, Schiefer D, Schwarzer G, Schulz H, Engert A (2008) High-dose chemotherapy with autologous stem cell transplantation in the first line treatment of aggressive nonHodgkin lymphoma (NHL) in adults. Cochrane Database of Systematic Reviews CD004024. Gyan E, Foussard C, Bertrand P et al. (2009) High-dose therapy followed by autologous purged stem cell transplantation and doxorubicin-based chemotherapy in patients with advanced follicular lymphoma: a randomized multicenter study by the GOELAMS with final results after a median follow-up of 9 years. Blood 113: 995–1001. Haioun C, Itti E, Rahmouni A et al. (2005 ) [18F]fluoro-2-deoxyd-glucose positron emission tomography (FDG-PET) in aggressive lymphoma: an early prognostic tool for predicting patient outcome. Blood 106: 1376–81. Hoster E, Dreyling M, Klapper W et al. (2008) A new prognostic index (MIPI) for patients with advanced-stage mantle cell lymphoma. Blood 111: 558–65. Illerhaus G, Muller F, Feuerhake F, Schafer AO, Ostertag C, Finke J (2008) High-dose chemotherapy and autologous stem-cell transplantation without consolidating radiotherapy as first-line treatment for primary lymphoma of the central nervous system. Haematologica 93: 147–8. Ishitsuka K, Tamura K (2008) Treatment of adult T-cell leukaemia/ lymphoma: past, present, and future. European Journal of Haematology 80: 185–96. Kaplan LD, Lee JY, Ambinder RF et al. (2005) Rituximab does not improve clinical outcome in a randomized phase 3 trial of CHOP with or without rituximab in patients with HIV-associated nonHodgkin lymphoma: AIDS-Malignancies Consortium Trial 010. Blood 106: 1538–43. Kyriakou C, Canals C, Goldstone A et al. (2008) High-dose therapy and autologous stem-cell transplantation in angioimmunoblastic lymphoma: complete remission at transplantation is the major determinant of Outcome-Lymphoma Working Party of the European Group for Blood and Marrow Transplantation. Journal of Clinical Oncology 26: 218–24. Lenz G, Staudt LM (2010) Aggressive lymphomas. The New England Journal of Medicine 362: 1417–29. Lenz G, Wright GW, Emre NC et al. (2008) Molecular subtypes of diffuse large B-cell lymphoma arise by distinct genetic pathways. Procedings of the National Academy of Sciences USA 105: 13520–5. McMillan A (2005) Central nervous system-directed preventative therapy in adults with lymphoma. British Journal of Haematology 131: 13–21. Matthews GV, Bower M, Mandalia S, Powles T, Nelson MR, Gazzard BG (2000) Changes in acquired immunodeficiency syndrome-related lymphoma since the introduction of highly active antiretroviral therapy. Blood 96: 2730–4. Matutes E, Oscier D, Montalban C et al. (2008) Splenic marginal zone lymphoma proposals for a revision of diagnostic, staging and therapeutic criteria. Leukemia 22: 487–95.

Non-Hodgkin lymphoma

Montoto S, Davies AJ, Matthews J et al. (2007) Risk and clinical implications of transformation of follicular lymphoma to diffuse large B-cell lymphoma. Journal of Clinical Oncology 25: 2426–33. Morris E, Thomson K, Craddock C et al. (2004) Outcomes after alemtuzumab-containing reduced-intensity allogeneic transplantation regimen for relapsed and refractory non-Hodgkin lymphoma. Blood 104: 3865–71. Morschhauser F, Radford J, Van Hoof A et al. (2008) Phase III trial of consolidation therapy with yttrium-90-ibritumomab tiuxetan compared with no additional therapy after first remission in advanced follicular lymphoma. Journal of Clinical Oncology 26: 5156–64. Navarro WH, Kaplan LD (2006) AIDS-related lymphoproliferative disease. Blood 107: 13–20. Oksenhendler E, Boulanger E, Galicier L et al. (2002) High incidence of Kaposi sarcoma-associated herpesvirus-related non-Hodgkin lymphoma in patients with HIV infection and multicentric Castleman disease. Blood 99: 2331–6. Parker A, Bowles K, Bradley JA (2010) Diagnosis of post-transplant lymphoproliferative disorder in solid organ transplant recipients – BSCH and BTS guidelines. British Journal of Haematology 149: 675–92. Pfreundschuh M, Trumper L, Osterborg A et al. (2006) CHOP-like chemotherapy plus rituximab versus CHOP-like chemotherapy alone in young patients with good-prognosis diffuse large-Bcell lymphoma: a randomized controlled trial by the MabThera International Trial (MInT) Group. Lancet Oncology 7: 379– 91. Pfreundschuh M, Schubert J, Ziepert M et al. (2008) Six versus eight cycles of bi-weekly CHOP-14 with or without rituximab in elderly patients with aggressive CD20+ B-cell lymphomas: a randomized controlled trial (RICOVER-60). Lancet Oncology 9: 105–16. Rezvani AR, Storer B, Maris M et al. (2008) Nonmyeloablative allogeneic haematopoietic cell transplantation in relapsed, refractory, and transformed indolent non-Hodgkin lymphoma. Journal of Clinical Oncology 26: 211–17. Rodriguez J, Conde E, Gutierrez A et al. (2007) The results of consolidation with autologous stem-cell transplantation in patients with peripheral T-cell lymphoma (PTCL) in first complete remission: the Spanish Lymphoma and Autologous Transplantation Group experience. Annals of Oncology 18: 652–7.

Rohatiner AZ, Nadler L, Davies AJ et al. (2007) Myeloablative therapy with autologous bone marrow transplantation for follicular lymphoma at the time of second or subsequent remission: long-term follow-up. Journal of Clinical Oncology 25: 2554–9. Savage KJ, Harris NL, Vose JM et al. (2008) ALK− anaplastic largecell lymphoma is clinically and immunophenotypically different from both ALK+ ALCL and peripheral T-cell lymphoma, not otherwise specified: report from the International Peripheral T-Cell Lymphoma Project. Blood 111: 5496–504. Schulz H, Bohlius J, Skoetz N et al. (2007) Chemotherapy plus rituximab versus chemotherapy alone for B-cell non-Hodgkin lymphoma. Cochrane Database of Systematic Reviews CD003805. Sehn LH, Berry B, Chhanabhai M et al. (2007) The revised International Prognostic Index (R-IPI) is a better predictor of outcome than the standard IPI for patients with diffuse large B-cell lymphoma treated with R-CHOP. Blood 109: 1857–61. Thomson KJ, Morris EC, Bloor A et al. (2009) Favourable longterm survival after reduced-intensity allogeneic transplantation for multiple-relapse aggressive non-Hodgkin lymphoma. Journal of Clinical Oncology 27: 426–32. Tsukasaki K, Hermine O, Bazarbachi A et al. (2009) Definition, prognostic factors, treatment, and response criteria of adult T-cell leukaemia-lymphoma: a proposal from an international consensus meeting. Journal of Clinical Oncology 27: 453–9. van Oers MH, Klasa R, Marcus RE et al. (2006) Rituximab maintenance improves clinical outcome of relapsed/resistant follicular non-Hodgkin lymphoma in patients both with and without rituximab during induction: results of a prospective randomized phase 3 intergroup trial. Blood 108: 3295–301. Vidal L, Gafter-Gvili A, Leibovici L et al. (2009) Rituximab maintenance for the treatment of patients with follicular lymphoma: systematic review and meta-analysis of randomized trials. Journal of the National Cancer Institute 101: 248–55. Whittaker SJ, Marsden JR, Spittle M, Russell Jones R (2003) Joint British Association of Dermatologists and UK Cutaneous Lymphoma Group guidelines for the management of primary cutaneous T-cell lymphomas. British Journal of Dermatology 149: 1095–107. Willemze R, Jaffe ES, Burg G et al. (2005) WHO-EORTC classification for cutaneous lymphomas. Blood 105: 3768–85. Yeo W, Chan TC, Leung NW et al. (2009) Hepatitis B virus reactivation in lymphoma patients with prior resolved hepatitis B undergoing anticancer therapy with or without rituximab. Journal of Clinical Oncology 27: 605–11.

685

CHAPTER 36

36

Myeloproliferative neoplasms Peter J Campbell1 and Anthony R Green2 1

Cancer Genome Project, Wellcome Trust Sanger Institute, Cambridge, UK Cambridge Institute for Medical Research, Cambridge, UK

2

Introduction, 686 The polycythaemias, 686 Polycythaemia vera, 686 Other causes of erythrocytosis, 690 Essential thrombocythaemia, 693 Pathophysiology, 693 Clinical features, 694 Investigations, 694 Treatment, 696 Prognosis, 696 Essential thrombocythaemia and pregnancy, 696 Primary myelofibrosis, 697 Pathophysiology, 697

Clinical features, 697 Investigations, 698 Treatment, 699 Prognosis, 701 Mastocytosis, 701 Pathophysiology, 701 Clinical features, 702 Investigations, 702 Treatment, 702 Prognosis, 704 Clonal hypereosinophilic syndromes, 704 Pathophysiology, 704 Clinical features, 705

Introduction For the purposes of this chapter, the term ‘myeloproliferative neoplasm’ (MPN) refers to clonal disorders of haemopoiesis that lead to an increase in the numbers of one or more mature blood cell progeny. The chronic myeloid leukaemias would fit this definition and share pathogenetic features with some of the MPNs, but have historically (since the discovery of the Philadelphia chromosome) been studied separately from the MPNs and are described in another chapter. Myelodysplastic syndrome (MDS) can also, in a minority of cases, fit our working definition of MPN, in being associated with increased numbers of mature cell progeny, but dysplasia is a major feature and there are, usually, coexisting cytopenias. Not surprisingly, a small number of patients do not fit neatly into a single category and exhibit features of both MPN and MDS. This chapter focuses on the classical MPNs: polycythaemia (rubra) vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF), three related disorders originally grouped together by William Dameshek in 1951. They share clinical, morphological and molecular features and can transform, in their course, into one another. They are clonal disorders of the pluripotent haemopoietic stem cell and have, to varying degrees, the potential to transform into acute myeloid leukaemia (AML). Secondary (non-clonal) polycythaemias and Postgraduate Haematology: 6th edition. Edited by A. Victor Hoffbrand, Daniel Catovsky, Edward G.D. Tuddenham, Anthony R. Green © 2011 Blackwell Publishing Ltd.

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Investigations, 705 Treatment, 706 Prognosis, 706 Chronic neutrophilic leukaemia, 706 Pathophysiology, 706 Clinical features and treatment, 707 Neutrophilic chronic myeloid leukaemia, 707 Transient abnormal myelopoiesis of Down syndrome, 707 Incidence, clinical features and treatment, 707 Pathophysiology, 708 Selected bibliography, 708

thrombocytoses are also discussed in this chapter, as they often enter the differential diagnosis of their clonal counterparts. In addition, some of the less common MPNs are described, namely mastocytosis and its variants, the clonal eosinophilic syndromes and chronic neutrophilic leukaemia. The ontogeny of the target cell for transformation is less well established in these disorders, but there is accumulating evidence implicating the pluripotent haemopoietic stem cell in at least some cases.

The polycythaemias True polycythaemia refers to an absolute increase in total body red cell volume (or mass), which usually manifests itself as a raised haemoglobin concentration (Hb) and/or packed cell volume (PCV). A raised Hb (or PCV) can also be secondary to a reduction in plasma volume, without an increase in total red cell volume; this is known as apparent (or relative) polycythaemia. True polycythaemia is further subdivided into primary polycythaemia (namely PV), a clonal haematological disorder, and secondary polycythaemia, which results from an increased erythropoietin drive, either in the presence or absence of hypoxia (Figure 36.1).

Polycythaemia vera The central pathological feature of PV is an expansion in the total red cell mass, although elevations in the platelet and/or neutrophil counts are relatively common. The first description

Myeloproliferative neoplasms

of PV was by Vaquez in 1892. Osler, in 1903, published the first series of patients, identifying salient clinical features setting PV apart from other erythrocytoses. Considerable information has been gathered about PV since the work of these pioneers, much of it due to the work of the Polycythaemia Vera Study Group (PVSG), which was set up in 1967 with the aim of optimizing diagnosis and management of PV. The recent discovery of the V617F mutation in the pseudokinase domain of the tyrosine kinase JAK2, in nearly all cases of PV, represents a major advance in our understanding of this disorder. Pathophysiology PV is a stem cell disorder characterized by hyperplasia of all three major myeloid cell lineages. The first line of evidence in support of the stem cell origin of PV came in the form of clonality studies. Using X-chromosome inactivation patterns in the mid-1970s Fialkow and colleagues showed that neutrophils, erythrocytes and platelets originated from the same clone. Large studies have since confirmed these findings.

Raised Hb

Red cell mass estimation Raised

Not raised

True polycythaemia

Primary polycythaemia

Polycythaemia vera

Apparent polycythaemia

Secondary polycythaemia

Idiopathic erythrocytosis

Primary familial and congenital polycythaemia

Figure 36.1 An aetiological classification of polycythaemia.

FERM

Human Dog Mouse Rat Chicken

SH2

Erythropoiesis in PV is autonomous and does not rely on erythropoietin (Epo). Plasma levels of this hormone are reduced in PV patients, and PV progenitor cells, unlike normal ones, can survive in vitro and give rise to erythroid colonies (BFU-E) in the absence of added Epo (endogenous erythroid colonies). PV erythroid progenitors show increased sensitivity to Epo but also to several other growth factors, including insulin-like growth factor (IGF)-1, thrombopoietin, interleukin (IL)-3 and granulocyte/monocyte colony-stimulating factor. Germline mutations in the Epo receptor (EpoR) are known to occur in inherited polycythaemia, but such mutations are absent in patients with PV. In 2005, several groups identified a unique acquired mutation in the cytoplasmic tyrosine kinase JAK2 in myeloid cells from the great majority of patients with PV. JAK2 lies downstream of several cell surface receptors including EpoR. Upon Epo binding to EpoR, JAK2 becomes phosphorylated and in turn phosphorylates downstream targets, most important of which are the STATs (signal transducers and activators of transcription), leading to stimulation of erythropoiesis. Valine 617 is located in the JH2 domain of JAK2, which acts to repress its kinase activity (Figure 36.2). The V617F mutation leads to increased kinase activity, confers cytokine independence and results in erythrocytosis in a mouse transplant model. The mutation appears to be fairly specific to the classical MPNs and although it has been reported in small numbers of patients with related myeloid neoplasms, it is not present in lymphoid or non-haemopoietic cancers. Intriguingly, the mutation is homozygous in a large proportion of patients with PV and PMF but this is rare in ET. It appears that the moderate familial predisposition to MPNs can be largely attributed to an inherited haplotype block surrounding the JAK2 gene, although the mechanism underlying this interaction between somatic and germline genetics has not been identified. When appropriately sensitive methods are used for the detection of the V617F mutation, about 95% of PV patients are positive. Recently, mutations elsewhere in JAK2 have been described in most of the V617F-negative patients who have PV by strict diagnostic criteria. These mutations cluster in exon 12,

Val617Phe JH2

JH1

FFEAASMMSKLSHKHLVLNYGVCVCGDENILVQEFV FFEAASMMSQLSHKHLVLNYGVCVCGEENILVQEFV FFEAASMMSQLSHKHLVLNYGVCVCGEENILVQEFV FFEAASMMSQLSHKHLVLNYGVCVCGEENILVQEFV FFEAASMMSQLSHKYLVLNYGVCVCGEENILVQEYV

Figure 36.2 Diagrammatic representation of JAK2 indicating the location of valine 617 and the very high degree of cross-species amino acid homology in its JH2 domain. The JH2 domain

normally acts to repress the kinase activity of JAK2, but its ability to do so is impaired in the presence of the JAK2 V617F mutation. (Modified from Baxter et al. 2005 with permission.) 687

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and can be of several different types, but all seem to affect the pseudokinase domain, leading to constitutive activation of the JAK2 kinase. Interestingly, compared with PV patients with the V617F mutation, those carrying exon 12 mutations tend to be younger and have a more isolated erythrocytosis, with less frequent thrombocytosis and leucocytosis. The molecular basis for these differences in phenotype has not been identified. Recently, mutations in a novel tumour-suppressor gene, TET2, have been identified in myeloid disorders of all types, including MPNs. In particular, 20–25% of patients with MDS, 50% of patients with chronic myelomonocytic leukaemia, 12– 15% of patients with MPN and about 20% of patients with AML carry somatically acquired mutations of the gene. Little is known about how the gene functions in normal cells, nor how disruption of the gene promotes myeloid disorders. However, the mutation is found in early haemopoietic progenitors, can either pre-date or post-date JAK2 V617F mutation, and allows stable engraftment of haemopoietic cells in immunodeficient mice. Clinical features

Epidemiology The annual incidence of PV is reported to be around 2–3 per 100 000 population with a male to female ratio of 1.2 : 1. The median age at onset is 55–60 years and although incidence increases with age, PV can occur at any age even, rarely, in childhood. Thrombotic complications Thrombosis is the most common serious complication of PV. Untreated PV patients run a greatly increased risk of thrombosis, which can be arterial, venous or microvascular. The increased PCV leads to an increased blood viscosity, rheological abnormalities and abnormal platelet–endothelial contact. Additionally, procoagulant changes in platelets (e.g. decreased response to prostaglandin D2), thrombocytosis and pre-existing vascular disease can all conspire to dramatically increase thrombotic risk. Arterial occlusions can lead to myocardial infarcts, strokes, transient ischaemic attacks, amaurosis, scotomata, and mesenteric and limb ischaemia. Less commonly, microvascular occlusions affecting the extremities and erythromelalgia can occur. In the venous circulation, unusual sites such as the splanchnic vessels can be involved. As a result, mesenteric, splenic and hepatoportal thromboses (Figure 36.3) are recognized presenting features of PV. Recent data indicate that this propensity to venous thrombosis in atypical sites is particularly strongly correlated with presence of the JAK2 V617F mutation, and indeed patients presenting with otherwise unexplained splanchnic vein thrombosis will often be found to have the mutation, even in the absence of an overt MPN. Superficial thrombophlebitis, 688

Figure 36.3 Polycythaemia vera presenting with Budd–Chiari syndrome in a 28-year-old man: contrast computed tomography showing reduced enhancement of the right lobe of the liver (arrow) with characteristic sparing of the caudate lobe (arrowhead). On this occasion, the left lobe was also relatively spared. Marked ascites and a bulky spleen are also seen.

conventional deep venous thromboses and pulmonary emboli are also seen.

Neurological features Over and above the consequences of occlusive vascular lesions, the sluggish cerebral blood flow secondary to the increased PCV is thought to underlie features such as headaches, drowsiness, insomnia, amnesia, tinnitus, vertigo, chorea and even depression. Transient visual disturbances also occur. Pruritus This symptom occurs in about one-quarter of PV patients and in some it may be severe. It is characteristically aquagenic, precipitated by warm baths, and can be associated with erythema, swelling or even pain. Pruritus is often relieved by controlling the PCV, but its aetiology remains elusive. Basophilia, hyperhistaminaemia and iron deficiency may have a role and there is an increased incidence in patients with a lower mean corpuscular volume. Skin Plethora, dilated conjunctival vessels and rosacea-like facial skin changes are not uncommon at presentation. Brown discoloration of the skin, erythromelalgia and, rarely, Sweet syndrome may be seen. Splenomegaly Palpable splenomegaly is seen in 30–50% of cases of PV. It is unclear if its presence affects prognosis, but it may be associated with an increased risk of progression to myelofibrosis.

Myeloproliferative neoplasms

Hypertension and gout Hypertension is probably more common in patients with PV as is hyperuricaemia, with gout seen in about 5% of cases. Leukaemic transformation This is perhaps the most feared complication of PV, but the risk of developing acute leukaemia in PV patients treated only with venesection is very small (1–3%). However, this risk increases dramatically (more than 10-fold) when radioactive phosphorus (32P), chlorambucil or irradiation are used as treatment. The median time interval between first starting such therapy and developing acute leukaemia is 5–8 years. Interestingly, in about half of patients transforming to acute leukaemia from a preceding JAK2-positive MPN, the leukaemia is JAK2 negative. Myelofibrosis Progression to myelofibrosis (Figure 36.4) occurs in around 10–20% of PV cases at 15 years after diagnosis. This figure is approximate, not least because different studies have used distinct criteria to define myelofibrotic transformation. Transformation often occurs gradually over many years and is thought to be associated with an increased risk of leukaemic conversion. The management of these patients is similar to that of PMF. Investigations The diagnosis of PV requires both the identification of features in support of this diagnosis and the exclusion of secondary and apparent polycythaemia. The original set of diagnostic criteria was formulated by the PVSG in the 1970s. The British Committee for Standards in Haematology (BCSH) guidelines

for diagnosing PV, taking into account the latest advances in the molecular understanding of these disorders, are shown in Table 36.1. Most patients can now be diagnosed on the basis of a raised haematocrit together with the presence of the JAK2 V617F mutation. It is also important to emphasize that the V617F mutation has been identified in a small proportion of other hematological malignancies (e.g. AML, chronic myelomonocytic leukaemia and myelodysplasia). However, none of these disorders is associated with a raised red cell mass and clinical distinction from PV is rarely an issue. The role of bone marrow biopsy in the evaluation of PV remains controversial, but it may be useful as a baseline investigation for later comparison in younger patients with the disorder, or if there are atypical clinical and laboratory features at presentation. A minority of patients with clinical PV is negative for the V617F mutation, even when tested using sensitive detection methods. Many reference laboratories now offer highly sensitive molecular testing for JAK2 exon 12 mutations, and this should be considered in patients who have a typical clinical and laboratory presentation of PV but who are negative for the V617F mutation. Rarely, patients will be negative for both Table 36.1 Diagnostic criteria for polycythaemia vera, as recommended in the BCSH guidelines. JAK2-positive polycythaemia vera A1 High haematocrit (>0.52 in men, >0.48 in women) or raised red cell mass (>25% above predicted)* A2 Mutation in JAK2 Diagnosis requires both criteria to be present JAK2-negative polycythaemia vera A1 Raised red cell mass (>25% above predicted) or haematocrit >0.60 in men, >0.56 in women. A2 Absence of mutation in JAK2 A3 No cause of secondary erythrocytosis A4 Palpable splenomegaly A5 Presence of an acquired genetic abnormality (excluding BCR–ABL1) in haemopoietic cells B1 Thrombocytosis (platelet count >450 × 109/L) B2 Neutrophil leucocytosis (neutrophil count >10 × 109/L in non-smokers, >12.5 × 109/L in smokers) B3 Radiological evidence of splenomegaly B4 Endogenous erythroid colonies or low serum erythropoietin Diagnosis requires A1 + A2 + A3 + either another A or two B criteria

Figure 36.4 Blood film from a case of post-polycythaemic myelofibrosis after splenectomy. Note the presence of nucleated erythrocytes, giant platelets and features of splenectomy including target cells, spherocytes and acanthocytes.

*Dual pathology (coexistent secondary erythrocytosis or relative erythrocytosis) may rarely be present in patients with a JAK2positive MPN. In this situation, it would be prudent to reduce the haematocrit to the same target as for polycythaemia vera. 689

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V617F and exon 12 JAK2 mutations. In these patients, the diagnosis of PV can be made if the other criteria set out in Table 36.1 are met, but it should be remembered that this group of truly JAK2-negative PV is very rare, and secondary erythrocytosis, idiopathic erythrocytosis and relative erythrocytosis are all much more likely diagnoses in this clinical setting. Treatment Identification of the JAK2 V617F mutation has generated much interest in the development of therapeutic JAK2 inhibitors. However, as patients with PV currently have a very good prognosis, new agents will have to display an excellent safety profile. For the time being, the treatment of PV should employ existing treatment modalities whose effectiveness has been validated, as described below. In the absence of thrombocytosis, regular venesection remains the mainstay of treatment for PV in patients who can tolerate it. A target haematocrit of 0.45 or less is widely used, following the demonstration that in patients with PV, higher haematocrits are associated with a significantly increased risk of thrombosis. Venesection has little impact on the haematocrit in the short term: the purpose of regular venesection is to induce iron deficiency such that the haematocrit remains chronically below the target threshold of 0.45. Thus, typical venesection regimens start with phlebotomy every 2–3 weeks until the haematocrit is controlled, and thereafter phlebotomy is generally needed every 1–3 months, depending of factors such as dietary iron intake and erythropoietic activity. Cytoreductive therapy is recommended for patients unable to undergo venesection and those with thrombocytosis, in view of the probable increased risk of thrombosis. Hydroxycarbamide (hydroxyurea) is the most commonly used drug. It is orally bioavailable and generally very well tolerated. It will reduce both the haematocrit and the platelet count. The commonest complications are leucopenia or thrombocytopenia, which are dose dependent and can usually be avoided by close monitoring of the blood count when the drug is first introduced. In susceptible patients it can cause photosensitivity, painful leg ulcers and gastrointestinal side-effects. The usual dose is 0.5–2 g daily. It has been suggested that hydroxycarbamide may increase the inherent leukaemogenic risk associated with PV. This concern is largely based on studies involving small numbers of patients or patients who have also required other cytotoxic agents (and may therefore represent a subgroup with more aggressive disease). At present, there are no convincing data to show that hydroxycarbamide, when used as a single agent, significantly increases the risk of leukaemia, although a small effect cannot be excluded. Preliminary data from studies of hydroxycarbamide in sickle cell disease are reassuring, but longer follow-up is required. Interferon alfa is effective in controlling both the platelet count and the PCV, and there are some data that it may induce impressive reductions in the burden of V617F-positive cells in 690

the blood. It is not widely used because of its cost, route of administration (subcutaneous injection) and its side-effects (including fatigue, flu-like symptoms and depression). However, it can be useful in young patients who are reluctant to take other cytotoxic agents, in pregnancy and in patients with intractable pruritus. The usual dose range is 3–5 million units three times per week. Anagrelide is a newer agent that lowers the platelet count by inhibiting megakaryocyte differentiation. It can be useful in controlling the platelet count of patients being treated with venesection and can also be combined with hydroxycarbamide to allow the use of lower doses of both agents. Approximately 10% of patients are completely refractory to anagrelide. The usual dose is 1–2 mg daily, but occasional patients may require doses of up to 8 mg daily. Its side-effects are mainly secondary to its inotropic and vasodilatory properties and include headaches, palpitations and fluid retention. Busulfan is sometimes used in elderly patients or when all other treatments are not tolerated. It is very convenient as it need only be administered intermittently, but may increase the risk of leukaemia. The usual dosage is 25–75 mg as a single dose every 2–3 months. Low-dose aspirin (75–100 mg daily) reduces thrombotic complications in PV and is used in most patients without contraindications to this drug. Pruritus often improves with control of the PCV but paroxetine, antihistamines and aspirin (in some cases) can help. There is also evidence that interferon can be useful in intractable cases. In view of the age of most patients and the relatively benign natural history of treated PV, bone marrow transplantation is not advocated for stable disease. The role, if any, of transplantation employing reduced-intensity conditioning regimens is not yet clear. Prognosis In the first half of the 20th century, untreated polycythaemia had a dismal prognosis with a 50% survival of less than 2 years. However, adequately treated PV now has a relatively benign natural history with a life expectancy of over 11 years, bearing in mind that the average age of onset is 60 years. Factors predictive of poorer prognosis and increased complication rates in PV include JAK2 mutation burden and white cell count at diagnosis, both factors probably serving as surrogate markers of disease activity.

Other causes of erythrocytosis All disorders with an increased red cell mass that are not due to clonal proliferation of haemopoietic progenitors are included under this heading. They are most conveniently subclassified into primary and secondary causes. In primary polycythaemia, the defect is intrinsic to the red cell precursors, which are hypersensitive to Epo. In secondary polycythaemia, the defect is upstream of the red cell precursors. The latter group can be

Myeloproliferative neoplasms

Table 36.2 Causes of erythrocytosis other than polycythaemia vera. Primary erythrocytosis Primary familial and congenital polycythaemia Secondary erythrocytosis Erythrocytosis in the presence of systemic hypoxia Chronic hypoxia Lung disease Hypopnoea High altitude Congenital cyanotic heart disease Defective oxygen transport High-affinity haemoglobins Red cell metabolic defects (low 2,3-DPG) Methaemoglobinaemia Heavy smoking (carboxyhaemoglobinaemia) Erythrocytosis in the absence of systemic hypoxia Inherited/congenital erythrocytosis Chuvash polycythaemia Inherited mutations in EPOR and oxygen signalling Abnormal erythropoietin secretion Renal tumours Cystic/polycystic kidney disease Renal transplantation Renal hypoxia (e.g. renal artery stenosis) Cerebellar haemangioma Hepatocellular carcinoma Endocrine syndromes Administration of androgenic steroids Cushing syndrome Conn syndrome Phaeochromocytoma (rarely) Bartter syndrome Idiopathic erythrocytosis

further subdivided into polycythaemias in the presence or absence of systemic hypoxia. A small group of patients do not fall into any of these categories and are given the diagnosis of idiopathic erythrocytosis (Table 36.2). The clinical management of many of these syndromes is not well defined. The term ‘apparent polycythaemia’ refers to a raised haematocrit in the absence of a raised red cell mass and is discussed later in this chapter. Primary erythrocytosis

Inherited/congenital erythrocytosis Chuvash polycythaemia is an autosomal recessive condition that is endemic in the Russian mid-Volga river region of

Chuvashia. Patients have increased levels of circulating Epo but do not carry mutations of EpoR. Chuvash polycythaemia was recently shown to be associated with a C→T mutation at nucleotide 598 (leads to an Arg200Trp substitution) in the von Hippel–Lindau (VHL) gene. The VHL gene is pivotal for ubiquitination and subsequent degradation of HIF-1 transcription factor, which is central to the oxygen-sensing pathway. This VHL mutant leads to a reduced rate of degradation of HIF-1 and upregulation of downstream targets including Epo, leading to polycythaemia. Recently, some of the rare non-Russian families with inherited polycythaemia were also shown to carry VHL mutations, and occasional pedigrees with mutations elsewhere in the oxygen-sensing pathway have been described, including the PHD2 and HIF2A genes. Primary familial and congenital polycythaemia (PFCP) is a rare disorder in which erythropoiesis is intrinsically overactive. The disorder is usually transmitted in an autosomal dominant manner with some cases appearing sporadically. Clinical features include the presence of isolated erythrocytosis without evolution into leukaemia or other MPNs, absence of splenomegaly, normal white blood cell and platelet counts, low or normal plasma Epo levels, normal haemoglobin–oxygen dissociation curve/P50, and hypersensitivity of erythroid progenitors to Epo. Mutations in the gene encoding EpoR have been described in several (but not all) families with PFCP. In most cases, the mutations lead to a C-terminal truncation of the EpoR protein, with increased sensitivity to Epo. Secondary erythrocytosis

Erythrocytosis in the presence of systemic hypoxia Chronic lung disease and hypopnoea Lung disease is the predominant cause of chronic systemic hypoxia at sea level. Hypoxaemic chronic obstructive pulmonary disease is the commonest syndrome, but any lung–airway disease leading to chronic hypoxia could cause polycythaemia. Syndromes such as obstructive sleep apnoea and hypoventilation due to muscle weakness or paralysis can also occasionally be associated with secondary erythrocytosis. Where possible, hypoxia should be ameliorated by treating the lung disease or with home oxygen therapy. Erythrocytosis has opposing effects on oxygen delivery as it increases the oxygen-carrying capacity while also increasing blood viscosity. Unfortunately, there is little evidence from clinical trials to guide management. In practice, many specialists suggest that venesection should be performed for haematocrits above 0.55. High altitude Residents at altitudes above 4000 m compensate for the ambient hypoxia by multisystem adaptation, including mild erythrocytosis, increase in capillary perfusion and lung diffusion capacity as well as biochemical changes in metabolic enzymes and myoglobin. Excessive altitude polycythaemia (PCV ≥ 0.65) is 691

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seen in a proportion of cases and is often accompanied by hyperuricaemia and proteinuria. Some of these individuals eventually decompensate and develop chronic mountain sickness (Monge disease). Such people deteriorate steadily and develop extreme polycythaemia (sometimes PCV ≥ 0.75), arterial desaturation and right heart failure. Resettlement at lower altitudes halts disease progression and can partly reverse it. Treatment with angiotensin-converting enzyme (ACE) inhibitors has been shown in randomized studies to reduce the PCV and proteinuria seen in excessive altitude polycythaemia. Congenital cyanotic heart disease Congenital heart defects leading to a right-to-left shunt can cause dramatic erythrocytosis (up to PCV of 0.80). Surgery to correct the cardiac defect should be undertaken when possible. A few inoperable patients survive to adulthood and the management of their erythrocytosis is not straightforward. As in patients with chronic lung disease, the increase in oxygen-carrying capacity afforded by erythrocytosis is countered by an increased viscosity and associated haemodynamic changes. Here, however, we are often dealing with young patients with responsive vasculatures, which can usually accommodate such changes. Some experts advocate venesection when the PCV is greater than 0.60, whereas others allow the haematocrit to rise further and venesect for symptoms such as recurrent haemoptysis, marked fatigue or deteriorating exercise tolerance. High-affinity haemoglobins High-affinity haemoglobins release less oxygen for a given oxygen partial pressure, and may thus give rise to tissue hypoxia. This leads to an Epo-driven erythrocytosis, which tends to renormalize Epo levels. The pathognomonic anomaly is a left shift in the oxygen dissociation curve (Figure 36.5). The precise

Oxygen saturation (%)

1.0 Hb San Diego 0.8 0.6

HbA

0.4 0.2 0 0

50 100 % Oxygen partial pressure (torr)

150

Figure 36.5 Haemoglobin–oxygen dissociation curves from a 27-year-old man with a raised red cell mass and a normal person (HbA) showing the presence of a high-affinity haemoglobin (‘left shift’). Mass spectrometric analysis showed the man to be heterozygous for Hb San Diego, a high-affinity β-chain variant. 692

variant can be identified by mutational screening of DNA or by protein mass spectrometry. There are over 40 haemoglobin variants with an increased affinity for oxygen, all dominantly inherited. Most are due to mutations in β-globin, with a small number due to mutations in α-globin. Mutations are clustered in regions of the globin chains involved in the regulation of the transition between tense (T) and relaxed (R) states of haemoglobin. Normally, oxy-HbA is in the T state and has low affinity for oxygen, and deoxy-HbA is in the R state and has high affinity for oxygen. Thus, mutations at the αβ contact site (e.g. Hb San Diego), the C-terminus (e.g. Hb Bethesda) and the 2,3-diphosphoglycerate (2,3-DPG)-binding site (e.g. Hb Helsinki) are the commonest. Most people with a high-affinity haemoglobin are in good health and are either diagnosed coincidentally or after being noticed to be plethoric. Some experience excessive muscle fatigue after vigorous exercise. Hyperviscosity is rarely a problem and surveys have failed to identify increased cardiovascular morbidity or mortality. Pregnancy is not adversely affected, even in mothers with haemoglobin affinity that exceeds that of HbF. Generally, therefore, no management is required, unless symptomatic. Red cell metabolic defects Very rare cases of erythrocytosis are due to abnormalities in red cell metabolism that lead to a reduction in intraerythrocytic 2,3-DPG. The best characterized defect is a mutant 2,3-DPG mutase. The only well-characterized family with this disorder showed autosomal recessive inheritance, although heterozygous family members had a decreased P50 and, in some cases, a moderate erythrocytosis. This disorder is excluded by the finding of a normal P50 in a fresh blood sample. Methaemoglobinaemia Oxidation of haem iron converts it from its normal ferrous (Fe2+) to the ferric (Fe3+) form and, correspondingly, haemoglobin (HbA) becomes methaemoglobin (Met-HbA). This constitutes an important antioxidant mechanism for the red cell, and conversion of Met-HbA back to HbA requires the generation of NADH from glycolysis. The rate of HbA auto-oxidation is about 20 times slower under normal circumstances than the rate of Met-HbA reduction, thus preventing Met-HbA accumulation. Methaemoglobin has an increased affinity for oxygen and a left-shifted oxygen dissociation curve. Pathological acquired methaemoglobinaemia can result from exposure to strong oxidants (e.g. dapsone, paraquat, benzocaine) and can be lifethreatening when severe but is rarely sufficiently long-lived to give rise to polycythaemia. Hereditary methaemoglobinaemias can be due to haemoglobin mutations involving amino acids around the haem pocket (HbM disease), or secondary to enzymatic deficiencies

Myeloproliferative neoplasms

that interfere with the generation of NADH, which is required for day-to-day methaemoglobin reduction (namely NADH reductase and cytochrome b5 reductase). In view of the chronic nature of the methaemoglobinaemia in these disorders, secondary erythrocytosis can develop in the same manner as for highaffinity haemoglobin mutants. Heavy smoking Heavy smoking can lead to mild polycythaemia in the absence of hypoxic lung disease. The underlying cause of this is a raised carboxyhaemoglobin (CO-Hb) level resulting from chronically raised carbon monoxide (CO) levels. CO-Hb levels in urban dwelling non-smokers are rarely higher than 2%, with levels ranging from 3 to 20% in smokers. The short half-life of CO in the body (3–5 hours) leads, in smokers, to a rise in CO-Hb during the day and a fall during sleep, making it difficult to compare measurements taken at different times. Binding of CO to Hb, as well as displacing oxygen, leads to a conformational change similar to that seen in methaemoglobinaemia, with a similar left shift in the oxygen dissociation curve and a fall in P50.

Erythrocytosis in the absence of systemic hypoxia Abnormal erythropoietin secretion Abnormal Epo secretion is a well-recognized cause of secondary erythrocytosis and is most commonly secondary to renal pathologies such as renal tumours (benign and malignant), polycystic kidney disease and diseases associated with local hypoxia such as renal cysts, hydronephrosis and renal artery stenosis. The polycythaemia usually responds to treatment of the underlying renal pathology. Erythrocytosis occurs in 20–30% of patients after renal transplantation. The biggest risk to such patients is hypertension, strokes and cardiovascular complications. In many cases, the erythrocytosis and associated hypertension respond to ACE inhibitors. Theophylline may also be effective in some cases. Patients who remain polycythaemic despite such therapy should be treated with repeated venesection to maintain their haematocrit below 45%; 30–40% of cases resolve spontaneously. Non-renal tumours can rarely be associated with polycythaemia. The commonest reported ones are hepatocellular carcinoma, cerebellar and other haemangiomas and large uterine fibromyomas. Polycythaemia responds to removal of the tumour in most of these cases. Endocrine disorders The mechanism underlying the development of polycythaemia in most endocrine disorders lies in the overproduction of androgens, which can produce polycythaemia by increasing Epo levels and also, probably, through a direct action on bone marrow progenitors.

Idiopathic erythrocytosis In a small proportion of patients with polycythaemia the criteria for the diagnosis of PV are not met and no other aetiology for the raised red cell mass can be identified. This group is heterogeneous and likely to include patients with germline mutations causing polycythaemia as well as some that will go on to develop overt PV. With the advent of increasingly sophisticated diagnostic tests and the identification of the molecular lesions in many inherited forms of polycythaemia, idiopathic erythrocytosis is becoming a rare entity. Apparent polycythaemia Apparent polycythaemia refers to a raised PCV in the presence of a normal red cell volume (< 25% above the predicted mean normal value). The raised haematocrit is due to a reduction in the plasma volume. Smoking, hypertension, obesity, excessive alcohol and diuretic therapy have all been associated with apparent polycythaemia. Pathogenesis is uncertain and almost certainly heterogeneous. It is not clear whether apparent polycythaemia is associated with increased rates of thrombosis, but it seems sensible to encourage affected individuals to avoid known predisposing factors. There are no convincing data that routine venesection is beneficial.

Essential thrombocythaemia The fundamental pathological feature of ET is a persistent elevation in the platelet count. However, ET has been poorly understood largely because of a lack of positive diagnostic criteria together with the fact that cases labelled as ET are likely to be pathogenetically heterogeneous. In 1934 Epstein and Goedel first described a patient with persistent elevation of the platelet count in association with megakaryocyte hyperplasia and tendency for venous thromboses and haemorrhage. Subsequently, Ozer and Gunz independently described two series of patients in 1960, thus confirming ET as a specific clinical entity. The recent discovery that approximately half the cases of ET carry the JAK2 V617F mutation, as do half the cases of PMF and nearly all cases of PV, has enhanced our understanding of the relationship between the three disorders.

Pathophysiology X-chromosome inactivation patterns provided the first evidence that ET may be a clonal stem cell disorder involving granulocytes, platelets and red cells but not T cells. For about 60% of patients a molecular basis for the clonality can be found, with about 55% of ET patients positive for the JAK2 V617F mutation, and a further 5% carrying activating mutations near the transmembrane domain of the thrombopoietin receptor gene MPL. 693

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Clinical and pathological features vary significantly between patients with ET, suggesting that the disease is heterogeneous. Prospective data from over 800 patients with ET has demonstrated that the presence or absence of the JAK2 V617F mutation divides ET into two biologically distinct disorders. Mutation-positive ET exists along a continuum with PV as it displays multiple features of the latter, with significantly increased haemoglobin levels, neutrophil counts and bone marrow erythropoiesis, more venous thromboses and a higher incidence of polycythaemic transformation. Mutation-negative patients do nonetheless exhibit many clinical and laboratory features characteristic of an MPN including the presence of endogenous erythroid colonies and a risk of transformation to acute leukaemia. Patients with MPL-positive ET appear to have a phenotype that more closely resembles JAK2-negative ET, with an isolated thrombocytosis and less hypercellular bone marrow.

Clinical features Epidemiology The annual incidence of ET is similar to that of PV at around 1.5–2.0 cases per 100 000 population. The median age at onset is 50–55 years and although it can occur at any age, it is rare in childhood. Thrombotic complications As with PV, thrombotic complications are the main cause of morbidity and mortality in ET. Thromboses are present in around 15–20% of patients at presentation and may be arterial or venous. The range of clinical syndromes is similar to PV, but the frequency of splanchnic thromboses is probably lower and strongly correlated with presence of the JAK2 V617F mutation. A number of risk factors are associated with an increase in the risk of thrombosis in patients with ET. The best characterized are age over 60 years and a prior history of thrombosis. Other risk factors for thrombosis in ET are likely to include diabetes, hyperlipidaemia, hypertension and cigarette smoking. More recently, it has been reported that white cell count and reticulin levels at diagnosis have predictive value for thrombosis (and indeed other complications) in ET. The exact role these and other predictors have in individualizing treatment regimens remains unclear. Haemorrhagic complications Bleeding is less common and less well studied than thrombosis in ET, but can be dramatic when it happens. Efforts to correlate the thrombotic risk with platelet function abnormalities have generally been fruitless and this investigation is also unable to predict haemorrhagic risk. However, bleeding is more common in patients with platelet counts above 1000 × 109/L and, in at least some cases, this is due to acquired von Willebrand 694

disease, with a decrease in circulating high-molecular-weight multimers caused by adsorption to the surface of the excessive platelets. Splenomegaly and hyposplenism Splenomegaly is present in about 5% of ET patients at diagnosis and is rarely more than mild. Progressive enlargement of the spleen during the course of ET should raise suspicion of evolving myelofibrosis. It has been suggested that, over time, some patients with ET develop splenic atrophy secondary to silent microinfarcts in the splenic microcirculation. However, frank hyposplenism and its complications are rare. Transformation to myelofibrosis or polycythaemia vera Transformation to myelofibrosis and, more rarely, to PV are recognized complications of ET. Some, but not all, cases of apparent polycythaemic transformation may represent resolution of prior iron deficiency, as can happen with iron supplementation and after the menopause. The insidious onset of myelofibrotic transformation and the reluctance to serially study bone marrow trephine biopsies have hampered attempts to define its nature and frequency. Nonetheless, recent studies have shown that progression of reticulin levels over time shows extensive interindividual variability and can be influenced by choice of therapy, being more marked in patients treated with anagrelide (see below). From the available data, myelofibrotic transformation occurs in less than 10% and polycythaemic transformation in less than 1–2% of ET patients over 10 years. Leukaemic transformation ET can evolve into MDS or AML even in untreated cases, but only rarely. The presence of cytogenetic abnormalities and treatment with alkylating agents increase this risk. Approximately 3% of patients treated with hydroxycarbamide alone develop MDS or AML if followed for a median time of 8 years. As with PV, there are no data to demonstrate that hydroxycarbamide as a single agent significantly increases the risk of leukaemia inherent to this disease, but a small effect cannot be excluded.

Investigations The lack of pathognomonic features and the existence of many other causes of a raised platelet count have posed significant hurdles in the diagnosis of ET. The identification of the JAK2 V617F mutation now provides a very useful positive diagnostic criterion for approximately 50% of ET patients. However, for V617F-negative patients, ET remains a diagnosis of exclusion and can only be made after other clonal blood disorders and reactive thrombocytosis have been ruled out. A proposed diagnostic schema for ET, as outlined by the BCSH guidelines, is given in Table 36.3.

Myeloproliferative neoplasms

Table 36.3 Diagnostic criteria for essential thrombocythaemia as recommended in the BCSH guidelines. A1 A2 A3 A4 A5

Sustained platelet count > 450 × 109/L Presence of an acquired pathogenetic mutation (e.g. in the JAK2 or MPL genes) No other myeloid malignancy, especially PV*, PMF†, CML‡ or MDS§ No reactive cause for thrombocytosis and normal iron stores Bone marrow aspirate and trephine biopsy showing increased megakaryocyte numbers displaying a spectrum of morphology with predominant large megakaryocytes with hyperlobated nuclei and abundant cytoplasm. Reticulin is generally not increased (grades 0–2/4 or grade 0/3)

Diagnosis requires A1–A3 or A1 + A3–A5 *Excluded by a normal haematocrit in an iron-replete patient. Indicated by presence of significant bone marrow fibrosis (≥2/3 or 3/4 reticulin) and palpable splenomegaly, blood film abnormalities (circulating progenitors and teardrop cells) or unexplained anaemia. ‡ Excluded by absence of BCR–ABL1 fusion from bone marrow or peripheral blood. § Excluded by absence of dysplasia on examination of blood film and bone marrow aspirate. †

An alternative set of diagnostic criteria, based largely around salient bone marrow morphological features, have been proposed as part of the WHO classification of tumours. Included in these is the concept of ‘prefibrotic myelofibrosis’, a putative group of patients previously labelled as ET who reportedly have increased rates of transformation to myelofibrosis over time. These proposals remain highly controversial in the MPN literature. In particular, studies of interobserver reliability for the histological component of this classification have shown it to be poorly reproducible, and a prospective multicentre study of the prognostic discrimination achieved by such a label found it to be minimal. Nevertheless, bone marrow histological features such as giant multilobated megakaryocytes and megakaryocyte clustering (Figure 36.6) can be of value in making the diagnosis of ET. Reactive thrombocytosis Thrombocytosis is most commonly reactive and secondary to increased levels of circulating cytokines that stimulate thrombopoiesis. Inflammatory, vasculitic and allergic disorders, acute and chronic infections, malignancies, haemolysis, iron deficiency and blood loss can all lead to an increased platelet count (Table 36.4). Reactive thrombocytosis can sometimes be marked and, occasionally, the platelet count can be greater than

Table 36.4 Causes of a reactive thrombocytosis. Iron deficiency Blood loss (acute or chronic) Hyposplenism/splenectomy Surgery Acute bacterial infection Pneumonia, septicaemia, meningitis, diverticular abscess, etc. Chronic inflammation Vasculitides Inflammatory bowel disease Connective tissue disorders Rheumatoid arthritis Chronic infections Malignancies Rebound thrombocytosis Following treatment of immune thrombocytopenic purpura Recovery from chemotherapy Drugs Vincristine

Figure 36.6 Bone marrow trephine section (haematoxylin and eosin, H&E) from a 60-year-old man with essential thrombocythaemia. Note the hypercellularity and marked increase in megakaryocyte numbers, consisting largely of clusters of mature multilobated forms.

1000 × 109/L. There is usually evidence of ongoing inflammation in the form of a raised erythrocyte sedimentation rate or C-reactive protein but this is not always the case. Other clonal thrombocytoses A number of other haematological malignancies can be associated with thrombocytosis. Chronic myeloid leukaemia (CML) can be excluded by demonstrating the absence of the 695

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BCR–ABL1 fusion transcript. Iron deficiency can mask the typical raised red cell mass of PV, although the clinical utility of distinguishing iron-deficient PV from ET is probably minimal. Established PMF can be excluded by the absence of significant bone marrow fibrosis and other characteristic laboratory features such as leucoerythroblastic blood film and splenomegaly. Lastly, MDS can also be associated with a thrombocytosis in a minority of cases, but there are usually coexisting cytopenias, dysplastic features or specific cytogenetic abnormalities (e.g. deletion 5q). Sometimes, refractory anaemia with ringed sideroblasts can be associated with thrombocytosis and the presence of the JAK2 V617F mutation; this illustrates the overlap among these chronic myeloid disorders.

Treatment When considering the management of patients with ET, it is helpful to stratify them into risk groups, according to their risk of vascular complications, and take treatment decisions on this basis. Patients may be assigned to a high-, intermediate- or lowrisk category. High-risk patients High-risk patients are those over 60 years old or those with one or more high-risk features, i.e. a platelet count of 1500 × 109/L or more, a prior history of thrombosis or significant thrombotic risk factors such as diabetes or hypertension. In high-risk patients, control of the platelet count with hydroxycarbamide reduces thrombotic events compared with no cytoreductive therapy. The MRC PT-1 trial has recently compared treatment with hydroxycarbamide plus low-dose aspirin to anagrelide plus low-dose aspirin. Patients receiving anagrelide plus aspirin were significantly more likely to reach the composite primary end point (arterial thrombosis, venous thrombosis or major haemorrhage) and more likely to discontinue their allocated treatment. Compared with hydroxycarbamide plus aspirin, treatment with anagrelide plus aspirin was associated with a significantly increased rate of arterial thrombosis, major haemorrhage and myelofibrotic transformation, but a decreased rate of venous thromboembolism. These results suggest that hydroxycarbamide plus aspirin should remain first-line therapy for high-risk patients. Anagrelide is a useful second-line agent but the decision to use concurrent aspirin should depend on the relative risks of arterial thrombosis and haemorrhage in the individual patient. Analysis of patients from the PT-1 trial according to JAK2 status has shown that compared with V617F-negative patients, mutation-positive patients share many features with PV including a higher risk of venous thrombosis. Moreover, V617Fpositive patients were more sensitive to hydroxycarbamide than anagrelide, raising the possibility that hydroxycarbamide is particularly effective in these patients. 696

Interferon alfa can give good control of the platelet count in ET, but as discussed under PV, its significant side-effect profile, subcutaneous administration and cost prevent its widespread use. It has a clearer role in the management of ET in pregnancy (see later) and some favour it in young patients. Busulfan can achieve good control of the platelet count but is only rarely used because of concerns over its long-term leukaemogenic potential. This is true for other alkylating agents and for radioactive phosphorus (32P). Intermediate-risk patients Intermediate-risk patients are those between 40 and 60 years old who lack any of the high-risk features listed above. It is not clear whether it is beneficial to lower the platelet count in this group. Most receive either aspirin alone, or hydroxycarbamide and aspirin. Low-risk patients Low-risk patients are those younger than 40 years old who lack any high-risk features. Low-risk patients are usually given lowdose aspirin alone unless there is a contraindication such as previous peptic ulceration or allergy to salicylates. Antiplatelet agents such as dipyridamole or clopidogrel should be considered in these cases. In the setting of ET-associated haemorrhage, it is probably best to avoid antiplatelet agents.

Prognosis Few studies have directly addressed survival in ET and these have reached different conclusions. Some suggest that mortality at 10 years is that of age-matched controls, whereas others found it to be worse. In high-risk patients, hydroxycarbamide reduces vaso-occlusive events from 10.7 to 1.6 per 100 patient-years.

Essential thrombocythaemia and pregnancy ET is the MPN encountered most frequently in women of childbearing age. In pregnancy, the commonest complication of ET is first-trimester miscarriage, which occurs in up to 30% of pregnancies. This is thought to be secondary to placental microinfarcts and insufficiency. The reported increased incidence of antiphospholipid antibodies in ET may contribute to this. Other less frequent complications include intrauterine death, growth retardation, premature delivery and pre-eclampsia. The risk of maternal thrombosis and haemorrhage is higher than in normal pregnancy, although a successful outcome (live birth) is achieved in around 60% of cases and no maternal deaths were seen in a recent review of 220 pregnancies. The optimal management of ET in pregnancy has not yet been fully defined. There is conflicting evidence about the effectiveness of aspirin but, given the good documentation of its safety in a large unrelated study of pre-eclampsia, it should

Myeloproliferative neoplasms

probably be given to most patients who are pregnant or planning a pregnancy. Pregnancies deemed at high risk of thrombosis either on the basis of a prior history of thrombosis or a platelet count over 1500 × 109/L should be considered for therapy with a combination of interferon and aspirin. Hydroxycarbamide and anagrelide should not be used on account of their teratogenic potential. In addition to lowering the platelet count, cases at the highest risk of thrombosis, such as those with previous thrombosis, hypertension or diabetes, should be considered for antithrombotic prophylaxis, normally in the form of low-molecular-weight heparin. The platelet count may rise dramatically in the postpartum period but can normally be controlled with hydroxycarbamide or anagrelide. Hydroxycarbamide and anagrelide are excreted in breast milk so that breast-feeding is contraindicated while a patient is receiving either of these agents. Although interferon alfa is also excreted in breast milk, it is unlikely to be absorbed intact by the baby and there are anecdotal reports of successful breast-feeding while the mother was receiving interferon.

Primary myelofibrosis Also known as agnogenic myeloid metaplasia and idiopathic myelofibrosis, PMF has the poorest prognosis of the MPNs. PV and ET can develop into a condition that resembles PMF, usually after a latency of many years. The first reported case of PMF is probably that described by Hueck in 1879 as a ‘peculiar leukaemia’. It was not until Dameshek’s seminal work in 1951 that PMF was recognized as an MPN. The identification of the JAK2 V617F mutation in approximately half the cases of PMF and ET and nearly all cases of PV has revolutionized our understanding of the relationship between the three disorders. In fact, analogous to the concept of chronic and accelerated phase in CML, it may well be that PMF represents an accelerated phase of MPN, associated with the acquisition of further genetic abnormalities. In this model, patients presenting with apparently de novo PMF may have had a preceding undiagnosed disorder such as ET or PV.

Pathophysiology PMF is a clonal MPN of the pluripotent haemopoietic stem cell, in which the proliferation of multiple cell lineages is accompanied by progressive bone marrow fibrosis. This is true for both JAK2 V617F-positive and -negative cases while the precise effect of the mutation on the PMF phenotype awaits further studies. Marrow fibrosis is thought to be secondary to the release of proinflammatory cytokines from abnormal clonal cells (primarily megakaryocytes), which act to stimulate fibroblast proliferation and fibrosis. In support of this premise, transgenic mice expressing high levels of thrombopoietin rapidly develop mye-

lofibrosis in association with increased megakaryocyte numbers. Additionally, mice expressing reduced levels of the transcription factor GATA-1, which impairs the ability of their megakaryocytes to differentiate into platelets, also develop myelofibrosis in association with increased expression of cytokines such as transforming growth factor (TGF)-β1, platelet-derived growth factor (PDGF) and vascular endothelial growth factor in the bone marrow. In the peripheral circulation there is an increase in the number of CD34-positive cells together with increased numbers of progenitors capable of giving rise to a variety of haemopoietic colonies. As with PV and ET, erythroid and megakaryocytic colonies can also be derived in the absence of exogenous growth factors. The same molecular abnormalities seen in chronic-phase MPNs, such as mutations in JAK2, MPL and TET2, are found in patients with PMF, underscoring the interrelated nature of these disorders. Nonetheless, other mutations and epigenetic abnormalities are more frequently found in PMF, in keeping with the more aggressive phenotype, poorer prognosis and later stage of the disease. For example, cytogenetic abnormalities are found in up to 60% of cases. The commonest are deletions of 20q and 13q, trisomy 8, and abnormalities of chromosomes 1, 5, 7 and 9. Oncogene mutations are not infrequently found, and include point mutations in the RAS, KIT and TP53.

Clinical features Epidemiology The estimated annual incidence of PMF is around 0.5–1.5 per 100 000 population, with most patients diagnosed in the sixth decade and roughly equal involvement of the two sexes. Up to one-third of patients are asymptomatic at diagnosis and many of these are discovered after unrelated blood tests show modest abnormalities such as anaemia and thrombocytopenia. Splenomegaly An enlarged spleen is found in almost all patients at presentation and splenic pain/discomfort is a common presenting symptom of PMF. Most cases develop moderate to marked splenomegaly during the course of the disease and about 10% of cases develop massive splenomegaly, with the spleen extending to the right iliac fossa (Figure 36.7). This dramatic increase in splenic mass (up to 20–30 times normal) can lead to a substantial increase in splenic blood flow which, in the most severe cases, can lead to portal hypertension with oesophageal varices and ascites. Painful and painless splenic infarcts are common sequelae of splenomegaly in PMF. Extramedullary haemopoiesis The spleen is the commonest site of extramedullary haemopoiesis in PMF. The liver is also usually involved and this can lead to significant hepatomegaly. Unusual sites can sometimes be 697

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Figure 36.7 Massive splenomegaly in a 53-year-old man with an 8-year history of primary myelofibrosis.

affected, leading to haemopoietic tumours surrounded by a capsule of connective tissue. Such sites include lymph nodes, central nervous system, skin, pericardium, peritoneum, pleura, ovaries, kidneys, adrenals, gastrointestinal tract and lungs. Many such cases remain asymptomatic, but involvement of the central nervous system can be a cause of serious morbidity. Treatment with radiotherapy or surgery, when required, almost always leads to resolution of these masses. Systemic symptoms A hypermetabolic state presenting with fevers, anorexia, weight loss and night sweats develops in many cases of PMF, sometimes early in the disease. The presence of such symptoms is associated with a poor prognosis. Anaemia Mild to moderate anaemia is found in most patients at presentation and worsens as myelofibrosis progresses. The anaemia is in large part due to reduced erythropoiesis, but may be compounded by hypersplenism, bleeding and iron or folate deficiency. Acquired HbH disease is a rare complication. Platelet abnormalities Platelet counts are raised in up to half of the cases at presentation and can be associated with thrombotic complications. However, progressive thrombocytopenia is a frequent occurrence and becomes increasingly troublesome as the disease progresses. Dysmegakaryopoiesis and abnormal platelet function further add to the risk of haemorrhagic complications. White cells and leukaemic transformation The presence of immature myeloid as well as erythroid progenitors is a characteristic feature of PMF (Figure 36.8). Neutrophilia is common, as are modest elevations in basophil and eosinophil counts. As the disease progresses, leucopenia increases in frequency and is believed to be secondary to progressive hyper698

Figure 36.8 Peripheral blood film in primary myelofibrosis showing a blast, an abnormal myelocyte, teardrop red cells and marked anisopoikilocytosis.

splenism, dysmyelopoiesis and progressive replacement of the bone marrow by fibrotic tissue. In end-stage PMF, myeloid precursors become increasingly common relative to mature cells, as do circulating blasts. Transformation to AML occurs in 20–30% of cases of PMF and is usually rapidly fatal.

Investigations Diagnostic criteria for PMF are shown in Table 36.5. Other causes of bone marrow fibrosis are listed in Table 36.6. Peripheral blood The presence of myeloid and erythroid precursors in the peripheral blood (leucoerythroblastic blood picture) is common in PMF (see Figure 36.8). Other causes of a leucoerythroblastic blood film include bone marrow infiltration, severe sepsis, severe haemolysis and a sick neonate. Teardrop poikilocytes (dacryocytes), basophilic stippling, macrocytosis (which may or may not be secondary to folate deficiency), giant platelets and megakaryocyte fragments may also be present. Bone marrow Attempts at bone marrow aspiration often yield a dry tap or a haemodilute sample, making aspirate morphology of limited diagnostic value. Sufficient material can often be obtained from either bone marrow or peripheral blood to assess the karyotype, which can help exclude diagnoses such as CML. Other chromosomal abnormalities may be found in up to 60% of cases as

Myeloproliferative neoplasms

Table 36.5 Diagnostic criteria for primary myelofibrosis.

Table 36.6 Differential diagnosis of marrow fibrosis.

JAK2-positive primary myelofibrosis A1 Reticulin ≥ grade 3 (on a 0–4 scale) A2 Mutation in JAK2 B1 Palpable splenomegaly B2 Otherwise unexplained anaemia (Hb < 11.5 g/L for men, < 10 g/L for women) B3 Teardrop red cells on peripheral blood film B4 Leucoerythroblastic blood film (presence of at least two nucleated red cells or immature myeloid cells in peripheral blood film) B5 Systemic symptoms (drenching night sweats, weight loss > 10% over 6 months or diffuse bone pain) B6 Histological evidence of extramedullary haemopoiesis Diagnosis requires A1 + A2 and any two B criteria

Haematological malignancies Primary myelofibrosis Chronic myeloid leukaemia Acute myelofibrosis (AML M7) Myelodysplasia Myeloma Hairy-cell leukaemia Non-Hodgkin lymphoma Hodgkin disease Systemic mastocytosis Metastatic carcinoma Infections Tuberculosis Leishmaniasis Drugs/toxins Benzene Thorotrast Irradiation Bone disease Paget disease Osteopetrosis Hyperparathyroidism Hypoparathyroidism Inflammatory diseases Systemic sclerosis Systemic lupus Other Grey platelet syndrome

JAK2-negative primary myelofibrosis A1 Reticulin ≥ grade 3 (on a 0–4 scale) A2 Absence of mutation in JAK2 A3 Absence of BCR–ABL1 fusion gene B1 Palpable splenomegaly B2 Otherwise unexplained anaemia (Hb < 11.5 g/L for men, < 10 g/L for women) B3 Teardrop red cells on peripheral blood film B4 Leuckoerythroblastic blood film (presence of at least two nucleated red cells or immature myeloid cells in peripheral blood film) B5 Systemic symptoms (drenching night sweats, weight loss >10% over 6 months or diffuse bone pain) B6 Histological evidence of extramedullary haemopoiesis Diagnosis requires A1 + A2 + A3 and any two B criteria

Treatment detailed above. Abnormalities of chromosomes 5 and 7 are usually found in patients with prior exposure to genotoxic agents and are associated with a poor prognosis. Bone marrow trephine biopsy is essential to make a diagnosis of PMF. Initial stages are characterized by an increase in bone marrow cellularity in association with disorganization of marrow architecture and the presence of abnormal large megakaryocytes often occurring in clusters (Figure 36.9). Bone marrow fibrosis becomes increasingly dominant and progressively replaces haemopoiesis. Intrasinusoidal haemopoiesis can sometimes be seen at this stage (Figure 36.10). The degree of fibrosis is best demonstrated using silver impregnation, which stains reticulin fibres (Figure 36.9). Collagen fibres are best demonstrated using a trichrome stain. The degree of fibrosis can be graded from 0 to 4 according to severity. In a minority of cases of advanced PMF, osteosclerosis ensues with thickening of the trabecula and extensive deposition of osteoid. Such changes may be evident on plain radiography.

The only curative treatment for PMF is allogeneic stem cell transplantation, but this is only appropriate for a small proportion of patients. In the remaining cases therapy remains supportive and aimed at alleviating symptoms, but has little impact on the relatively poor survival of PMF patients. It is for this reason that the future development of therapeutic JAK2 inhibitors is most eagerly awaited for these patients, rather than for those with PV or ET who have a much better prognosis. Conventional allogeneic bone marrow transplantation is only a realistic option in young patients, who represent perhaps 10% of all cases. The decision to proceed to transplantation should always be made in light of the patient’s specific prognosis, age and general fitness. Only small series have been reported and the long-term survival of patients 45 years and younger is approximately 50%, with a 30% transplant-related mortality. For patients 45 years and older, outcomes are much worse, with long-term survival of 10–20%. Small numbers of reducedintensity transplants have been reported, which show that this 699

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(a)

Figure 36.10 Bone marrow trephine section in primary myelofibrosis (H&E). Note the presence of two dilated sinusoids (arrows) containing immature haemopoietic cells, including megakaryocytes.

(b)

Figure 36.9 Bone marrow trephine sections from a patient with early-stage primary myelofibrosis. The H&E stain (a) shows hypercellularity, disorganized architecture, increase in megakaryocyte numbers and prominent sinusoids. The silver stain (b) also shows a marked increase in reticulin fibres.

modality is feasible and safe in patients over 50 years old, although its efficacy is not yet clear. Anaemia responds to treatment with androgens in up to onethird of cases, with the best responses seen in patients without massive splenomegaly and with a normal karyotype. The drugs most commonly used are oxymetholone (50–150 mg daily) and danazol (400–600 mg daily). Both of these can have virilizing effects and can lead to abnormal liver function. Patients with a reduced red cell survival may respond to treatment with corticosteroids. Human recombinant Epo has recently shown promise in small clinical studies of anaemia in PMF. Splenectomy also has a role (see below). Despite these treatments, most patients become transfusion dependent eventually. 700

Cytoreductive therapy can be useful in the management of some aspects of PMF, such as hepatosplenomegaly, constitutional symptoms and troublesome thrombocytosis. Hydroxycarbamide is the most widely used agent but anagrelide has also been used for thrombocytosis. The indications for splenectomy include splenic pain, constitutional symptoms, portal hypertension and transfusiondependent anaemia. In contrast, there is no good evidence that thrombocytopenia responds to splenectomy. The procedure has significant mortality (around 10%) and morbidity, particularly in elderly patients. Problems include perioperative bleeding, infection and thrombosis as well as rebound thrombocytosis and progressive hepatomegaly. It is particularly important to correct any coagulation abnormalities prior to surgery. Splenic irradiation is an alternative to splenectomy in some cases and can significantly reduce splenic size, albeit transiently. This procedure is not without complications as it can lead to life-threatening cytopenias. Radiotherapy can also be useful for treating pockets of extramedullary haemopoiesis involving vital organs or body cavities. The anti-angiogenic drug thalidomide has been reported to improve anaemia, thrombocytopenia or splenomegaly in approximately one-third of patients, but these changes are clinically significant only in a small proportion (20%). Thalidomide is poorly tolerated at conventional doses (> 100 mg daily) with more than half of the patients being unable to tolerate it beyond 3 months. Furthermore, thalidomide increases the risk of extreme thrombocytosis and probably that of venous thrombosis, and needs special precautions for use in view of its extreme teratogenicity. Trials of lower doses of the drug alone or in combination with prednisolone suggest that such doses are

Myeloproliferative neoplasms

Table 36.7 Prognostic scoring systems in myelofibrosis: the Lille scoring system. Number of adverse prognostic factors

Risk group

Cases (%)

Median survival (months)

0 1 2

Low Intermediate High

47 45 8

93 26 13

Adverse prognostic factors: Hb 10 g/dL, WBC < 4 or > 30 × 109/L. Source: Dupriez et al. (1996) with permission.

Table 36.9 Classification of mastocytosis. Cutaneous mastocytosis Urticaria pigmentosa and variants Mastocytoma of the skin Systemic mastocytosis Indolent Aggressive Systemic mastocytosis with associated haematological non-mast cell disorder Mast cell leukaemia Mast cell sarcoma Source: modified from Valent et al.(2001) with permission.

Table 36.8 Prognostic scoring systems in myelofibrosis: the Sheffield prognostic system. Age (years)

Hb (g/dL)

Karyotype

Median survival in months (95% CI)

10

Normal Abnormal Normal Abnormal

180 (6–354) 72 (32–112) 54 (46–62) 22 (14–30)

Normal Abnormal Normal Abnormal

70 (61–79) 78 (26–130) 44 (31–57) 16 (5–27)

≤10 ≥68

>10 ≤10

Source: Reilly et al.(1997) with permission.

better tolerated and may be similarly efficacious. Bisphosphonates can help with bone pain.

Prognosis The median survival is 3–5 years, but the range is very wide. As a result, efforts have been made to devise algorithms to individualize prognosis. The two most widely used algorithms are shown in Tables 36.7 and 36.8. Recently, an elevation in CD34 cell count above 300 × 106/L and the presence of JAK2 V617F mutation were also identified as adverse prognostic features.

Mastocytosis Mastocytosis comprises a rare group of disorders characterized by a pathological increase in mast cells in tissues including the skin, bone marrow, liver, spleen, lymph nodes and gastrointestinal tract. Mastocytosis can be an isolated finding or can form part of other haematological disorders, including MDS, MPNs or AML. Some cases involve just the skin (cutaneous mastocytosis) whereas others involve multiple tissues and are associated

with systemic symptoms (systemic mastocytosis). Paediatric mast cell disease is generally a reactive condition rather than a clonal MPN and is not further discussed here. A classification of mast cell diseases has been proposed (Table 36.9). The first case of urticaria pigmentosa (a form of cutaneous mastocytosis) was described in 1869 by Nettleship, and systemic disease due to increased mast cells was first documented by Ellis in 1949. The observation that stem cell factor (SCF) is an essential growth factor for mast cell development has led to significant advances in our understanding of this group of diseases.

Pathophysiology After a search for abnormalities of SCF failed to identify any pathological changes, researchers turned to c-Kit, the tyrosine kinase receptor for SCF. A point mutation in the KIT gene leading to a single amino acid substitution (Asp816Val) in c-Kit was thus identified. This mutation leads to ligand-independent phosphorylation of c-Kit and consequent clonal expansion of mast cells. Asp816Val was originally identified in cases of mastocytosis with associated haematological disorders but is now known to be present in the majority of adults presenting with urticaria pigmentosa or indolent systemic mastocytosis. More recently, other activating mutations affecting the same codon have been identified in a minority of adult cases of cutaneous mastocytosis (Asp816Tyr, Asp816Phe). A different mutation has been reported in a small number of cases of paediatric mastocytosis, namely Lys839Glu, which surprisingly gives rise to a dominantnegative (inactivating) form of c-Kit; the significance of this observation remains unresolved. Recent reports have suggested that in some cases of mastocytosis, the c-Kit mutation is found in other haemopoietic cells such as B cells, myeloid cells and T cells. These results suggest that mastocytosis, as with the classical MPNs, may be a clonal disorder of the haemopoietic stem cell. 701

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Table 36.10 Criteria for the diagnosis of systemic mast cell disease. Major Multifocal dense infiltrates of mast cells in bone marrow and/or other extracutaneous tissues

Figure 36.11 Urticaria pigmentosa in a 55-year-old woman. Note the widespread pink or brownish macules that become confluent in areas.

Clinical features Cutaneous manifestations Urticaria pigmentosa is the usual presenting feature in children and adults with isolated mastocytosis. Yellowish-brown lesions, usually macular and sometimes papular, appear in a patchy distribution. Less commonly, there is diffuse involvement of the skin, which becomes thickened and darker brown (Figure 36.11). Pruritus is common, as is flushing, and some cases develop haemorrhagic bullous disease. Whealing of lesions on rubbing is known as the Darier sign. Systemic disease Systemic manifestations are very heterogeneous and are thought to be largely secondary to mast cell mediator release. Episodes of flushing, angio-oedema, or even anaphylaxis with or without any specific trigger, can arise as a result of systemic histamine release. Gastrointestinal symptoms include abdominal pain, diarrhoea, nausea and vomiting. Gastritis and peptic ulceration may occur secondary to hyperhistaminaemia and severe cases may develop malabsorption. Osteoporosis is well recognized and can sometimes lead to pathological fractures. Peripheral blood cytopenias may arise secondary to mast cell infiltration of the bone marrow. Hepatosplenomegaly is more common in cases associated with another clonal haematological disorder. Fever, fatigue and weight loss can sometimes ensue and may result from the release of cytokines such as tumour necrosis factor (TNF)-α and IL-1. Symptoms of organ failure due to infiltration are characteristic of aggressive systemic mastocytosis. Depending on the organs involved, cytopenias, pathological fractures, impaired liver function, ascites and malabsorption can all be seen.

Investigations Clinical features of mastocytosis can be highly suggestive of the disease but diagnosis usually requires histological and biochem702

Minor More than 25% of mast cells on bone marrow smears or tissue biopsies are atypical or spindle-shaped Identification of a codon 816 KIT point mutation in blood, bone marrow or lesional skin Mast cells in bone marrow, blood or other lesional tissues expressing CD25 or CD2 Baseline total serum tryptase greater than 20 ng/mL Major and one minor or three or more minor criteria needed for diagnosis Source: modified from Valent et al.(2001) with permission.

ical confirmation. An algorithm has been proposed for the diagnosis of systemic mast cell disease and is shown in Table 36.10. Routine investigations should include a full blood count, liver function tests and a random serum tryptase. Tests for histamine metabolites in 24-hour urine specimens are probably no more useful than measurements of serum tryptase. Plasma levels of soluble CD25 and CD117 (c-Kit) have shown promise as novel markers of mast cell disease. Bone marrow aspiration and trephine biopsy allow assessment of bone marrow involvement. Mast cell aggregates can be visualized on conventional haematoxylin and eosin-stained sections (Figure 36.12) but stand out much more clearly with stains such as toluidine blue (Figure 36.13). Immunochemistry using anti-tryptase antibodies can also be very useful, being highly specific for mast cells. Flow cytometry for expression of CD2 and CD25 in bone marrow mast cells may be useful as this phenotype is not seen in normal mast cells. Abdominal ultrasound or computed tomography should be performed to look for hepatosplenomegaly and lymphadenopathy. Plain radiography and bone densitometry can be used to determine if bone involvement and osteoporosis are present. Endoscopy and biopsy can be useful if gut involvement is suspected.

Treatment Despite significant advances in the understanding of its pathophysiology, no curative treatment exists for mastocytosis, the management of which remains symptomatic. There are four main components to the management of mastocytosis: 1 avoidance of factors that can trigger mediator release from mast cells;

Myeloproliferative neoplasms

Figure 36.12 Systemic mastocytosis involving the bone marrow (H&E). Malignant whorls of rounded and spindle-shaped mast cells are seen infiltrating the bone marrow in a paratrabecular distribution (arrow). A lymphoid aggregate and areas of hypocellularity are seen interspersed between the mast cell infiltrate and neighbouring haemopoietic islands (arrowhead). (Courtesy of Dr Wendy Erber.)

2 treatment of acute mediator release; 3 treatment of chronic mediator release; 4 reduction of the mast cell burden/organ infiltration. Avoidance of triggers of mast cell mediator release is primarily an exercise in patient education. Severe reactions due to systemic mast cell mediator release are difficult to predict in patients with mastocytosis and do not correlate well with disease category, mast cell burden or severity of other symptoms. All patients and relevant healthcare workers should be warned of particular triggers, including general anaesthesia, contrast radiography and insect stings. Known mast cell activators such as morphine and dextran should only be introduced with great caution. Patients with previous anaphylaxis or severe hypotension should carry injectable adrenaline and they, their family and friends should be instructed in its intramuscular administration. Local mediator release in cutaneous mastocytosis can be moderated by avoidance of triggering factors such as friction and heat. Acute systemic mast cell mediator release should be treated in much the same way as other forms of anaphylaxis. Treatment with adrenaline and intravenous fluids should be started as soon as possible, with early involvement of intensive care specialists in severe cases. Antihistamines (H1 and H2 blockers) should be introduced and continued long term if the episode is particularly severe or recurrent. Symptoms of chronic mediator release are the commonest clinical problem in mastocytosis. Symptomatic cutaneous disease should be managed with the help of a dermatologist. Treatments include H1 and H2 blockers, topical corticosteroids and PUVA (psoralen with UVA irradiation) for severe disease.

(a)

(b)

Figure 36.13 Toluidine blue stain of bone marrow aspirate (a) and trephine biopsy section (b) in systemic mastocytosis. Abnormal spindle-shaped mast cells containing metachromatic granules are seen.

Non-life-threatening systemic symptoms such as flushing, abdominal pain and diarrhoea should be treated with H1 and H2 blockers, sodium cromoglycate and corticosteroids. Inhibitors of prostaglandin synthesis, such as aspirin and nonsteroidal anti-inflammatory drugs, can also be useful. Aspirin should always be started with caution as it can initially lead to acute mediator release. Such drugs can be used prophylactically if symptoms recur frequently. Gastrointestinal disease usually responds to the drugs used to treat chronic systemic symptoms. 703

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Anecdotally, leukotriene antagonists help with abdominal cramps and diarrhoea. Peptic ulcer and reflux disease should be treated with proton pump inhibitors. Osteoporosis should be treated with bisphosphonates and may be prevented with bisphosphonates or calcium and vitamin D supplementation. Bone density should be recorded and monitored according to the severity of osteoporosis. Radiotherapy can help with severe localized pain. For patients in whom adequate symptomatic control cannot be achieved and for those with aggressive mastocytosis, interferon alfa, usually given in combination with oral corticosteroids, should be considered. Splenectomy may help reduce the mast cell burden and associated systemic symptoms. Cladribine has been found effective in isolated cases of aggressive systemic mastocytosis. Treatment with chemotherapy is usually reserved for cases of rapidly progressive aggressive mastocytosis, mast cell leukaemia and mast cell sarcoma, but published data are not encouraging. Mast cell sarcoma may also respond to local radiotherapy when appropriate. Allogeneic bone marrow transplantation should also be considered. Treatment of any associated haematological disorder should be undertaken as appropriate for that disorder and the overall prognosis is usually that of the latter. Future treatments Treatments that target the mutant c-Kit tyrosine kinase have attracted a lot of interest recently. Imatinib is known to inhibit wild-type c-Kit in vitro and to be active against juxtamembrane mutants of c-Kit found in gastrointestinal stromal tumours. In contrast, the drug does not have the same effect on malignant mast cells carrying codon 816 mutations, probably because the mutant c-Kit does not allow access of imatinib to the site, hence conferring resistance to this drug in a similar way to acquired imatinib resistance in CML. In keeping with this, there have been early reports of its lack of efficacy in the presence of codon 816 mutations. A recent report showing that imatinib was effective in patients with mastocytosis with associated eosinophilia but without demonstrable KIT mutations awaits confirmation, particularly as these cases may represent variants of chronic eosinophilic leukaemia (CEL). Novel tyrosine kinase inhibitors that can inhibit the Asp816Val mutant c-Kit in vitro are under investigation.

Prognosis Age and disease category are the most important determinants of outcome. The most benign syndrome is paediatric mastocytoma, which disappears with time in over 50% of cases. Paediatric urticaria pigmentosa also has a good prognosis and resolves in about half of the cases. In adult mastocytosis, urticaria pigmentosa is usually associated with mast cell deposits in the marrow or other tissues, making this a systemic syndrome. Indolent systemic mastocy704

tosis carries a favourable prognosis and usually persists as a chronic low-grade disorder, although it rarely progresses to aggressive mastocytosis or mastocytosis associated with another haematological malignancy. Aggressive systemic mastocytosis can show a slowly progressive or a rapid clinical course but its overall prognosis has not been well defined in clinical studies. Mast cell leukaemia is rare but has a grave prognosis, with a median survival of less than 6 months.

Clonal hypereosinophilic syndromes The term ‘hypereosinophilic syndrome’ (HES) was coined by Hardy and Anderson in 1968, who gave it the definition still in use today. Recently, major progress has been made in elucidating the molecular pathogenesis of clonal eosinophilia as described below.

Pathophysiology Eosinophilia can be divided into three categories: reactive, idiopathic and clonal (Table 36.11). Reactive eosinophilia, which is by far the most common, is discussed in Chapter 17. Idiopathic eosinophilias are those in which the cause is obscure. Within this category, HES describes patients with an unexplained elevation of peripheral blood eosinophils (> 1.5 × 109/L) for more than 6 months associated with endorgan damage (see Chapter 17). Many of these are probably cases of CEL for which the molecular defect has not been identified. Clonal eosinophilias are those in which the eosinophilia is part of a clonal haematological malignancy. CEL is defined as an eosinophil count in excess of 1.5 × 109/L, with evidence of eosinophil clonality or an increased blast count in blood or bone marrow. The distinction between this entity and HES is blurred as it relies on the availability of a clonal marker. Indeed it has been shown recently that 25–50% of cases labelled as HES in fact have a microdeletion on chromosome 4, which results in the fusion of the FIP1L1 and PDGFRA genes and the generation of a constitutively active tyrosine kinase. Importantly, patients carrying this fusion respond well to the tyrosine kinase inhibitor imatinib. Some patients with HES who do not carry this fusion gene also respond to imatinib, suggesting that in such cases other tyrosine kinases may be dysregulated. In addition to CEL, a number of other haematological malignancies may be associated with increased numbers of clonal eosinophils, and in many cases this reflects tyrosine kinase dysregulation. PDGFRA rearrangements (e.g. TEL–PDGFRB) may present as chronic myelomonocytic leukaemia or atypical CML. The 8p11 myeloproliferative syndrome (EMS) is associated with rearrangements in the FGFR1 gene (e.g. ZMYM2–FGFR1) and leads to a chronic MPN that frequently presents with eosinophilia and associated T-cell lymphoblastic lymphoma. CML,

Myeloproliferative neoplasms

Table 36.11 Causes of eosinophilia. Reactive eosinophilia Infections Parasitic Others (rarely) Vasculitides Polyarteritis nodosa Churg–Strauss syndrome Connective tissue disorders Rheumatoid arthritis Systemic sclerosis Systemic lupus erythematosus Allergic and inflammatory disorders Asthma Eczema Bullous skin diseases Inflammatory bowel disease Drug reactions Hypersensitivity l-Tryptophan Immunodeficiencies Wiskott–Aldrich syndrome Job syndrome (hyper-IgE) Neoplasia Hodgkin disease Non-Hodgkin lymphoma Peripheral blood T-cell clones Some cases of acute lymphoblastic leukaemia Non-haematological cancers (rare) Clonal eosinophilia Chronic eosinophilic leukaemia Atypical chronic myeloid leukaemia (PDGFRA fusions) 8p11 Myeloproliferative syndrome (FGFR1 fusions) Chronic myeloid leukaemia (BCR–ABL1 fusion) Acute myeloid leukaemia, e.g. carrying inv(16) Acute lymphoblastic leukaemia (occasionally) Idiopathic eosinophilia Hypereosinophilic syndrome

a consequence of the BCR–ABL1 tyrosine kinase fusion protein, may also be associated with clonal eosinophilia. Eosinophilia as part of the malignant clone also occurs in patients with AML associated with inversion of chromosome 16 and the SMMHC–CBFB rearrangement. It has been reported that rare cases of acute lymphoblastic leukaemia may be associated with clonal eosinophilia but in this disease the eosinophilia is more usually secondary to growth factor release. Growth factor release is also believed to underlie the reactive eosinophilia seen in Hodgkin disease and in cases with clonal T cells in the peripheral blood.

Sustained hypereosinophilia can lead to symptomatology and end-organ damage regardless of its aetiology, but does not always do so. The reasons for this are unclear but may lie in the heterogeneity of eosinophilia and genetic differences between individuals that affect the propensity of eosinophils and other granulocytes to inflict tissue damage.

Clinical features (see also Chapter 17) Much of the tissue damage in eosinophilia is believed to be secondary to eosinophil degranulation and release of mediators such as eosinophil cationic protein and major basic protein. Eosinophil mediators act mainly locally in tissues infiltrated by eosinophils to cause tissue damage. The recent finding that a raised serum tryptase in a subset of cases with clonal eosinophilia hints at a role for other cells (mast cells) in some cases. Patients can present with constitutional symptoms such as fatigue, muscle aches or fevers. Pruritus, angio-oedema, diarrhoea and cough may also be present. Many tissues can be involved, but cardiac disease is the major cause of mortality. The heart can be affected by endomyocardial fibrosis, pericarditis, myocarditis and intramural thrombus formation. Death is usually due to dilated cardiomyopathy. Involvement of the central and peripheral nervous systems can result in mononeuritis multiplex, paraparesis, encephalopathy and even dementia. Pulmonary involvement can take the form of pulmonary infiltrates, fibrosis or pleural disease with effusions. Gastrointestinal involvement can manifest as diarrhoea, gastritis, colitis, hepatitis or Budd–Chiari syndrome. The skin can be affected by pruritus, angio-oedema, papules or plaques. Rarely, other tissues such as the kidneys and bones can be involved.

Investigations There are two aims in the investigation of eosinophilia: one is to establish its aetiology and the other to look for evidence of end-organ damage. As regards the former, given the diverse nature of the aetiologies of eosinophilia, a full history including family history, drug history and travel history can provide valuable clues. Investigations will usually aim to exclude reactive causes and will be guided by the clinical picture. Bone marrow aspiration will reveal morphological abnormalities associated with haematological malignancies and allows cytogenetic analysis. It is also important to look for clonal T-cell receptor (TCR) gene rearrangements, the FIP1L1– PDGFRA and BCR–ABL1 fusion genes, as well as rearrangements of the PDGFRB and FGFR1 genes. It has been reported that serum tryptase is raised in patients with the FIP1L1– PDGFRA fusion. Investigations to assess end-organ damage will depend on the clinical presentation. However, echocardiography should be 705

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Figure 36.15 Abundance of mature neutrophils and band forms in a blood film from a patient with chronic neutrophilic leukaemia. Figure 36.14 Cardiac MRI scan in a 65-year-old man with hypereosinophilic syndrome showing a rim of subendocardial fibrosis (arrow).

performed and repeated annually in patients with sustained eosinophilia, particularly as cardiac disease correlates poorly with the eosinophil count. If there is strong clinical suspicion of cardiac damage, then cardiac magnetic resonance imaging (MRI) can be useful, as this is more sensitive in detecting early disease (Figure 36.14). If there is doubt as to the aetiology of cardiac disease, endomyocardial biopsy may demonstrate eosinophil infiltration. Serial monitoring of pulmonary function may be required if there is evidence of lung involvement.

Treatment Treatment should be used to halt or reverse organ damage. Eosinophilia without evidence of end-organ damage does not usually require treatment. When underlying clonal or non-clonal disorders are identified they should be treated appropriately. Patients with rearrangements of the PDGFRA or PDGFRB genes respond well to imatinib, with normalization of eosinophil counts within weeks. A trial of imatinib is also reasonable in patients with HES who lack a clonal marker, as a proportion of these patients also respond. For patients who do not respond to imatinib, prednisolone is the initial treatment of choice. Steroids reduce blood eosinophilia and the inflammation resulting from tissue infiltration. Cardiac disease may respond even in the absence of a significant reduction in the eosinophil count. Hydroxycarbamide and interferon alfa may benefit patients resistant to steroids. Cladribine and ciclosporin were also found to be of use in some cases. 706

Prognosis The reported prognoses of CEL and HES are highly variable, with estimates of 3-year survival ranging from 23 to 96%. This is likely to reflect heterogeneity within these two categories of patients. In patients with HES, indicators of a poor prognosis include lack of response to steroids, a markedly elevated eosinophil count, normal IgE levels, splenomegaly, dysplastic features and male sex. Many of these adverse prognostic indicators may simply be markers of clonal (versus reactive) eosinophilia.

Chronic neutrophilic leukaemia Chronic neutrophilia is a very common entity, and is usually secondary to chronic infections, chronic inflammation or malignancy. A very small subgroup of patients with chronic neutrophilia have chronic neutrophilic leukaemia (CNL), a clonal haematological disorder (Figure 36.15). Given the absence of a specific marker for this disease, CNL, like ET, is a diagnosis of exclusion (see also Chapter 27).

Pathophysiology Cases can be subdivided into two main groups: ‘true’ CNL and neutrophilic CML. The two groups are only distinguishable by the presence in the latter of a rare type of BCR–ABL1 rearrangement that produces a 230-kDa fusion protein (p230). In addition, rare patients with MDS can closely mimic CNL but exhibit dysplastic features and there are anecdotal reports of PV evolving into a disorder indistinguishable from CNL. The literature also includes reports of what was thought to be clonal chronic neutrophilia in association with plasma cell dyscrasias. However, data are accumulating that this type of neutrophilia is non-

Myeloproliferative neoplasms

Table 36.12 Diagnostic features of chronic neutrophilic leukaemia. Peripheral blood leucocytosis >25 × 109/L Segmented neutrophils and bands >80% of white blood cells Immature granulocytes < 10% of white blood cells Myeloblasts < 1% of white blood cells Hypercellular bone marrow biopsy Neutrophilic granulocytes increased in percentage and number Myeloblasts < 5% of nucleated marrow cells Normal neutrophil maturation pattern Hepatosplenomegaly No identifiable cause of reactive neutrophilia No evidence of another haematological malignancy No Philadelphia chromosome or BCR–ABL1 fusion No evidence of another myeloproliferative disorder (i.e. normal PCV, platelets < 600 × 109/L, no bone marrow fibrosis or other features of PMF) No evidence of a myelodysplastic syndrome (i.e. no dysplasia, monocytes < 1 × 109/L) Source: modified from Vardiman et al.(2001) with permission.

The optimal treatment of CNL remains unclear. Oral cytoreductive agents such as hydroxycarbamide and busulfan can control the neutrophil count, as can interferon. The only potentially curative modality is allogeneic bone marrow transplantation and this option should be considered in younger patients.

Neutrophilic chronic myeloid leukaemia This entity is probably even more rare than CNL, with only a handful of documented cases in the literature. The reported cases followed a more benign course than conventional CML, with a lower white cell count, lower proportion of immature granulocytes, milder anaemia, less marked splenomegaly and a lower propensity to acute transformation. Given recent advances in the treatment of BCR–ABL1-related diseases, it is important to consider and exclude neutrophilic CML during the investigation of chronic neutrophilia.

Transient abnormal myelopoiesis of Down syndrome

clonal and probably a result of cytokine release from clonal plasma cells. Diagnostic features of CNL are shown in Table 36.12. Neutrophilic CML usually exhibits these features, save for its association with the p230 BCR–ABL1 rearrangement.

Children born with Down syndrome show a 10- to 20-fold increased risk of leukaemia, despite not showing an increased incidence of other cancer types. One haematological disorder, characteristically found only in neonates with Down syndrome, is transient abnormal myelopoiesis (TAM), also known as transient myeloproliferative disorder or transient leukaemia.

Clinical features and treatment

Incidence, clinical features and treatment

A recent review of the literature identified only 33 cases that fulfilled criteria for CNL. These cases exhibited a male to female ratio of 2 : 1 and a median age at diagnosis of 62.5 years (range 15–86 years). The median survival was 30 months, with only 28% of patients surviving to 5 years. Transformation to AML ensued in 21% (7 of 33) and this was invariably lethal. Other causes of death included sepsis and haemorrhage. Haemoglobin was normal with platelet counts above 100 × 109/L in most cases. The mean leucocyte count at diagnosis was 54.3 × 109/L with mature and band forms in the peripheral blood. Vitamin B12 levels were raised and neutrophil alkaline phosphatase levels were not low in most cases. Hyperuricaemia and gout were common. The spleen may be moderately enlarged. Bone marrow biopsies were markedly hypercellular and showed marked granulocytic proliferation, as did the bone marrow aspirates. Cytogenetic abnormalities were seen in about one-third, with del(20q), del(11q), del(12p), +8, +9 and +21 being mentioned in the WHO classification of tumours. In a small number of cases, X-chromosome inactivation patterns were used to demonstrate the clonal nature of CNL in patients lacking a cytogenetic marker.

About 10% of neonates with Down syndrome are thought to develop TAM, although this may be underestimated since many otherwise healthy babies with trisomy 21 do not routinely have blood tests performed. Additional problems with recognition arise from the fact that many normal neonates with Down syndrome show mild abnormalities of blood counts, including polycythaemia and thrombocytopenia. About 25% of neonates with TAM are asymptomatic, with the blood film showing sometimes significantly elevated numbers of circulating immature myeloid cells, including basophilic blasts, nucleated red cells, megakaryocyte fragments and thrombocytosis or thrombocytopenia. In symptomatic babies, the clinical features are variable but can include neonatal jaundice, bleeding problems, respiratory distress and rarely liver failure. The natural history of TAM is intriguing. In the majority of babies, the disorder resolves spontaneously by 3 months of age, without the need for treatment. Severely symptomatic infants, especially those with respiratory or hepatic dysfunction, can be treated very effectively with low-dose cytarabine chemotherapy. About 20% of neonates with TAM will subsequently develop acute megakaryoblastic leukaemia (AMKL) before the age of 4 707

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years, although sometimes the leukaemia develops without antecedent TAM.

Pathophysiology The fact that TAM only occurs in neonates points to it being a disorder of fetal haemopoiesis, and this presumably also explains why it is self-limiting in most cases. The characteristic association between TAM and AMKL indicates that a multistep model of mutation is operative in this clinical progression. Firstly, the disorder only occurs in trisomy 21, suggesting that there are genes on this chromosome that somehow predipose to abnormal fetal haemopoiesis. Several ETS family members and RUNX1 are found on chromosome 21, but the key molecular pathways here have not been identified. Secondly, the development of TAM is tightly linked with the acquisition of characteristic mutations in the gene for the transcription factor GATA-1, located on the X chromosome. These mutations cluster in the first two coding exons of the gene and tend to be disruptive (frameshift insertions or deletions, splice-site and nonsense mutations). They all have the end result of generating a transcript in which protein translation is initiated downstream of the usual start site, leading to a GATA-1 protein lacking the first 84 amino acids. This prevents its interaction with another transcription factor, FOG1, leading to the characteristic phenotype of TAM. Thirdly, the progression to AMKL is probably driven by acquisition of further mutation(s). In particular, JAK3 activating mutations are found in a small percentage of patients with AMKL in Down syndrome, although the mutations for most cases have not yet been identified.

Selected bibliography Barbui T, Carobbio A, Rambaldi A, Finazzi G (2009) Perspectives on thrombosis in essential thrombocythemia and polycythemia vera: is leukocytosis a causative factor? Blood 114: 759–63. Barosi G, Ambrosetti A, Finelli C et al. (1999) The Italian Consensus Conference on Diagnostic Criteria for Myelofibrosis with Myeloid Metaplasia. British Journal of Haematology 104: 730–7. Baxter EJ, Scott LM, Campbell PJ et al. (2005) Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 365: 1054–61. Campbell PJ, Green AR (2006) The myeloproliferative disorders. New England Journal of Medicine 355: 2452–66. Campbell PJ, Scott LM, Buck G et al. (2005) Definition of subtypes of essential thrombocythaemia and relation to polycythaemia vera based on JAK2 V617F mutation status: a prospective study. Lancet 366: 1945–53. Dupriez B, Morel P, Demory JL et al. (1996) Prognostic factors in agnogenic myeloid metaplasia: a report on 195 cases with a new scoring system. Blood 88: 1013–18.

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Finazzi G, Caruso V, Marchioli R et al. (2005) Acute leukaemia in polycythemia vera: an analysis of 1638 patients enrolled in a prospective observational study. Blood 105: 2664–70. Gotlib J, Cross NC, Gilliland DG (2006) Eosinophilic disorders: molecular pathogenesis, new classification, and modern therapy. Best Practice and Research. Clinical Haematology 19: 535–69. Harrison CN, Gale RE, Machin SJ, Linch DC (1999) A large proportion of patients with a diagnosis of essential thrombocythemia do not have a clonal disorder and may be at lower risk of thrombotic complications. Blood 93: 417–24. Harrison CN, Bareford D, Butt N et al. (2010) Guideline for investigation and management of adults and children presenting with a thrombocytosis. British Journal of Haematology 149: 352–75. James C, Ugo V, Le Couedic JP et al. (2005) A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 434: 1144–8. Kralovics R, Passamonti F, Buser AS et al. (2005) A gain-of-function mutation of JAK2 in myeloproliferative disorders. New England Journal of Medicine 352: 1779–90. Landolfi R, Marchioli R, Kutti J et al. (2004) Efficacy and safety of low-dose aspirin in polycythemia vera. New England Journal of Medicine 350: 114–24. Levine RL, Wadleigh M, Cools J et al. (2005) Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 7: 387–97. Levine RL, Pardanani A, Tefferi A, Gilliland DG (2007) Role of JAK2 in the pathogenesis and therapy of myeloproliferative disorders. Nature Reviews. Cancer 7: 673–83. McMullin MF, Bareford D, Campbell P et al. (2005) Guidelines for the diagnosis, investigation and management of polycythaemia/ erythrocytosis. British Journal of Haematology 130: 174–95. McMullin MF, Reilly JT, Campbell P et al. (2007) Amendment to the guideline for diagnosis and investigation of polycythaemia/ erythrocytosis. British Journal of Haematology 138: 821–2. Metcalfe DD (2008) Mast cells and mastocytosis. Blood 112: 946–56. Passamonti F, Cervantes F, Vannucchi AM et al. (2010) A dynamic prognostic model to predict survival in primary myelofibrosis: a study by the IWG-MRT (International Working Group for Myeloproliferative Neoplams Research Treatment), Blood 115: 1703–8. Pikman Y, Lee BH, Mercher T et al. (2006) MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia. PLoS Medicine 3: e270. Reilly JT, Snowden JA, Spearing RL et al. (1997) Cytogenetic abnormalities and their prognostic significance in idiopathic myelofibrosis: a study of 106 cases. British Journal of Haematology 98: 96–102. Scott LM, Scott MA, Campbell PJ, Green AR (2006) Progenitors homozygous for the V617F mutation occur in most patients with polycythemia vera, but not essential thrombocythemia. Blood 108: 2435–7. Scott LM, Tong W, Levine RL et al. (2007) JAK2 exon 12 mutations in polycythemia vera and idiopathic erythrocytosis. New England Journal of Medicine 356: 459–68.

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Spivak JL, Silver RT (2008) The revised World Health Organization diagnostic criteria for polycythemia vera, essential thrombocytosis, and primary myelofibrosis: an alternative proposal. Blood 112: 231–9. Swerdlow SH, Campo E, Harris NL et al. (eds) (2008) WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. IARC, Lyon. Tefferi A, Thiele J, Orazi A et al. (2007) Proposals and rationale for revision of the World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis: recommendations from an ad hoc international expert panel. Blood 110: 1092–7.

Valent P, Horny HP, Escribano L et al. (2001) Diagnostic criteria and classification of mastocytosis: a consensus proposal. Leukemia Research 25: 603–25. Vardiman JW, Pierre R, Thiele J et al. (2001) Chronic myeloproliferative disorders. In: World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of the Haematopoietic and Lymphoid Tissues (ES Jaffe, NL Harris, H Stein, JW Vardiman, eds), pp. 15–44. IARC Press, Lyon. Wilkins BS, Erber WN, Bareford D et al. (2008) Bone marrow pathology in essential thrombocythemia: interobserver reliability and utility for identifying disease subtypes. Blood 111: 60–70.

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CHAPTER 37

Histocompatibility Ann-Margaret Little1, Steven GE Marsh2 and J Alejandro Madrigal2

37

1

Histocompatibility and Immunogenetics, Gartnavel General Hospital, Glasgow, UK The Anthony Nolan Research Institute, Royal Free Hospital, London, UK

2

The major histocompatibility complex and human leucocyte antigens, 710 Structure of HLA proteins, 711 Antigen processing and presentation, 711 HLA function, 712

HLA polymorphism, 712 HLA associations with disease, 713 HLA nomenclature, 713 HLA matching in transplantation, 714 HLA matching required for haemopoietic stem cell transplantation, 717

The major histocompatibility complex and human leucocyte antigens In the 1950s three independent scientists, Jean Dausset, Rose Payne and Jon van Rood, described the presence of alloantibodies in the sera of individuals exposed to genetically non-identical tissues such as the fetus in pregnancy and/or blood cells after transfusion. These alloantibodies were subsequently shown to react against protein antigens, encoded by a genetic region called the major histocompatibility complex (MHC). The MHC had also been independently identified in animal models of skin transplantation and tumour immunology, where genetically different strains of mice were shown to reject transplants from one another. Genetically identical mice could accept such transplants without rejection. These observations led to theories of self/non-self discrimination, and the elucidation of the genetics of MHC genes encoding the protein ‘histocompatibility’ (or transplantation) antigens. Analysis of the structure and function of histocompatibility antigens has led to the establishment of routine and successful transplant protocols for both solid organ and haemopoietic stem cells between genetically disparate individuals. The MHC in humans is located on the short arm of chromosome 6 (6p21.3). This region of the genome has been extensively studied and fully sequenced as part of the Human Genome Project (www.sanger.ac.uk/HGP/Chr6/). The MHC is an extremely gene-dense region of the genome, and it can be divided into ‘three’ subregions based on the type of genes found.

Postgraduate Haematology: 6th edition. Edited by A. Victor Hoffbrand, Daniel Catovsky, Edward G.D. Tuddenham, Anthony R. Green © 2011 Blackwell Publishing Ltd.

710

Histocompatibility testing procedures, 717 Serology, 717 DNA-based methods, 717 Cellular assays, 719 Other genetic polymorphisms, 720 Selected bibliography, 720

The genes encoding histocompatibility antigens are located within the MHC class I and II regions (Figure 37.1). These genes are called HLA genes (defined as human leucocyte antigens, although originally called human locus-A). HLA genes found in the class I region differ in structure from those found in the class II region and as a result the encoded proteins also differ. HLA class I genes encode a polypeptide of about 340 amino acids. This polypeptide is found as a cell-surface transmembrane glycoprotein and is associated with the soluble protein β2-microglobulin encoded by a gene on chromosome 15. There are three classical class I genes: HLA-A, HLA-B and HLA-C. Their encoded proteins are expressed on virtually all nucleated cells within the body and also on platelets. The expression of HLA class I proteins varies for different types of tissues. For example, expression is high on lymphocytes but low on hepatocytes and tissues comprising the nervous system. In addition there are class I genes encoding non-classical class I molecules called HLA-E, HLA-F and HLA-G. The tissue distribution of these molecules is more restricted than that of HLA-A, -B and -C, and this is a reflection of their differing function. These molecules are not considered as transplantation antigens. HLA class II molecules are similar in structure to HLA class I molecules. They are composed of two MHC-encoded polypeptide chains (α and β). HLA class II molecules, unlike class I, have a restricted tissue distribution. They are mainly expressed on antigen-presenting cells such as B cells, dendritic cells, macrophages and also activated (but not resting) T cells. There are three classical class II molecules: HLA-DR, HLA-DQ and HLADP. In addition, there are other non-classical class II molecules encoded by the HLA-DM and HLA-DO genes. These nonclassical class II molecules are not transplantation antigens but do contribute to the antigen-presenting function of class II molecules.

Histocompatibility

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Figure 37.1 Diagrammatic representation of the major histocompatibility complex. Approximate location of HLA-A, -B, -C, -DRA, -DRB, -DQA, -DQB, -DPA and -DPB genes are indicated. Other related genes are also present.

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Antigen processing and presentation Peptide binding to HLA class I and II proteins plays an important role in the function of these molecules. The way in which peptides are derived and the binding procedure differs for the two classes of molecules and this is reflected in their different function.

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Structure of HLA proteins The X-ray crystallographic structure of the extracellular domains of several HLA class I and II proteins has been resolved. The structures are divided into four domains as illustrated in Figure 37.2. The two most membrane-distal domains, α1 and α2 for HLA class I and α1 and β1 for HLA class II, form a βpleated sheet surrounded on both sides by two α-helices. In both class I and II structures a short peptide is found bound in the cleft. Thus both class I and class II proteins can be considered trimolecular proteins consisting of three subunits: HLA heavy chain, β2-microglobulin and peptide form class I molecules, and HLA α- and β-chains and peptide form class II molecules.

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Figure 37.2 Diagrammatic representation of HLA class I and II molecules showing protein domains and bound peptide.

HLA class I molecules are assembled within the endoplasmic reticulum (ER). The heavy chain is directed to the ER via a leader peptide sequence. Within the lumen of the ER, the extracellular domains of the heavy chain associate with β2microglobulin. This association is mediated by ER-resident chaperones including calnexin and calreticulum. Association with peptide also occurs within the ER. Peptides are derived from the cytoplasmic degradation of molecules that takes place in the proteasome, a multicatalytic protein complex. Peptides produced by the proteasome are actively transported into the ER via a transmembrane peptide pump called TAP 711

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(transporter associated with antigen processing). Association between HLA class I heavy chain/β2-microglobulin complex and peptide is catalysed by another ER-resident chaperone called tapasin associated with the thiol oxidoreductase ERp57. Once the class I trimolecular complex is formed, the molecule can leave the ER and complete its journey to the cell surface via the Golgi apparatus. The journey of a class II molecule differs from that of a class I molecule. Both α- and β-chains are directed to the ER, where a complex of three αβ chains and three invariant chains is formed. The invariant chain is also a transmembrane protein. Association of HLA class II molecules with the invariant chain effectively blocks the peptide-binding site on the class II molecule, thus preventing binding of ERresident peptides. Invariant chain cytoplasmic tail sequences direct the class II molecule through the Golgi apparatus, but unlike class I molecules the class II molecules take a detour to endosomal vesicles before arriving at the cell surface. The nonclassical HLA-DM and HLA-DO molecules are located within endosomal vesicles and it is here that the invariant chain is cleaved, leaving a peptide, called CLIP, blocking the class II peptide binding cleft. Specific peptide binding is catalysed by HLA-DM, and may also be aided by HLA-DO. The peptides that bind to class II molecules are derived from proteins that have been processed by the endocytic pathway. These proteins can be derived from the internalization of cell surface or extracellular proteins, or from endogenous cytosolic proteins via processes of autophagocytosis. Thus the antigen presentation pathway for HLA class II molecules differs from that of HLA class I molecules by directing the class II molecules to a location where they can bind peptides that are in the majority, differently sourced from those presented by class I molecules. Peptides derived from exogenous sources may also be presented by HLA class I molecules via cross-presentation pathways. The detailed mechanisms involved in these pathways are not fully elucidated but such events likely play a significant role in ensuring an adequate immune response is elicited by the ability to present antigenic peptides from the same source on both class I and II molecules.

HLA function Once presented at the cell surface, HLA molecules, together with bound peptide, are subject to surveillance by circulating T cells and natural killer (NK) cells. Both these cell types possess receptors that recognize and interact with HLA molecules. Typically, T cells expressing the CD4 molecule recognize HLA class II molecules and T cells expressing CD8 recognize HLA class I molecules. The specificity of interaction is determined during development of T cells within the thymus, such that circulating T cells should not interact with HLA molecules presenting peptides derived from normally expressed self proteins. However, the presentation of non-self peptides, for example 712

peptides derived from viral or bacterial proteins, or aberrant expression of tumour antigens can initiate, in appropriate conditions, activatory signals mediated by the T-cell receptor and coreceptors, resulting in the generation of an immune response against the cells expressing the target HLA protein–peptide complex. NK cells have a determining role in the immune response to tumours and viral infections. NK cells function in innate immune responses and also contribute to the development of adaptive immune responses. The interactions between HLA molecules and NK cell receptors also impact on alloimmune responses occurring after transplantation and pregnancy. NK cells express a highly diverse repertoire of receptors, including killer cell immunoglobulin-like receptors (KIRs), that elicit either activatory or inhibitory signals. Some HLA class I molecules have been identified as ligands recognized by KIRs. Thus the interaction between an NK cell and target cell is influenced by the interaction between HLA on the target cell and KIRs on the NK cell. Typically, an interaction between HLA class I molecule and corresponding KIR will result in a negative signal and this will outweigh any additional activatory signals; thus the NK cell will not attack the target cell. However, if a cell has lost expression of HLA molecules as a result of malignancy or viral infection, the absence of the inhibitory signal will result in activatory signals being dominant and allow NK cell-mediated attack on the target cell. Interactions between HLA and KIR have been implicated in various medical conditions, including infectious disease, autoimmunity, cancer, reproduction and transplantation. KIRs are encoded within a multigene complex on chromosome 19, the gene organization of which varies within different haplotypes. KIR genes are also functionally polymorphic, thus making analysis of KIRs as challenging as that of HLA, if not more so.

HLA polymorphism The outstanding feature of HLA genes and the proteins encoded is the extensive polymorphism exhibited. At each of the genes there are multiple possible variants (Table 37.1). The variants are called alleles. The nucleotide sequence differences between HLA alleles at a given locus can be translated into the protein sequence and analyses of the polymorphism has demonstrated that most variation exists within the peptide-binding domains. Experimental data support this by showing that different HLA proteins bind peptides with different sequences. Thus one of the functions of HLA polymorphism is to allow the presentation of numerous different peptides to the immune system. As there are six antigen-presenting ‘classical’ HLA molecules (HLA-A, -B, -C, -DR, -DQ, -DP) and most individuals are heterozygous for these loci, potentially each individual has 12 different HLA molecules, each of which can bind thousands of different peptides. The existence of a polymorphic polygenic

Histocompatibility

Table 37.1 Number of HLA alleles and serologically defined antigens by April 2010. HLA gene

No. of alleles

No. of antigens

A B C DRA DRB1 DRB3 DRB4 DRB5 DQA1 DQB1 DPA1 DPB1

1001 1605 690 3 785 52 14 19 35 108 28 133

24 50 9 – 20 1 1 1 – 7 – –

antigen-presenting system gives each individual the capacity to elicit immune responses against a wide variety of protein antigens. A complete listing of currently defined HLA alleles is given at www.ebi.ac.uk/imgt/hla/. HLA polymorphism also varies within different populations. Variation in climate, geography and infectious pathogens are factors considered to have influenced the evolution of HLA polymorphism. It is therefore not surprising to find that HLA allele frequencies vary for different ethnic groups. This has an impact on unrelated donor transplantation: an HLA-matched donor is most likely to be found within a donor pool of the same ethnicity as the patient. HLA polymorphism therefore functions as an advantage at the level of both a single individual and a population in terms of defence against pathogens. The extreme diversity found makes it very likely that someone somewhere possesses an HLA molecule that can be effective in generating an immune response against any infectious agent. However, HLA polymorphism is a significant obstacle to finding a matched unrelated donor for transplantation.

HLA associations with disease Because both HLA class I and class II molecules control the positive and negative selection of T-cell receptors in the thymus and modulate the activity of NK cells, it is not surprising to find many associations between HLA and immune system disorders such as autoimmune disease. The first strong association described was that of HLA-B27 with ankylosing spondylitis and related seronegative spondylarthropathies, where over 95% of affected individuals possess an HLA-B*27 allele. Despite tremendous effort to discover the immunological basis for the association between B27 and ankylosing spondylitis, the answer

is still unknown. Other factors must also be involved because only 3–4% of individuals with HLA-B27 will develop disease. HLA type has also been associated with susceptibility to, and protection from, infectious diseases. Individuals possessing HLA-B*53:01, an allele prevalent within African populations, are more likely to be resistant to severe malaria. This association is supported by experiments showing that the B53 allotype can present a peptide derived from the malaria parasite. With HIV infection, the HLA type of the infected individual can influence the rate of disease progression. HLA-B35 is associated with more rapid progression to AIDS, whereas HLA-B27 and HLAB57 are associated with slower disease progression. These associations reflect the function of different HLA molecules in the presentation of HIV peptides to CD8+ T cells in order to evoke an immune response against HIV-infected cells. HLA class II molecules are restricted in their tissue distribution. However, appropriate stimulation can induce the expression of class II molecules on tissues where they are not normally expressed. This aberrant HLA class II expression may contribute to an autoimmune reaction by providing HLA–peptide complexes not encountered during T-cell receptor education in the thymus. Among the many associations between HLA class II molecules and autoimmune and/or inflammatory disease is that between rheumatoid arthritis and HLA-DR4. This association has been linked to the presence of an epitope contributed by residues 67, 70, 71 and 74 of the β-chain that contributes to a pocket (P4) in the peptide-binding site, thus supporting the role of peptide binding in the disease. For other associations between HLA and disease, the answer has been found. Haemochromatosis has been associated with haplotypes possessing HLA-A*03. Genetic mapping has identified the gene, HFE, responsible for this association. Thus associations between HLA and disease may not be directly related to the HLA allotype but may be due to the presence of other genes that are closely linked or which are hitch-hiking within particular HLA haplotypes. Associations between HLA type and hypersensitivity to drugs have also been reported, leading to the application of HLA typing to patient treatment. HLA-B*57:01 typing is used to identify HIV-infected patients at risk from life-threatening hypersensitivity to the drug abacavir, an inhibitor of HIV-1 reverse transcriptase. The mechanism of this interaction is thought to involve an interaction between the drug and a peptide normally presented by the HLA-B*57:01 encoded protein, resulting in a highly immunogenic CD8+ T-cell immune response.

HLA nomenclature The naming of HLA specificities falls under the remit of the WHO Nomenclature Committee for Factors of the HLA System. The Committee names HLA genes, alleles and serologically 713

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defined antigenic specificities. The names of the antigens, which were originally defined using either serological or cellular techniques, are a combination of letters which indicate the gene encoding the antigen, and numbers assigned in chronological order of their description. An example of an individual’s HLA type determined by cellular and serological methods is A1, A2; B7, B13; Cw6, Cw7; Dw1, Dw13; DR1, DR4; DR53; DQ5, DQ8; DPw1, DPw4. The numbering of antigens encoded by HLA-A and -B genes is in a single series for historical reasons, as these antigens were originally believed to represent the products of a single gene. The use of a lower case ‘w’ between the gene name and antigen number indicates a provisional specificity, although in most cases these have been removed. The exceptions are Bw4 and Bw6, which represent public epitopes rather than distinct antigens, HLA-C antigens (e.g. Cw1), to distinguish them from complement factors, and the HLA-D and HLA-DP antigens defined by cellular techniques. Many HLA antigens have been characterized by serological methods into two or more subtypes, which are called ‘splits’, with the parent antigen called ‘broad’. Thus both A23 and A24 are splits of the broad antigen A9. It is convention to indicate the broad antigen specificity in parentheses following the designation of the split,for example A23(9) or A24(9). A full listing of all the serologically and cellularly defined HLA antigens is given in Table 37.2. Since 1987 the WHO Nomenclature Committee for Factors of the HLA System has assigned official names to HLA allele sequences. It was recognized that a single antigenic specificity, such as HLA-A2, defined by serology could be subdivided still further by DNA sequencing. Each allele is given a numerical designation that may be up to eight digits in length. The gene name is followed by an asterisk (*) and then the numerical designation. Each allele name consists of at least two, and maximally four, fields. The first field indicates the allele group, which often corresponds to the broad serological antigen encoded by the allele. The second field is used to list subtypes, numbers

assigned in the order in which the DNA sequences have been determined. Alleles whose numbers differ in the first two fields must differ in one or more nucleotide substitutions that change the amino acid sequence of the encoded protein. The third field is used to name alleles that differ only by synonymous nucleotide substitutions (also called silent or non-coding). The fourth field is used to name alleles that differ in either intron, or 3′ or 5′ regions of the gene. Lastly, an allele may have a suffix indicating aberrant expression; for example N indicates that it is a null allele with no protein being expressed, L indicates low cell surface expression and S indicates that the molecule is expressed only in a soluble form. For example, the A*24:02:01:01 and A*24:02:01:02L alleles differ only by a single nucleotide which lies within an intron at a splice site. A*24:02:01:01 is expressed at normal levels on the cell surface, whereas A*24:02:01:02L is expressed at very low levels on the cell surface due to the splice mutation. Many new HLA alleles are reported each year (Figure 37.3). Details of the most recent advances in HLA nomenclature can be found in the latest WHO Nomenclature Committee for Factors of the HLA System Report or by accessing the IMGT/HLA Sequence Database, the official database of the Nomenclature Committee (www.ebi.ac.uk/imgt/hla).

HLA matching in transplantation The benefits of HLA polymorphism in allowing the generation of immune responses against a wide range of antigens is completely negated when transplantation of organs and cells between genetically disparate individuals is considered. Typically, an individual will possess T cells capable of reacting against a foreign antigen at a frequency of 1 in 104–105. However, if cells from two HLA disparate individuals are mixed, the frequency of responding cells can be as high as 1–10%. These responding cells are called alloreactive cells.

A

B

C

D

DR

DQ

DP

A1 A2 A203 A210 A3 A9 A10 A11 A19 A23(9) A24(9) A2403 A25(10)

B5 B7 B703 B8 B12 B13 B14 B15 B16 B17 B18 B21 B22

Cw1 Cw2 Cw3 Cw4 Cw5 Cw6 Cw7 Cw8 Cw9(w3) Cw10(w3)

Dw1 Dw2 Dw3 Dw4 Dw5 Dw6 Dw7 Dw8 Dw9 Dw10 Dw11(w7) Dw12 Dw13

DR1 DR103 DR2 DR3 DR4 DR5 DR6 DR7 DR8 DR9 DR10 DR11(5) DR12(5)

DQ1 DQ2 DQ3 DQ4 DQ5(1) DQ6(1) DQ7(3) DQ8(3) DQ9(3)

DPw1 DPw2 DPw3 DPw4 DPw5 DPw6

714

Table 37.2 Complete listing of recognized serological and cellular HLA specificities.

Histocompatibility

Table 37.2 Continued

A

B

A26(10) A28 A29(19) A30(19) A31(19) A32(19) A33(19) A34(10) A36 A43 A66(10) A68(28) A69(28) A74(19) A80

B27 B2708 B35 B37 B38(16) B39(16) B3901 B3902 B40 B4005 B41 B42 B44(12) B45(12) B46 B47 B48 B49(21) B50(21) B51(5) B5102 B5103 B52(5) B53 B54(22) B55(22) B56(22) B57(17) B58(17) B59 B60(40) B61(40) B62(15) B63(15) B64(14) B65(14) B67 B70 B71(70) B72(70) B73 B75(15) B76(15) B77(15) B78 B81 B82 Bw4 Bw6

C

D

DR

Dw14 Dw15 Dw16 Dw17(w7) Dw18(w6) Dw19(w6) Dw20 Dw21 Dw22 Dw23 Dw24 Dw25 Dw26

DR13(6) DR14(6) DR1403 DR1404 DR15(2) DR16(2) DR17(3) DR18(3) DR51 DR52 DR53

DQ

DP

715

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2600 2400 Class I alleles Class II alleles

2200 2000 Number of alleles

1800 1600 1400 1200 1000 800 600 400 200 8 19 9 9 19 0 91 19 9 19 2 9 19 3 9 19 4 9 19 5 96 19 97 19 9 19 8 9 20 9 00 20 0 20 1 0 20 2 0 20 3 0 20 4 0 20 5 0 20 6 07 20 08 20 09

19

19

87

0 Year

Alloreactions are responsible for the inability to transplant organs and cells between genetically different individuals. To overcome alloreactivity, it is necessary to define the HLA type of donor and recipient and to select an HLA-matched donor wherever possible. In solid organ transplantation, HLA matching is encouraged for kidney transplants, where outcome data support a beneficial role of HLA matching. However, immunosuppressive therapy is extremely effective in renal transplantation and this allows for imperfect matching to be performed and optimum usage of all available organs. Nevertheless, it is critical to avoid transplantation of mismatched donor kidneys possessing HLA types against which the patient has raised alloantibodies as a result of a previous mismatched transplant, pregnancy or blood transfusion. The presence of such alloantibodies can lead to hyperacute rejection, but immunodepletion protocols may be used to remove such donor-specific antibodies. HLA matching for other solid organs such as liver and heart and lung is not usually performed. The liver is an immunoprivileged site that tolerates HLA mismatches, whereas heart and lung transplants are usually performed on patients for whom no other chance of life is possible, thereby disallowing selection of HLA-matched donors. Alloreactions mediated following solid organ transplantation are usually only directed at the transplanted organ and with effective post-transplant immune monitoring can be managed with appropriate immunosuppression. Patients receiving blood products, for example random pooled platelets, can produce antibodies against the HLA molecules present on transfused platelets. The presence of HLAspecific antibodies can lead to platelet refractoriness, and for 716

Figure 37.3 Increase in number of HLA alleles identified by year.

these patients it is advisable to seek HLA-matched platelet donors. Alloreactions occurring after haemopoietic stem cell transplantation (SCT) can result in failure of the cells to engraft or graft-versus-host disease (GVHD), in which donor cells mediate attack on various tissues within the host’s body. The risks of graft rejection and GVHD are significantly reduced if the donor cells are histocompatible with the patient. The perfect donor is an identical twin. However, this will only be the case for very few patients. The chances of finding an HLA-matched sibling donor for a patient is theoretically one in four (25%). Therefore, again, the majority of patients will not be able to find a suitable donor within their family. Another source of HLA-matched donors are the various volunteer donor registries that exist throughout the world. The first volunteer donor register, the Anthony Nolan Trust, was established in London in 1974 at a time when unrelated donor transplants were viewed as experimental. Since this time, the outcome of unrelated donor transplantation has vastly improved and the use of unrelated volunteer donors is now accepted practice. There are now over 60 volunteer donor registers throughout the world. In addition, the successful use of cord blood-derived stem cells for transplantation has driven the establishment of cord blood banks in many countries. An advantage of using cord blood cells in clinical transplantation is that a reduced degree of HLA matching is acceptable. Thus patients who fail to find a matched unrelated donor may receive mismatched cord blood stem cells. There are a number of factors that may negate the use of cord blood stem cells, such as the number of nucleated cells to be infused, particularly for

Histocompatibility

adult patients, as these may be insufficient to enable engraftment, and the inability to obtain donor cells for future donor lymphocyte infusion. However, the use of double cord units in adults can overcome the problem of cell numbers and it is certain that cord blood will be an important player in widening the application of haemopoietic SCT. There are over 13 million potential volunteer adult donors and cord blood units registered for use in haemopoietic SCT worldwide. Unrelated adult and cord blood donations take place as a result of the successful international networking of the worldwide adult donor registries and cord blood banks, under the auspices of the World Marrow Donor Association. In 2007, 42% of haemopoietic stem cell donations were sourced in a country different to that of the patient. Details on both volunteer donor and cord blood registries can be found on www.bmdw.org.

HLA matching required for haemopoietic stem cell transplantation The importance of HLA matching in haemopoietic SCT is well accepted. However, the degree of resolution required at each locus for an optimum match is not fully understood. Data obtained from large multicentre registry studies support the importance of optimum matching, which consists of allele-level matching for HLA-A, HLA-B, HLA-C and HLA-DRB1. The role of HLA-DQB1 is still not defined. Mismatching for HLADQB1 does not appear detrimental in studies published by the National Marrow Donor Program; however, an HLA-DQB1 mismatch with another HLA mismatch (HLA-A, -B or -C) was more detrimental than a single HLA-A, -B or -C mismatch. Furthermore, a hierarchy of beneficial mismatching has not been proven in terms of antigen-level mismatching over allelelevel mismatching. Any type of mismatch can have a negative effect on transplant outcome. Because HLA-B and HLA-C are in linkage disequilibrium (i.e. matching for HLA-B increases the chance that HLA-C will also be matched), it is more difficult to mismatch for one and not the other. Similarly, HLA-DRB1 and -DQB1 genes are in linkage disequilibrium. HLA-A is the most telomeric of the HLA genes and in less linkage disequilibrium with other HLA class I genes; therefore, when selecting an HLA-mismatched donor, HLA-A is usually the easiest locus to mismatch. Therefore most mismatching targets HLA-DQB1 and then HLA-A. However there are no data to support HLA-A mismatching as being clinically more acceptable than mismatching for other single loci. The role of HLA-DPB1 is emerging in analysis of large patient and donor cohorts, including the International Histocompatibility Workshop studies. In these studies, HLA-DPB1 mismatching is associated with reduced disease relapse, together with an increase in acute GVHD. The association between HLA-DPB1 and disease relapse suggests that HLA-DP molecules may mediate a functional role in a graft-versus-leukaemia response.

Histocompatibility testing procedures Detection of HLA polymorphisms can be performed by either targeting the DNA sequence of the HLA gene or by analysis of the expressed protein.

Serology Serology describes the use of antibodies to detect epitopes on target antigens. HLA polymorphism results in the presence of different antigenic epitopes on the protein molecule. Antibodies used to define these antigenic epitopes are usually obtained from multiparous women, and can also be created using monoclonal antibody technology. The latter has the advantage of being available in unlimited supply. Serology involves the incubation of peripheral blood mononuclear cells (PBMCs), or separated T and B cells for distinguishing HLA class I from class II, with serum containing antibodies of known antiHLA specificity. If the PBMCs express the appropriate HLA molecules, then binding between the antibodies in the serum and the PBMCs will occur. This binding will result in lysis of the PBMCs after the addition of complement components. The cells can be stained in order to detect which cells are alive (no reaction) and which cells are dead (positive reaction). The pattern of positive and negative reactions is then interpreted to give an HLA type. In order to obtain a full HLA type, panels of antisera have to be screened to cover the range of recognized HLA specificities. Despite the number of serological reagents that have been analysed, there are many HLA class I and II polymorphisms which remain undetected by serology (see Table 37.1). Many of these polymorphisms are not present on the surface of the HLA molecules but are found within the peptide-binding site, and could alter the peptide-binding specificity of the HLA molecule and hence the antigen presentation function of the molecule. Thus serological typing does not detect all the functional polymorphisms of HLA molecules.

DNA-based methods The introduction of the polymerase chain reaction (PCR) for analysis of DNA has led to the establishment of rigorous DNAbased methodologies that can be applied for genetic diagnostics. DNA-based methods are now used routinely for histocompatibility typing. The most frequent HLA class I antigen in white populations and the first HLA antigen to be defined, HLA-A2, encompasses three serological variants. Sequencing analyses have shown that at least 199 alleles encode the HLA-A2 specificity. Of these 199 alleles, 155 differ by substitutions that are predicted to influence the antigen presentation function of the HLA-A2 molecule, and 10 alleles (A*02:15N, A*02:32N, A*02:43N, A*02:53N, A*02:82N, 717

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A*02:83N, A*02:88N, A*02:94N, A*92:13N and A*92:25N) contain mutations which prevent expression of the HLA-A2 antigen. For HLA class I molecules the polymorphism is mostly localized within the α1 and α2 domains, which are encoded by exons 2 and 3 respectively of the class I gene. For HLA class II, the α1 domain of the β-chain, encoded by exon 2, is most polymorphic for DRB1, whereas for DQ and DP, the polymorphism extends to both the α1 and β1 domains. Thus DNA methods for HLA class I typing focus predominantly on exons 2 and 3, whereas for HLA class II exon 2 is targeted. Sequence-specific oligonucleotide and sequencespecific primer methods The most widely used DNA-based methods for HLA typing are PCR sequence-specific primers (PCR-SSP) and PCR sequencespecific oligonucleotides (PCR-SSO). These methods use DNA primers (SSP) or DNA probes (SSO) that react with polymorphic sequence motifs present within the nucleotide sequence of HLA alleles. The presence of a positive reaction indicates that the polymorphism defined by the primer or probe is present in the DNA sample, whereas a negative reaction defines the absence of that particular sequence polymorphism. For PCR-SSO typing, the target DNA molecule is amplified by locus-specific PCR (e.g. all HLA-A alleles are amplified in one PCR) or by group-specific PCR (e.g. all DRB1*04 alleles are amplified in one PCR). The PCR product is immobilized directly onto a solid-phase matrix containing pre-immobilized HLA-specific oligonucleotide probes, for example nylon membrane or Luminex xMAP microspheres. Interaction between the immobilized probes and HLA PCR product is measured as positive, whereas no binding between probe and PCR product is measured as negative. Most methods utilize specific software to measure positive and negative reactions and to assign the interpreted HLA type.

Specific amplification products Control amplification products

Figure 37.4 Example of PCR-SSP test. Photograph of an ethidium bromide-stained agarose gel containing electrophoresed PCR products. In this example, 24 PCR primer mixes have been utilized to determine HLA-DRB1*11 subtype. Each lane on the gel contains the product from amplification of a nonpolymorphic region of the genome. The presence of this product indicates successful amplification. A second band is observed in 718

PCR-SSP typing (Figure 37.4) utilizes multiple PCR primer pairs all tested independently on the same sample DNA. The primer pairs are designed such that the 3′ end defines the specificity of the primer with the target sequence. To obtain a full HLA type requires numerous primer pairs which have been designed to operate under identical PCR amplification conditions. If the target sequence for a primer pair is present in the sample DNA, a PCR product will be produced, whereas if the target sequence is not present there will be no PCR product. The presence and absence of PCR products can be visualized after agarose gel electrophoresis. Because of the extensive HLA polymorphism, specialized software is usually required to interpret results. PCR-SSP can be used to perform a full HLA type or to target a particular group of HLA alleles to obtain a highresolution type (e.g. subtype A*02). The resolution of the typing results obtained by PCR-SSO and PCR-SSP methods can vary depending on the number of probes or primer mixes utilized, with an increasing number required for higher resolution. As SSO methods utilize less PCR steps, this method is more useful for high-throughput HLA genotyping, for example as required by laboratories with large sample numbers, including unrelated haemopoietic donor registry laboratories. SSP methods are more suitable for smaller volumes and are reserved for quick turnaround typing or for achieving higher resolution on particular samples. Regardless of the number of typing reagents used with PCRSSP and PCR-SSO methods, the end result is the presence or absence of a reaction between a probe or primer of a known sequence and the target HLA sequence. Thus the data generated can be directly related to the DNA sequence of the HLA alleles possessed by an individual. Direct sequencing The generation of a database of known nucleotide sequences of HLA alleles has had a tremendous impact on the transfer of

Molecular weight ladder

lanes 1, 3, 6 and 9, where specific amplification of a polymorphic region from an HLA gene is achieved. If no second band is present, then the individual being tested does not possess the complementary HLA polymorphism. The pattern of positive and negative reactions (presence and absence of second bands) is interpreted to give the HLA type. (Courtesy of Franco Tavarozzi, The Anthony Nolan Trust.)

Histocompatibility

methodologies from serology to DNA-based techniques. Improvements in automated instrumentation for DNA sequencing has also made this methodology an affordable alternative for HLA typing. The sequencing techniques utilized for HLA typing involve PCR amplification and direct sequencing of the PCR product. The strategies developed have focused on the amplification of DNA fragments containing the polymorphic exons, which for the majority of class I alleles extends to a fragment containing exons 2 and 3 at a minimum. The overall size of an HLA class I gene is about 3.2 kb and therefore it is feasible to target all exons (and introns) within the gene to define the polymorphisms present; this additional information greatly increases the resolving power of sequencing methods. HLA class II genes are much larger than HLA class I genes and the amount of sequence information available for exons outside exon 2 is limited. HLA class II sequencing methods minimally target exon 2, with additional data sought from exon 3 to resolve some exon 3 known polymorphisms. HLA-DQB1 also benefits from analysis of both exon 2 and exon 3. As most loci are heterozygous, assignment of HLA type is dependent on the use of software capable of assigning heterozygous positions. In addition an up-to-date database of all known HLA sequences is required (Figure 37.5). The advantages of DNA-based methods over serology are numerous and include the ease with which typing reagents (primers, probes) can be synthesized and the interpretation of the data as directly relating to nucleotide sequence, whereas the data obtained with polyclonal antisera requires extensive knowledge of cross-reactivities of sera with different HLA antigens. In addition, the storage of material is simplified for DNA typing, as live cells are not required. Ambiguities DNA typing methods can generate ambiguous results when both alleles in a heterozygous combination are analysed together. Therefore it can be difficult to determine whether a sequence motif is in the cis or trans orientation compared with a different motif. This problem is overcome by performing allele-specific amplifications on samples with ambiguities prior to analysis, usually by SSO or sequencing. Alternatively, sequencing primers can be designed to target particular polymorphisms, allowing separation of the two alleles in a heterozygous sample. Fewer ambiguities are obtained using PCR-SSP as the PCR primers used link cis polymorphisms.

Cellular assays Cellular methods for assessing potential alloreactivity between patient and donor can also be utilized for the selection of the most appropriate stem cell donor. The mixed lymphocyte culture (MLC) assay has been used since the 1960s. MLC meas-

(a) GGCTGCGACGTGGGGTCGGACGGGCGCTTCCTCCGCGGGTACCRSCAGKACGCCTACGACGGCAAGGATTACATCGCCCTGAAMGAGGA GGCTGCGACGTGGGGTCGGACGGGCGCTTCCTCCGCGGGTACCRSCAGKACGCCTACGACGGCAAGGATTACATCGCCCTGAAMGAGGA GENERIC_A_Ain3_2009-03-30_F04 Rev A: 254 C: 854 G: 443 T: 300

GGCTGCGACGTGGGGTCGGACGGGCGCTTCCTCCGCGGGTACCRSCAGTACGCCTACGACGGCAAGGATTACATCGCCCTGAAMGAGGA

a/g c/g

g/t

(b) GGCTGCGACGTGGGGTCGGACGGGCGCTTCCTCCGCGGGTACCACCAGTACGCCTACGACGGCAAGGATTACATCGCCCTGAAAGAGGA GGCTGCGACGTGGGGTCGGACGGGCGCTTCCTCCGCGGGTACCACCAGTACGCCTACGACGGCAAGGATTACATCGCCCTGAAAGAGGA

GGCTGCGACGTGGGGTCGGACGGGCGCTTCCTCCGCGGGTACCACCAGTACGCCTACGACGGCAAGGATTACATCGCCCTGAAAGAGGA

Group A_A_Aint2_2009-04-07_G09 A: 305 C: 944 G: 473 T: 454

a

c

t

(c) GGCTGCGACGTGGGGTCGGACGGGCGCTTCCTCCGCGGGTACCGGCAGGACGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGA GGCTGCGACGTGGGGTCGGACGGGCGCTTCCTCCGCGGGTACCGGCAGGACGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGA

GGCTGCGACGTGGGGTCGGACGGGCGCTTCCTCCGCGGGTACCGGCAGGACGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGA Group B_A_Aint2_2009-04-07_A09 A: 186 C: 488 G: 246 T: 261

g

g

g

Figure 37.5 HLA sequencing electropherogram. The electropherograms are from ASSIGN software analysis of sequencing performed on an HLA-A PCR product containing exons 2, 3 and 4. (a) Heterozygous loci. The sequence is read from left to right. For this short stretch of nucleotides (12 from 822 analysed for HLA-A), eight different results are possible for the heterozygous nucleotides: ACG, ACT, AGG, AGT, GCG, GCT, GGG and GGT. Overall analysis of all 822 nucleotides gives an ambiguous result of A*03:01, *24:02 or A*03:07, *24:56 or A*03:08, *24:07 or A*03:15, *24:53 or A*03:17, *24:21. (b, c) Separated loci. By sequencing the two HLA-A alleles after separation using allele-specific PCR primers, the heterozygous ambiguities are resolved. The result for (b) gives ACT and for (c) gives GGG. The final result for this ambiguity, after analysis of all 822 nucleotides sequenced, is HLA-A*03:01, *24:02. (Courtesy of Shem Wallis-Jones, The Anthony Nolan Trust.)

719

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ures the reactivity of donor T cells against alloantigens expressed by patient cells, i.e. graft versus host direction. By mixing both donor and patient cells together, any alloreactions that occur will cause the responding donor cells to proliferate and incorporate 3H-thymidine, which can be measured. The patient cells are prevented from proliferating by being gamma-irradiated prior to the mixing of cells. However, since the introduction of DNA-based methods for HLA class II typing, the MLC reaction is infrequently used. Indeed studies have indicated that positive MLC reactions in the absence of detectable HLA class II mismatches are not predictors of the development of GVHD after bone marrow transplantation. More sensitive cellular assays have been developed to measure HLA disparity. The cytotoxic T-lymphocyte precursor (CTLp) assay uses limiting dilution analysis to measure the frequency of donor CTLs responding to predominantly HLA class I mismatches on patient cells. High frequencies of CTLp have been shown to be strongly associated with HLA class I mismatches, which may have escaped detection by the low to medium level of resolution of conventional typings that were widely used for class I matching prior to the use of allele-level DNA typing. Similarly, the helper T-lymphocyte precursor (HTLp) assay measures the HTL response of the donor to HLA class II mismatches, with HLA-DR mismatches associated with high HTLp frequencies with no apparent contribution from HLA class I mismatches. Both CTLp and HTLp assays can be combined in a single limiting dilution analysis, thus minimizing the use of often valuable material. The drawback of cellular assays is that they take up to 2 weeks to perform, and they require viable cells from both donor and patient. With more widespread use of high-resolution HLA typing techniques, the number of laboratories performing cellular assays has decreased significantly.

Other genetic polymorphisms HLA polymorphisms clearly play an important role in determining the outcome of haemopoietic SCT. There are also other genetic polymorphisms that have been demonstrated to influence outcome. Minor histocompatibility antigens (mHA) are alloantigens that are also capable of initiating an immune response when their genes are mismatched for patient and donor, despite the presence of HLA matching. Minor histocompatibility antigens are peptides derived from normal self proteins that possess polymorphisms. A donor and patient may share HLA type, but differ in the polymorphism found in the mHA gene. Several human mHA have been defined, for example HA1, HA2, HA3. The genes encoding several of these mHA have been described and this has allowed the development of typing procedures for these genes using techniques such as PCR-SSP. Single-nucleotide polymorphisms (SNPs) in other genes, particularly cytokines, have also been shown to have an impact 720

on the outcome of SCT. Recent studies have described the significant correlation between transplant outcome in patients suffering from acute leukaemias and three SNPs in the CARD15 (caspase recruitment domain 15) gene, now called NOD2 (nucleotide-binding oligomerization domain 2). These may be defined by a variety of techniques that are able to target single mutations within a gene. There is therefore a choice of techniques that may be used for optimum donor selection for patients requiring SCT. Each methodology has its advantages and disadvantages. It is now accepted practice that DNA-based typing should be performed on all related and unrelated donors and patients. Serology and cellular assays may be used to complement the outcome of DNA typing. A major challenge for the future of histocompatibility typing for SCT will be to acquire a greater understanding of what types of mismatches are acceptable in that they do not result in adverse alloreactions, and also to know which mismatches are detrimental. It is likely that more sensitive and reproducible cellular assays may play a role in determining these mismatches. The use of ELISPOT assay and real-time PCR methods for detecting low numbers of cytokine-producing cells may in the future become additional tools for the histocompatibility laboratory, especially for the selection of best mismatched donors when no optimum matched donor is available.

Selected bibliography History Klein J (1986) Natural History of the Major Histocompatibility Complex. John Wiley & Sons, New York. HLA molecular structure Bjorkman PJ, Saper MA, Samraoui B et al. (1987) Structure of the human class I histocompatibility antigen, HLA-A2. Nature 329: 506–12. Brown JH, Jardetzky TS, Gorga JC et al. (1993) Three-dimensional structure of the human class II histocompatibility antigen HLADR1. Nature 364: 33–9. HLA and KIR Kulkarni S, Martin MP, Carrington M (2008) The Yin and Yang of HLA and KIR in human disease. Seminars in Immunology 20: 343–52. Parham P (2006) Taking license with natural killer cell maturation and repertoire development. Immunological Reviews 214: 155–60. HLA nomenclature Holdsworth R, Hurley CK, Marsh SGE et al. (2009) The HLA Dictionary 2008: a summary of HLA-A, -B, -C, -DRB1/3/4/5, -DQB1 alleles and their association with serologically defined HLA-A, -B, -C, -DR and -DQ antigens. Tissue Antigens 73: 95–170.

Histocompatibility

Marsh SGE, Albert ED, Bodmer WF et al. (2010) Nomenclature for factors of the HLA system, 2010. Tissue Antigens 75: 291–455. Marsh SGE, Parham P, Barber LD (2000) The HLA Facts Book. Academic Press, London. Robinson J, Waller MJ, Fail SC et al. (2009) The IMGT/HLA database. Nucleic Acids Research 37: D1013–D1017.

HLA polymorphism Little A-M, Parham P (1999) Polymorphism and evolution of HLA molecules. Reviews in Immunogenetics 1: 105–23. HLA matching in haemopoietic stem cell transplantation Bray RA, Huryley CK, Kamani NR et al. (2008) National Marrow Donor Program HLA matching guidelines for unrelated adult donor hematopoietic cell transplants. Biology of Blood and Marrow Transplantation 14: 45–53. Lee SJ, Klein J, Haagenson M et al. (2007) High-resolution donor– recipient HLA matching contributes to the success of unrelated donor marrow transplantation. Blood 110: 4576–83. Petersdorf EW (2008) Optimal HLA matching in hematopoietic cell transplantation. Current Opinion in Immunology 20: 588–93.

Shaw BE, Gooley TA, Malkki M et al. (2007) The importance of HLA-DPB1 in unrelated donor hematopoietic cell transplantation. Blood 110: 4560–6.

Minor histocompatibility antigens Spaapen R, Mutis T (2008) Targeting haematopoietic-specific minor histocompatibility antigens to distinguish graft-versustumour effects from graft-versus-host disease. Best Practice and Research. Clinical Haematology 21: 543–57.

Histocompatibility testing procedures Beksac M (2007) Bone Marrow and Stem Cell Transplantation. Humana Press, Totowa, NJ. Bidwell JL, Navarrete C (2000) Histocompatibility Testing. Imperial College Press, London. Powis SH, Vaughan RW (2003) MHC Protocols. Humana Press, Totowa, NJ. Useful URLs IMGT/HLA Database: www.ebi.ac.uk/imgt/hla/ HLA Nomenclature: hla.alleles.org World Marrow Donor Association: www.worldmarrow.org HLA population data: www.allelefrequencies.net

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CHAPTER 38

38

Stem cell transplantation Charles Craddock1 and Ronjon Chakraverty2 1

Centre for Clinical Haematology, Queen Elizabeth Hospital, Birmingham, UK Royal Free and University College Medical School, London, UK

2

Introduction, 722 Immunological basis of stem cell transplantation, 722 Antigens and cellular effectors, 722 Acute graft-versus-host disease, 724 Chronic graft-versus-host disease, 724 Graft-versus-leukaemia effect, 724 Immune reconstitution, 724 Stem cell engraftment, 726 Biology of stem cell engraftment, 726 Clinical factors determining stem cell engraftment, 726 Stem cell mobilization, 727 Biology of stem cell trafficking, 727 Stem cell mobilization in clinical practice, 727 Choice of stem cell source and dose, 727 Stem cell manipulation and expansion, 728

Conditioning regimens: basic principles, 729 Conditioning regimens in autologous SCT, 729 Myeloablative conditioning regimens in allogeneic SCT, 730 Comparison of myeloablative conditioning regimens, 730 Strategies for GVHD prophylaxis in myeloablative regimens, 731 Reduced-intensity conditioning regimens in malignant and non-malignant disease, 731 Choice of RIC regimen, 732 Strategies for GVHD prophylaxis in RIC regimens, 732 Clinical management of patients undergoing stem cell transplantation, 732 Practicalities of stem cell infusion and blood product support, 733

Introduction Haemopoietic stem cell transplantation (SCT) represents an important and increasingly utilized curative therapy in haematological malignancies and has an emerging role in the management of patients with haemoglobinopathies and bone marrow failure. Improvements in supportive care and treatment of post-transplant complications, a greater availability of alternative donors and advances in tissue typing have simultaneously improved transplant outcome and patient eligibility. The realization that an immunologically mediated graft-versus-leukaemia (GVL) effect makes a major contribution to the curative effect of an allograft has underpinned the concept that allogeneic transplantation can be used as a platform for antitumour immunotherapy. These advances have coincided with the advent of reduced-intensity conditioning (RIC) regimens, which have substantially reduced the morbidity and mortality of allografting and resulted in a marked increase in the number of stem cell transplants, particularly allografts, performed over this period in Europe and worldwide (Figure 38.1).

Complications of allogeneic SCT, 733 Complications of autologous SCT, 741 Patient factors determining outcome after allogeneic stem cell transplantation, 741 Indications for transplantation, 741 Factors determining the choice of an allogeneic stem cell donor: the donor algorithm, 742 Management of disease relapse, 743 Future developments in stem cell transplantation, 744 Autologous SCT, 744 Allogeneic SCT, 744 Selected bibliography, 745

Immunological basis of stem cell transplantation The major complications of allogeneic SCT are caused by the immunological responses triggered by the infusion of donor haemopoietic progenitors and lymphocytes into an immunosuppressed host. These can take the form of either a hostversus-graft (HVG) or a donor-derived graft-versus-host (GVH) response. Clinically, the HVG response can result in graft rejection, while a GVH response may manifest itself as either graft-versus-host disease (GVHD) or a GVL reaction. It is now possible to blunt the HVG reaction by optimizing the immunosuppressive properties of the conditioning regimen and consequently graft rejection is rare in most clinical settings. In contrast, GVHD and disease relapse remain the major complications of allogeneic transplantation and novel approaches that permit the induction of GVL without inducing host injury are required. Conversely, autologous SCT is a relatively unremarkable immunological event in which these allogeneic responses are absent.

Antigens and cellular effectors Postgraduate Haematology: 6th edition. Edited by A. Victor Hoffbrand, Daniel Catovsky, Edward G.D. Tuddenham, Anthony R. Green © 2011 Blackwell Publishing Ltd.

722

The antigens against which HVG and GVH responses are directed include the products of highly polymorphic genes lying

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within the major human leucocyte antigen (HLA) complex on chromosome 6 and minor histocompatibility antigens (minor H antigens) encoded by a number of disparate genes lying outside the HLA system. These antigens and the methods employed to identify them have been reviewed in Chapter 37. In very general terms, the degree of difference or ‘incompatibility’ between the donor and recipient will define the risk of graft rejection and/or GVHD. Thus, in situations where the donor is genetically identical to the recipient (syngeneic transplantation), graft rejection or significant GVHD is not observed. For patients undergoing allogeneic transplantation using an HLA-identical sibling, the relevant antigens are minor H antigens, which are polymorphic proteins recognized by T cells as processed peptides in the context of a ‘self ’ HLA molecule. To date, just over 20 such antigens have been identified and include antigens that are ubiquitously expressed on host tissue (e.g. HY antigens encoded by genes on the Y chromosome) or which have restricted tissue distribution (e.g. HA-1 or HA-2, which are only expressed by the haemopoietic system). Female donors, specifically multiparous individuals who have had male infants, may have been primed against HY antigens and as a consequence have circulating T cells that recognize male patient cells expressing these proteins. This is likely to underlie the increased risk of GVHD in male recipients of female grafts. Where a patient has no HLA-identical sibling donor, an alternative donor may be considered. In this case, the transplant centre will generally seek a donor who is matched with the recipient at each of the HLA-A, -B, -C, -DRB1 and -DQB1 loci (the so-called ‘10/10 match’). Even with a complete 10/10 match, the risk of GVHD is higher than for an HLA-identical sibling, presumably because the number of polymorphic minor H antigen differences is likely to be greater. In the situation where the donor and recipient are mismatched for one or more class I or II HLA antigens, the risk of transplant complications

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12 months) after transplantation or by using an escalating dose schedule rather than a single ‘bulk’ infusion. For the most part, the antigens recognized in a GVL reaction overlap with those recognized during GVHD. Clinical separation of GVL and GVHD may reflect increased sensitivity of normal or malignant haemopoietic tissue to an emerging GVH reaction compared with epithelial cells. Selective expression of antigenic targets of a GVH reaction on haemopoietic tissues (e.g. the minor H antigen HA-1, proteinase 3 or WT1) or leukaemic blasts (e.g. the product of the BCR–ABL1 fusion gene) may also underlie the development of a GVL reaction in the absence of GVHD. This concept forms the basis of novel strategies to deliver a GVL effect without a concomitant risk of GVHD, such as peptide vaccination or gene transfer of T-cell receptors (TCRs) specific for leukaemic antigens.

Immune reconstitution Allogeneic SCT is followed by a prolonged period of cellular and humoral immunodeficiency while donor-derived immune

Stem cell transplantation

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Figure 38.2 Pathophysiology of acute GVHD (a) and chronic GVHD (b). See text for for explanation of abbreviations.

Postgraduate Haematology

recovery occurs. Reconstitution of an immune response after transplantation can be evaluated in the clinic by monitoring the absolute numbers of T (CD4, CD8), B and NK cell numbers. NK cell numbers recover most rapidly while other subsets (especially CD4 cells and B cells) recover more slowly. In patients who have received a T-cell-depleted graft, T-cell recovery is substantially delayed. Analysis of thymic function after transplantation can be evaluated by measuring the episomal DNA excision circles of the TCR δ locus deleted during recombination of the TCR in functional αβ T cells (known as TREC, for T-cell receptor excision DNA circles). TREC levels are low for the first 6 months following transplantation and recover thereafter. As a consequence, the T-cell repertoire is limited and mostly dependent on expansion of donor memory T cells. Thymic function is reduced in adults and may be further compromised by the effects of chemoradiotherapy and GVHD. Significant HLA mismatching between donor and recipient may also lead to ‘holes’ within the T-cell repertoire due to perturbations in thymic selection. These defects reflect a failure of donor-derived thymic emigrants to interact with peptides presented in the context of ‘foreign’ host HLA molecules. Quantitative B-cell deficiency is present in virtually all patients in the first months after transplantation and may persist for a number of years post transplantation as a consequence of reductions in the number of marrow B-cell precursors, particularly in patients with chronic GVHD. This defect in B-cell production has multifactorial causes, including damage to the bone marrow stroma, the deleterious effect of inflammatory cytokines and the lympholytic effects of glucocorticoid therapy. The source of stem cells also influences the kinetics of immune reconstitution. Thus, peripheral blood stem cell (PBSC) grafts, which contain higher numbers of haemopoietic progenitors and mature T cells, are associated with more rapid immune reconstitution than bone marrow grafts. Cord blood transplantation may be affected by poor immune reconstitution in adults since the number of haemopoietic progenitors is often limited and the transferred T cells are naive. Thus, patients are at heightened risk of reactivation of cytomegalovirus (CMV) or Epstein–Barr virus (EBV).

Stem cell engraftment Biology of stem cell engraftment The establishment of durable donor haemopoiesis after SCT depends on the engraftment of long-term reconstituting haemopoietic stem cells (HSCs) (see Chapter 1). These cells, defined by their capacity for self-renewal as well as their ability to differentiate into all haemopoietic lineages, are normally resident in the bone marrow at low frequency but can be mobilized into the peripheral blood by cytokines or chemotherapy. A high 726

frequency of haemopoietic stem and progenitor cells is also present in UCB. The cell surface glycoprotein CD34 is expressed on haemopoietic progenitors and HSCs and is currently widely used as a stem cell marker in clinical transplantation. However, it is important to remember that xenograft studies have demonstrated that HSCs reside within a CD34+CD38− subpopulation and that many of the cells within the CD34+ population lack the properties of a long-term reconstituting stem cell. Murine transplant studies have established that in a syngeneic setting, where there is no HVG reaction, transplantation of very small numbers of HSCs can result in durable engraftment of lethally irradiated recipients. In contrast, host immunosuppression is required to blunt an HVG reaction capable of rejecting the transplanted stem cell inoculum when there is donor–host HLA disparity, whether the donor is an HLAidentical sibling or an alternative donor.

Clinical factors determining stem cell engraftment The sole determinant of durable engraftment after an autologous or syngeneic transplant is stem cell number and graft failure is exceedingly rare providing at least 2 × 106 CD34+ cells per kilogram are transplanted. Clinically durable engraftment in allograft recipients is determined by the degree of HLA disparity, the number of transplanted T cells and the size of the stem cell inoculum (Table 38.1). The immunosuppressive properties of the conditioning regimen play a critical role in blunting the HVG response and incorporation of total body irradiation (TBI) or drugs such as cyclophosphamide or fludarabine are highly effective in facilitating HSC engraftment. Historically, high rates of graft failure were observed when TCD was introduced as a form of GVHD prophylaxis in the late 1980s. However, the recognition that donor T cells play a critical role in facilitating engraftment led to redesign of conditioning regimens in patients receiving T-cell-depleted grafts so that their immunosuppressive properties were optimized. The widespread availability of PBSCs from sibling or unrelated donors (see below) allows transplantation of five to ten times more CD34-positive cells than if harvested bone marrow were used. Consequently, the use of PBSCs has played an important role in optimizing engraftment in settings such as TCD or transTable 38.1 Factors determining stem cell engraftment. Autologous transplantation Stem cell dose Allogeneic transplantation Intensity of host immunosuppression delivered by the conditioning regimen Numbers of donor T cells in the stem cell inoculum Degree of genetic disparity between donor and host

Stem cell transplantation

plantation of a mismatched donor where a significant risk of graft failure rate would be expected were bone marrow to be used as the stem cell source. Incorporating these principles into clinical practice has markedly reduced the risk of graft failure such that it occurs in fewer than 1% of patients undergoing an HLA-identical sibling allograft and fewer than 5% of those transplanted from an unrelated donor. For a long time graft failure was a major complication of cord blood transplantation (CBT), particularly in adult recipients, and this likely reflected the low stem cell dose. The recent demonstration that the simultaneous transplantation of two cord blood units delivers durable rates of engraftment in the region of 95% has dramatically improved outcome of UCB transplants in adults.

Stem cell mobilization Biology of stem cell trafficking In steady-state haemopoiesis only very small numbers of haemopoietic stem and progenitor cells are present in the peripheral blood. The localization of haemopoietic stem and progenitor cells within the bone marrow cavity is mediated through the binding of a range of adhesion molecules, including CXCR4 and VLA4, with their cognate ligands on bone marrow stroma. Recent studies have demonstrated that administration of haemopoietic growth factors such as granulocyte colony-stimulating factor (G-CSF) disrupts the adhesion of progenitors to the bone marrow stroma resulting in their mobilization into the peripheral blood in large numbers. This has had a dramatic impact on transplant practice, resulting in peripheral blood replacing bone marrow as the commonest source of haemopoietic progenitors in both autologous and, more recently, allogeneic transplants. Antagonists to CXCR4 have now been shown to have the potential to significantly augment G-CSF-mediated stem cell mobilization and this promises to increase further the effectiveness of cytokine-mediated stem cell mobilization. Adhesive interactions between haemopoietic progenitors and the bone marrow stroma also play a critical role in the homing of transplanted HSCs and stem cell engraftment can be augmented in animal models by manipulation of the levels of adhesion molecule expression on haemopoietic progenitors and bone marrow stroma. As yet these observations have not been translated into clinical practice.

Stem cell mobilization in clinical practice In patients undergoing autologous SCT, G-CSF administered alone or after myelosuppressive chemotherapy represents the commonest method of stem cell mobilization. Until recently G-CSF was only administered in conjunction with myelosuppressive doses of salvage chemotherapy or cyclophosphamide. While this represents a highly effective method of stem cell mobilization in the majority of patients, it is now clear that

administration of G-CSF alone, albeit at somewhat higher doses, can be equally effective and is substantially less toxic. As a result many units now utilize four daily subcutaneous injections of G-CSF, with stem cell harvesting on days 5 and 6. The minimum target cell dose of 2 × 106 CD34+ cells per kilogram can be harvested from more than 90% of patients using either G-CSF alone or G-CSF in combination with salvage chemotherapy or cyclophosphamide. In patients who fail to mobilize the target number of CD34+ cells, a number of possibilities exist. A second mobilization procedure can be performed using higher doses of G-CSF or, where appropriate, an additional chemotherapeutic drug such as cyclophosphamide or etoposide. Alternatively, the CXCR4 antagonist plerixafor can be used in conjunction with G-CSF. In the small minority of eligible patients who fail to mobilize adequate numbers of PBSCs using this approach, allogeneic transplantation should be considered. G-CSF-mobilized PBSCs are increasingly used as a stem cell source in allogeneic SCT. The apparent safety of G-CSF doses in the range routinely used for autologous stem cell mobilization (10–15 µg/kg for 4–6 days) has resulted in all major unrelated donor panels offering this as an alternative to bone marrow harvest. However, it is important that all donors are counselled about the attendant side-effects in the form of myalgia, bone pain and headache. Other reactions are less common, and there are rare reports of splenic rupture. Consequently, donors should be advised to report immediately any pain in the left upper quadrant or shoulder tip. Although the effects of G-CSF on the fetus are not known, all female donors of childbearing age should have a negative pregnancy test prior to its administration. Donors also need to be aware of the 2–3% chance that an additional bone marrow harvest will be required if G-CSF fails to mobilize sufficient PBSC.

Choice of stem cell source and dose Autologous transplantation The use of PBSCs as a stem cell source is associated with more rapid engraftment than would be observed with harvested bone marrow and this, coupled with their ease of procurement, has secured their role as the standard stem cell source in autologous transplants. The generally accepted minimum number of haemopoietic progenitors required for engraftment after autologous SCT is 2 × 106 CD34+ cells per kilogram. While increasing the number of CD34+ cells transplanted hastens neutrophil and platelet engraftment, there is little evidence that transplantation of more than 5 × 106 CD34+ cells per kilogram is beneficial and there remain concerns that higher stem cell doses may be associated with an increased risk of tumour contamination. In patients with myeloma, many centres aim to harvest a minimum of 4 × 106 CD34-positive cells per kilogram in order that sufficient cells are available so that a tandem transplant, or second autograft at the time of relapse, is possible. 727

Postgraduate Haematology

Sibling and unrelated donor transplantation PBSCs are the most frequently used stem cell source for patients undergoing a sibling allograft and are increasingly used in unrelated donor transplant recipients. While PBSCs are established as the preferred stem cell source in reduced-intensity allografts, for the reasons discussed above, the relative merits of PBSCs and bone marrow in patients transplanted using a myeloablative regimen remains a matter of debate. Thus while transplantation of PBSCs results in earlier neutrophil and platelet engraftment, which may reduce transplant-related mortality (TRM), particularly in patients with advanced leukaemia, there are emerging registry data that demonstrate an increased incidence of chronic GVHD compared with patients transplanted with bone marrow. This is likely to be consequent on the five- to tenfold greater dose of T cells in a PBSC harvest compared with bone marrow (Table 38.2). As yet it remains unclear whether the increased risk of chronic GVHD associated with the use of PBSC results in a reduction in relapse rate. Thus while PBSCs are routinely used as a stem cell source in patients being allografted for advanced leukaemia, where TRM is a major cause of treatment failure, bone marrow is still preferred in diseases such as aplastic anaemia where chronic GVHD is an important cause of treatment failure. Long-term follow-up studies comparing the outcome of patients transplanted using bone marrow and PBSCs will clearly be important in order to define the optimal stem cell source in standard-risk leukaemias. In paediatric transplantation, bone marrow remains the preferred stem cell source and the use of PBSCs is less common. Cell dose is an important factor determining outcome after both matched sibling and unrelated donor transplants and this effect is noted in recipients of both bone marrow and PBSCs. This effect is primarily mediated through a reduction in TRM consequent on accelerated immune reconstitution in recipients of a higher stem cell dose. The lowest acceptable stem cell dose to secure engraftment in an allogeneic setting is considered to be 2 × 106 CD34-positive cells per kilogram (2 × 108 mononuclear cells per kilogram if bone marrow is being used), although in practice the majority of patients transplanted using in excess of 1 × 106 CD34-positive cells per kilogram will engraft. Since outcome is improved with higher doses, most centres aim for a Table 38.2 Comparison of the cellular composition of a typical bone marrow harvest compared with G-CSF-mobilized in a peripheral blood stem cell (PBSC) (after two aphereses). CD34 cells (×106)

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dose in the region of 4 × 106 CD34-positive cells per kilogram. In patients transplanted from an HLA-identical sibling using a T-replete myeloablative regimen, there is some evidence that the incidence of chronic GVHD is increased if the stem cell dose exceeds 8 × 106 CD34-positive cells per kilogram. In this setting it may therefore be reasonable to have a target dose of 4–8 × 106 CD34-positive cells per kilogram. However, there is no evidence, as yet, that there should be an upper limit on cell dose in patients transplanted using an RIC regimen or from an unrelated donor. Cord blood transplantation UCB contains a high proportion of haemopoietic progenitors and HSCs and is an increasingly important stem cell source in paediatric and adult transplantation. Importantly, HLA disparity appears better tolerated in recipients of UCB and as a consequence the incidence of severe GVHD is lower with mismatched UCB than would be expected using a comparably mismatched unrelated donor. This has important implications in terms of donor identification in patients with uncommon HLA types for whom a suitably matched unrelated donor cannot be readily identified. A major factor limiting the uptake of CBT has been delayed or failed engraftment, which has been a common problem particularly in adult recipients. The two most important factors determining the likelihood of neutrophil and platelet engraftment after CBT are nucleated cell dose and HLA disparity. More recently, it has been shown that transplantation of two cord blood units, harvested from two separate donors, increases the speed of engraftment and is associated with a decreased risk of primary graft failure. This observation has substantially increased the number of CBT procedures performed in adults and encouraging results have been reported in patients with high-risk leukaemia using both myeloablative and, more recently, RIC regimens. Selection of cord blood units remains a complex area and consideration must be given to both stem cell dose and HLA disparity in identification of suitable unit(s) (Figure 38.3). Immune reconstitution is markedly delayed after CBT and viral infections, particularly CMV and adenovirus, coupled with fungal infections result in a substantial TRM. For all these reasons it is recommended that cord blood unit selection and transplantation should be performed by physicians with experience in this field.

Stem cell manipulation and expansion Immunophenotypic characterization of haemopoietic stem and progenitor cells has permitted the development of strategies by which PBSC grafts can be manipulated ex vivo. By conjugating antibodies that recognize CD34 with magnetic beads it is possible to achieve highly efficient enrichment of CD34+ cells from a PBSC or bone marrow harvest. This technology also allows, as a consequence of passive depletion of CD34− cells, effective depletion of donor T cells in allogeneic stem cell harvests and

Stem cell transplantation

High-resolution typing of a patient Simultaneous search of unrelated donor panels and cord blood registry Unrelated donor registry

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Urgent cord blood transplant Cord blood selection criteria Minimum accepted cell dose for a cord blood unit to be considered suitable for transplantation is 1.5 x 107 TNC/kg Matching criteria: serological typing class 1 (A, B) antigens, molecular typing class II (DRβ1) 4/6 is minimum accepted match In the majority of adults it will be necessary to use two cords to ensure optimal engraftment kinetics. However, it may on occasions be possible to identify a single cord unit with a sufficient TNC dose. For single cords the minimum cell dose is dependent on degree of HLA disparity: – recommended minimum cell dose for 6/6 match is 1.5 x 107 TNC/kg 5/6 match is 2.5 x 107 TNC/kg 4/6 match is 5.0 x 107 TNC/kg

Figure 38.3 Possible algorithm for identification of alternative donors in patients requiring urgent allogeneic transplantation who lack a sibling donor. TNC, total nucleated cell dose; VUD, volunteer unrelated donor.

can be used to provide an effective form of GVHD prophylaxis. The importance of cell dose in determining engraftment and outcome after SCT has led to studies aimed at expanding either the stem cell or progenitor population prior to transplantation. In the autologous setting this strategy has not been widely adopted, if only because collection of adequate numbers of cells to ensure rapid engraftment can now be reliably achieved in the great majority of patients. There is, however, significantly more interest in allografting, particularly in CBT, where engraftment can be delayed because of suboptimal stem cell or progenitor numbers.

Conditioning regimens: basic principles The combination of drugs and radiotherapy administered prior to stem cell infusion is termed the conditioning or preparative regimen. In autologous SCT, where there is no alloreactive

response, the sole purpose of the conditioning regimen is disease eradication. The most common conditioning regimens used in autologous transplantation utilize alkylating agents alone or in combination and other chemotherapeutic drugs. In the setting of allogeneic SCT the conditioning regimen has conventionally served two purposes: immunosuppression designed to abrogate an HVG reaction thereby preventing graft rejection, and myeloablation in order to achieve tumour eradication. A number of refinements have been made over the past three decades to the design and delivery of myeloablative conditioning regimens. However, they are still associated with significant toxicity which precludes their use in patients older than 50–55 years (45–50 years for recipients of unrelated donor transplants). The recent demonstration that durable donor engraftment can be reliably achieved using a non-myeloablative preparative regimen, coupled with increased awareness of the potency of the GVL reaction, has led to the development of a range of reduced intensity regimens. These protocols are associated with a substantially reduced TRM, allowing allogeneic transplantation to be safely performed in patients in whom it would previously have been contraindicated on the grounds of age or comorbidity. However, experience with RIC regimens is still relatively limited and longer follow-up and, where possible, randomized trials are required before their role in the management of older patients with haematological malignancies can be defined with precision. The encouraging results to date in older patients suggest that there may potentially be a role for their use in younger patients in preference to myeloablative regimens because of their reduced early mortality and possible longerterm benefits such as preserved fertility.

Conditioning regimens in autologous SCT Conditioning regimens in autologous SCT are designed with dose intensification in mind and are limited mainly by considerations of extramedullary toxicity. High-dose melphalan (200 mg/m2) is the standard conditioning regimen in myeloma autografts. BEAM (carmustine, etoposide, cytarabine, melphalan) is widely used in patients with lymphoma. Autologous transplants are rarely indicated in the management of AML and acute lymphoblastic leukaemia (ALL) but both busulfan/ cyclophosphamide and cyclophosphamide/TBI are effective preparative regimens in these diseases. A number of other drug combinations incorporating melphalan, busulfan and thiotepa are used in solid tumours. The major extramedullary toxicities of these regimens are mucositis and gastrointestinal toxicity. Disappointingly, there are few prospective randomized data on which to base the choice of conditioning regimen in autologous SCT. In myeloma a randomized comparison between a TBIcontaining regimen and high-dose melphalan alone demonstrated that no benefit was associated with the use of TBI. However, there are no large randomized studies of preparative regimens in lymphoma. 729

Postgraduate Haematology

Myeloablative conditioning regimens in allogeneic SCT The two commonest myeloablative conditioning regimens used in allogeneic SCT employ combinations of cyclophosphamide and either TBI or busulfan. TBI/cyclophosphamide Cyclophosphamide is an alkylating agent that, when administered in the doses routinely used in myeloablative conditioning regimens (120–200 mg/kg), has both immunosuppressive and antileukaemic properties. It is a prodrug that must be metabolized by the cytochrome P450 system in the liver to produce metabolically active derivatives, principally phosphoramide mustard, which exert their cytotoxic activity through the production of interstrand DNA links. The two major complications of cyclophosphamide at the doses employed in allogeneic transplantation are haemorrhagic cystitis and cardiac toxicity. Haemorrhagic cystitis results from the toxic effects of a cyclophosphamide metabolite, acrolein, on the uroepithelium and can be reduced by use of sodium 2-mercaptoethanesulfonate (MESNA), while cardiac toxicity is very rare at doses of cyclophosphamide below 150 mg/kg. TBI has dual immunosuppressive and antileukaemic properties when administered in myeloablative doses (typically 12– 14.4 Gy). Radiobiological principles predict that the toxicity of TBI can be reduced by either decreasing the overall dose of radiation or, as is now common, by giving it in fractionated form over a number of days (e.g 14.4 Gy divided into eight fractions over 4 days). The degree of immunosuppression produced by TBI-containing regimens is related to the total dose of irradiation delivered. The use of higher TBI doses is therefore an effective method of optimizing engraftment in myeloablative allografts where there is use of TCD or an alternative donor and consequently an increased risk of graft failure. Haematological malignancies are highly radiosensitive and the risk of disease relapse is reduced if a higher dose of TBI is used, although this benefit is blunted by increased transplant toxicity (see below). Early complications associated with the use of TBI include nausea, vomiting, diarrhoea and parotitis which can usually be managed symptomatically. Increased doses of TBI are also associated with pneumonitis and veno-occlusive disease (VOD) of the liver, both of which may be life-threatening. Long-term complications include cataract formation, hypothyroidism, infertility and, in children, growth retardation. Busulfan/cyclophosphamide Busulfan/cyclophosphamide is a myeloablative preparative regimen that has the practical advantage of not requiring the presence of irradiation facilities on site. Busulfan is an alkylating agent with potent activity against leukaemic progenitors and is a core component of both allogeneic and autologous transplant regimens. Historically, busulfan has only been available as an oral preparation, used at a dose of 14–16 mg/kg delivered 730

6-hourly over a period of 4 days. In this formulation, VOD and pulmonary and central nervous system (CNS) toxicity represent major complications. The pharmacokinetics of oral busulfan are highly variable because it undergoes first-pass metabolism in the liver and there is therefore substantial interpatient variability in plasma drug levels using a standard dosing schedule. Because the incidence of VOD is closely correlated with higher plasma busulfan levels and patients with low busulfan levels also have an increased risk of relapse, oral preparations of busulfan are far from ideal. Two approaches have been used to overcome this problem. A number of groups use frequent (6hourly) measurement of plasma busulfan levels during the first 24 hours of administration of an oral preparation followed by dose adjustment over the following 3 days in order to achieve a therapeutic busulfan level. Using such a targeted approach the Seattle group have achieved excellent results using busulfan/ cyclophosphamide with a very low risk of VOD and reduced relapse rates compared with those achieved using oral busulfan. Alternatively, a recently developed intravenous formulation of busulfan delivers therapeutic drug levels predictably and is associated with markedly reduced toxicity. It should be remembered that with both preparations prophylactic phenytoin or clonazepam should be used to prevent seizures, a complication associated with the administration of high doses of busulfan. Alternative myeloablative conditioning regimens Fludarabine augments alkylator-induced cell killing in vitro and regimens combining fludarabine with intravenous busulfan appear to be active and well tolerated. Of note, the incidence of VOD using this combination appears to be low and further studies of this regimen are indicated. In patients undergoing a sibling allograft for ALL, etoposide is often substituted for cyclophosphamide and a TBI/etoposide regimen has been shown in some studies to be associated with improved outcome. In non-malignant disorders, such as aplastic anaemia, cyclophosphamide alone can be used as a conditioning regimen and is sufficiently immunosuppressive to permit engraftment of allogeneic stem cells provided an adequate stem cell inoculum is transplanted. Addition of fludarabine is increasingly used in conjunction with cyclophosphamide in patients with sickle cell disease and thalassaemia.

Comparison of myeloablative conditioning regimens Sibling allografts There are two central questions in the design of myeloablative conditioning regimens: is there any survival benefit to be gained from intensifying the conditioning regimen and are cyclophosphamide/TBI and busulfan/cyclophosphamide equally effective preparative regimens? Prospective randomized trials in patients undergoing a sibling allograft for AML have failed to show any improvement

Stem cell transplantation

in survival using an increased dose of TBI. Studies performed in the past two decades have shown that while increasing the TBI dose reduces the risk of leukaemic relapse, this benefit is offset by a concomitant increase in TRM. Similarly, there is no evidence that addition of busulfan to a cyclophosphamide/TBI regimen has any impact on disease-free survival. The decision to use cyclophosphamide/TBI or busulfan/ cyclophosphamide as a conditioning regimen in sibling allografts has been studied in a number of randomized studies, all of which used oral busulfan without plasma level targeting. Given the substantial toxicity associated with the use of oral busulfan, the results of these studies need to be interpreted with caution since they may well underestimate the potential benefit of a busulfan/cyclophosphamide regimen utilizing targeted or intravenous busulfan. However, it is of interest that both regimens appeared equally effective for patients undergoing a sibling allograft for CML in first chronic phase, while there appeared to be some benefit attached to the use of a TBI-based regimen in patients with advanced leukemia. The advent of new delivery strategies for busulfan makes it important to reexamine the optimal myeloablative preparative regimen in patients with AML and myelodysplastic syndrome (MDS) and such studies are underway. As noted above, preliminary results with a combination of fludarabine and intravenous busulfan are also promising. Unrelated donor transplants The optimal conditioning regimen in patients undergoing an unrelated donor transplant has not been determined and there are few randomized trials in this setting. The greater degree of HLA disparity associated with the use of an unrelated donor results in a higher risk of graft failure than that observed using an HLA-identical sibling. For this reason many groups elect to use a cyclophosphamide/TBI-based regimen, with its greater immunosuppressive properties, although it should be noted that equivalent results have been reported in a number of large series using a busulfan/cyclophosphamide regimen. The other important factor determining the choice of conditioning regimen in unrelated donor transplant is whether TCD is employed. Regimens utilizing rigorous ex vivo TCD are associated with a higher risk of graft failure and the use of in vivo approaches, using antithymocyte globulin (ATG) or alemtuzumab, is preferred if TCD is to be used. In all patients receiving a TCD unrelated donor transplant, it is important to ensure that the conditioning regimen is sufficiently immunosuppressive if primary graft failure is to be avoided. Cord blood transplants The increased HLA disparity and high rates of primary graft failure in CBT makes it critical to optimize the immunosuppressive properties of the preparative regimen. In adults undergoing transplantation using a myeloablative regimen, engraftment rates in the region of 90–95% can be achieved by

the addition of fludarabine to a cyclophosphamide/TBI regimen, providing an adequate stem cell inoculum is used. ATG was initially used as additional GVHD prophylaxis in myeloablative CBT but is less commonly used now because of delayed immune reconstitution and an increased risk of post-transplant lymphoproliferative disorders.

Strategies for GVHD prophylaxis in myeloablative regimens Post-transplant immunosuppression using various combinations of ciclosporin, methotrexate, prednisolone and mycophenolate mofetil represent the commonest forms of GVHD prophylaxis in patients after a myeloablative sibling or unrelated donor transplant. Randomized trials from the Seattle group established the use of intravenous ciclosporin (2.5–5 mg/ kg daily) and short-course methotrexate (administered on days 2, 4, 8 and 12 post transplant) as the most effective form of GVHD prophylaxis in patients transplanted undergoing a T-replete allograft using either an HLA-identical sibling or volunteer unrelated donor. Rates of chronic extensive GVHD are in the region of 30% and 66% for recipients of T-replete sibling and unrelated donor transplants despite the use of ciclosporin/methotrexate GVHD prophylaxis. TCD is an additional, and highly effective method of reducing the risk of both acute and chronic GVHD. TCD can be achieved either by manipulating the stem cell inoculum ex vivo or by the in vivo administration of T-cell-depleting antibodies such as ATG or alemtuzumab (a humanized monoclonal antibody that recognizes CD52). Although a highly effective method of GVHD prophylaxis, TCD is associated with an increased risk of relapse and graft failure and delays immune reconstitution, increasing the risk of post-transplant infections such as CMV. There have been no randomized studies demonstrating a survival benefit for TCD. Clearly, the form of GVHD prophylaxis used for any particular patient should be selected with their individual risk of both GVHD and relapse in mind. Thus it may be desirable to avoid the use of TCD in patients with advanced leukaemia in whom the risk of relapse is high. In contrast, patients with a low risk of disease recurrence may benefit from more intensive GVHD prophylaxis. However, there remains no consensus concerning the use of TCD in allogeneic transplantation. Many UK and European groups, while performing T-replete sibling allografts, would choose to use in vivo TCD in unrelated donor transplant recipients. Compromise strategies in which TCD is used at the same time as further intensifying the conditioning regimen are of interest.

Reduced-intensity conditioning regimens in malignant and non-malignant disease As recently as 10 years ago it was believed that a myeloablative conditioning regimen was essential if durable engraftment of donor stem cells was to be achieved. A decade’s experience has 731

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confirmed that reduced-intensity regimens that incorporate either fludarabine or low-dose (200 cGy) TBI as a core immunosuppressive component consistently result in long-term donor stem cell engraftment with a remarkable reduction in toxicity, particularly in older patients or patients with significant comorbidities. At the same time there is now convincing evidence that such regimens provide a platform for a potentially curative GVL effect. As a result, numerous Phase II trials have demonstrated long-term disease-free survival after RIC allografts in patients whose outcome with conventional chemotherapy would be extremely poor. Currently the most important unresolved questions are, firstly, whether low-dose TBI/fludarabine or fludarabine in combination with an alkylating agent represents the optimal conditioning regimen and, secondly, whether a T-replete stem cell inoculum or some form of TCD should be used. Although there is no universally accepted definition of what constitutes an RIC regimen, the European Group for Blood and Marrow Transplantation (EBMT) definition of a regimen incorporating fludarabine with low-dose TBI (4 Gy or less) or other immunosuppressive or chemotherapeutic drug such as cyclophosphamide or melphalan is generally accepted.

Choice of RIC regimen Low-dose (200 cGy) TBI-based regimens Pioneering work in a canine model by Storb’s group demonstrated that durable engraftment could be achieved with a non-myeloablative conditioning regimen that used immunosuppressive doses of TBI (200–450 cGy) in conjunction with post-transplant immunosuppression in the form of ciclosporin and mycophenolate mofetil. In the late 1990s the Seattle group confirmed these results in patients with high-risk haematological malignancies and noted a substantial reduction in transplant toxicity compared with a myeloablative regimen. Early results from this group demonstrated a small but significant risk of graft failure with this regimen and fludarabine is now routinely incorporated into low-dose (200 cGy) TBI regimens. Accumulating data in the last decade confirms that this regimen now reliably achieves high rates of engraftment and full donor chimerism and is associated with a substantially reduced TRM compared with myeloablative preparative regimens. Its main drawback is a significant risk of acute and chronic GVHD. Recent data demonstrate that up to 66% of patients experience chronic extensive GVHD, the management of which is often very challenging, particularly in elderly and more frail patients. Non-TBI-based regimens At the same time as low-dose TBI regimens were being developed in Seattle, groups at the M.D. Anderson Cancer Center, Bethesda and Jerusalem demonstrated that durable engraftment of sibling and unrelated donor stem cells could be achieved with a similar reduction in transplant toxicity using a combination of fludarabine and an alkylating agent such as melphalan, 732

busulfan or cyclophosphamide. All groups reported a significant risk of severe acute and extensive chronic GVHD using ciclosporin with or without methotrexate as the sole form of GVHD prophylaxis. There are currently no data comparing the outcome of different RIC regimens. Given the significant risk of GVHD associated with the use of both regimens, a major area of research has focused on defining the optimal form of GVHD prophylaxis.

Strategies for GVHD prophylaxis in RIC regimens Currently, two contrasting approaches are used for GVHD prophylaxis in patients undergoing an RIC allograft. The first uses combinations of ciclosporin, methotrexate and mycophenolate mofetil as the sole form of GVHD prophylaxis and is used in patients transplanted using a T-replete stem cell inoculum. The alternative approach uses ciclosporin alone and is most common in patients transplanted using either ATG or alemtuzumab as in vivo TCD. Supporters of a T-depleted approach argue that the incidence of acute, and particularly chronic, GVHD is otherwise unacceptably high, particularly in older patients transplanted using an unrelated donor. It can also be argued that this strategy generates an effective platform for the subsequent delivery of DLI with less GVHD-related toxicity. They point to the effectiveness of ATG and alemtuzumab in reducing the risk of GVHD while at the same time securing high rates of durable donor engraftment. Those favouring a T-replete strategy highlight potential abrogation of a GVL effect by TCD. They also cite the delay in immune reconstitution associated with the use of TCD and consequent increased risk of viral, particularly CMV, infection. As with many debates in transplantation there are no randomized data to support either approach. Substantial numbers of patients achieve durable remissions with both T-replete and TCD regimens. While there may be an increased risk of disease relapse with a T-cell-depleted regimen, much would appear to depend on the intensity of TCD and the form of post-transplant immunosuppression employed.

Clinical management of patients undergoing stem cell transplantation The use of PBSCs coupled with improved supportive care has decreased the 100-day TRM of autologous transplantation to 1–3% in most centres. The morbidity and mortality of allogeneic SCT has also continued to fall over the last two decades but the 100-day TRM remains in the region of 10–20% depending on patient age, donor type, disease status and stem cell source. This progress reflects advances in supportive care and, in patients undergoing an unrelated donor transplant, more accurate tissue typing. Despite this organ toxicity, acute GVHD and

Stem cell transplantation

infections consequent on delayed immune reconstitution still represent major causes of morbidity and mortality in the first few months after allogeneic transplantation (Figure 38.4). In addition, chronic GVHD remains an increasing burden for patients and transplant services alike with the increasing use of mismatched unrelated and cord blood donors. The long-term complications of allogeneic transplantation, which can signifi-

Autologous Other 15%

Infection 8% IPn 1% Organ toxicity 6% Relapse 70%

HLA-identical sibling GVHD 13%

Relapse 41%

Other 16%

Infection 17% IPn 3%

Organ toxicity 10%

Unrelated donor GVHD 14%

Relapse 34% Other 16%

Infection 20%

Organ toxicity 10% IPn 6%

Figure 38.4 Causes of death after stem cell transplantation 2001–2006. (Courtesy of the International Bone Marrow Transplant Registry.)

cantly compromise a patient’s quality of life, are also increasingly recognized and have led to the introduction of specific late-effects clinics where multidisciplinary input from transplant physicians, endocrinologists, gynaecologists, ophthalmologists and psychologists can be provided.

Practicalities of stem cell infusion and blood product support Stem cell products are infused in the same way as other blood products except for the fact that online blood filters should not be used. Stem cell products must not be irradiated. They may either be infused once collected or, in the case of cryopreserved products, be infused immediately after being unfrozen, at the patient’s bedside. The most common side-effect of the cryopreservative (dimethyl sulphoxide, DMSO) is nausea but since it is excreted by the lungs, a garlic-like odour is also observed for 2–3 days after stem cell infusion. Damage to red blood cells releases free haemoglobin, which can precipitate acute renal failure, and thus patients should be adequately hydrated prior to stem cell infusion and for the following hours. In all patients urine output must be monitored closely. Patients will require red cell and platelet support during the immediate post-transplant period. In allograft recipients, particularly cord blood transplants and patients receiving myelotoxic drugs such as ganciclovir, platelet transfusion may be required for a number of months after transplantation. All cellular blood products should be irradiated (25 Gy) prior to administration in order to prevent transfusion-related GVHD (see Chapter 26). This should be commenced 6 weeks prior to transplant and is continued for 6 months after an autologous transplant or indefinitely for allogeneic transplants. In patients undergoing allogeneic SCT where there is a major ABO incompatibility between donor and recipient (e.g. a group O recipient receiving group A bone marrow), the graft must be depleted of red cells prior to administration unless PBSCs are being transplanted in which case red blood cells are usually effectively depleted during leucapheresis. The subsequent choice of blood group for platelets or red cells depends on the precise nature of the ABO incompatibility, the time from SCT and the results of blood grouping. Delayed erythroid engraftment or haemolysis caused by continuing synthesis of isohaemagglutinins by host lymphocytes may occur several weeks after stem cell infusion and is associated with the presence of a positive direct antiglobulin test and anti-donor red cell antibodies in serum or red cell eluates.

Complications of allogeneic SCT Early complications (days 0–90)

Graft failure Primary graft failure is defined as failure to achieve a neutrophil count above 0.5 × 109/L within 28 days of stem cell infusion and 733

Postgraduate Haematology

in the great majority of cases is caused by failure of the conditioning regimen to blunt an HVG reaction. It is rare unless there is marked donor–host HLA disparity, such as the use of a mismatched unrelated or family donor, or in vitro TCD has been deployed. Primary graft failure should be suspected in patients who fail to demonstrate any evidence of neutrophil engraftment by day +21 and should be investigated urgently with a bone marrow aspirate and trephine (which must be processed urgently), chimerism studies and virology to exclude parvovirus B19 or human herpesvirus (HHV)-6 infection. Patients with no morphological evidence of engraftment and an absence of donor chimerism require urgent intervention in the form of a second transplant, from the same donor if available. Such patients require further conditioning, and fludarabine with or without low-dose TBI provides potent immunosuppression in this setting. If there is no evidence of engraftment but peripheral blood lymphocytes type as donor, then an infusion of CD34positive selected PBSCs may be employed without prior conditioning therapy. Alternatively, cryopreserved autologous stem cells, if available, can be infused. The mortality of primary graft failure is in excess of 50% and it is therefore important to consider performing an autologous harvest in patients, such as unrelated donor recipients, where there is a significant risk of graft failure. Secondary graft failure is defined as the occurrence of sustained neutropenia and thrombocytopenia after donor engraftment. It is rare after sibling allografts and occurs predominantly in recipients of mismatched or unrelated donor transplants. The aetiology of secondary graft failure is often complex but causes that need to be considered include late graft rejection, drugs (co-trimoxazole and ganciclovir), viral infection (CMV, HHV-6 and parvovirus B19), disease relapse and rarely hypersplenism. Secondary graft failure is associated with significant mortality related to fungal infections and must be investigated urgently with a bone marrow aspirate and trephine and urgent chimerism studies. In patients with a hypocellular marrow and evidence of donor T-cell engraftment, a second infusion of donor stem cells is required.

Acute graft-versus-host disease Acute pattern GVHD usually occurs within the first 3 months post transplant, at or near the time of engraftment, and is characterized by the presence of rash, diarrhoea or abnormal liver function tests. Depending on the type of protocol employed, between 20% and 70% of patients will develop this disorder. Acute pattern GVHD occurring after 3 months, with no features of chronic GVHD, is also increasingly recognized in patients receiving RIC transplants, often folllowing withdrawal of immunosuppression. Risk factors for the development of acute GVHD include increased recipient age, unrelated or HLA-mismatched donor transplantation and the use of a female donor. Children have a lower risk of acute GVHD and recipients of CBT may have a lower incidence of GVHD compared 734

with adult bone marrow transplantation with similar degrees of HLA disparity. Skin GVHD typically presents as a maculopapular rash involving the face, neck, palms and soles but may extend to involve the whole body (Figure 38.5). In the worst cases it progresses to erythroderma, with bullae formation and painful blistering. Histology shows apoptosis at the base of dermal crypts, dyskeratosis, and evidence of lymphocytes in a perivas-

(a)

(b)

Figure 38.5 Acute skin GVHD: (a) acute cutaneous GVHD; (b) chronic oral GVHD.

Stem cell transplantation

cular distribution or adjacent to dyskeratotic keratinocytes. Gastrointestinal involvement presents with nausea, vomiting, secretory diarrhoea and/or abdominal pain. In more advanced disease, severe abdominal pain and distension in associated with voluminous, occasionally bloody, diarrhoea may occur. Gastric, antral and rectal biopsies have a high diagnostic yield, with diagnostic features including the presence of apoptotic cells in the base of crypts and a lymphocytic infiltrate. A well-defined manifestation of upper gut GVHD is the development of anorexia and nausea, both of which usually resolve rapidly if treated with low-dose methylprednisolone (1 mg/kg). A rising bilirubin and raised alkaline phosphatase are the initial features of liver GVHD, which typically develops later than skin or gut GVHD. Liver histology is diagnostic and demonstrates a portal tract lymphocytic infiltration, pericholangitis and bile duct loss. Accurate and early diagnosis of acute GVHD is essential for effective management of this potentially life-threatening disorder. Where possible diagnostic biopsies should be taken to both confirm the presence of GVHD and assist in the exclusion of other aetiologies. It is also important to stage GVHD accurately and the criteria devised by Glucksberg (and recently updated by the International Bone Marrow Transplant Registry) are widely used (Table 38.3). This staging system is a reliable indicator of prognosis and guides the intensity of treatment required. Grade 2–4 acute GVHD should be treated with high-dose methylprednisolone (typically 2 mg/kg daily), which is tapered according to response. In the setting of limited skin GVHD and upper gut GVHD, topical or oral steroids (1 mg/kg) coupled with optimization of ciclosporin levels may be sufficient to control symp-

toms. Approximately 70% of patients will improve significantly with oral or intravenous corticosteroid therapy, but a number will either fail to respond or relapse when immunosuppression is tapered. Failure to respond to 7 days of intravenous corticosteroid therapy defines steroid-resistant acute GVHD and these patients require the use of second-line therapies. Unfortunately, individuals with steroid-refractory acute GVHD have a poor prognosis, with a non-relapse mortality in excess of 70% secondary to persistent GVHD or as a result of infectious complications. Currently, robust evidence supporting efficacy of second-line treatments is lacking. Some patients with acute pattern GVHD affecting skin may respond to phototherapy. Patients with predominant gut involvement may respond to oral non-absorbable steroids or the monoclonal antibody infliximab. In other patients, treatment with other monoclonal antibodies such as daclizumab or pentostatin may be of benefit. More recently, encouraging results have been reported with the use of donor or third-party mesenchymal stromal cells, with a response rate of 60–70%. Application of this approach will require evaluation in prospective randomized studies. Patients with acute severe GVHD often require intensive supportive measures including replacement of gastrointestinal losses, parenteral nutrition, pain control and infectious prophylaxis.

Infectious complications Bacterial, fungal, protozoal and viral infections are a major cause of morbidity and mortality after allogeneic transplantation. Host factors include neutropenia, post-transplant immunosuppression and acute or chronic GVHD requiring steroid therapy. Numerous additional factors contribute to delayed

Table 38.3 Glucksberg staging of acute GVHD: (a) clinical staging; (b) clinical grading. (a) Stage

Skin

Liver bilirubin

Gut

+ ++ +++ ++++

Maculopapular rash 255 µmol/L

Diarrhoea 500–1000 mL/day or persistent nausea Diarrhoea 1000–1500 mL/day Diarrhoea >1500 mL/day Pain ± ileus

(b) Overall grade

Skin

Liver

Gut

Functional impairment

0 (none) I (mild) II (moderate) III (severe) IV (life-threatening)

0 + to ++ + to +++ ++ to +++ + to ++++

0 0 + ++ to +++ ++ to ++++

0 0 + ++ to +++ ++ to ++++

0 0 + ++ +++

Source: from Blume KG, Forman SJ, Appelbaum FR, eds (2004) Thomas’ Hemopoietic Stem Cell Transplantation, 3rd edn. Blackwell Science, Oxford, with permission. 735

Postgraduate Haematology

Viral Fungal Bacterial

HSV Candida

CMV

VZV/late CMV

Aspergillus

Gram negative and positive

Encapsulated

Neutropenia Acute GvHD Chronic GvHD

Days 0

50

100

150

Figure 38.6 Temporal pattern of infectious complications after allogeneic stem cell transplantation.

immune reconstitution after an allograft and include thymic atrophy in adults, the use of TCD and a suboptimal stem cell inoculum. Infectious complications are a particular challenge after CBT. The temporal pattern of infectious complications after allogeneic SCT is shown in Figure 38.6. Considerable progress has been made in the development of strategies to reduce the risk of infection after allogeneic SCT. All patients should be nursed in single rooms, preferably with laminar airflow or high-efficiency particulate air filtration. Evidence supports the use of triazole antifungals, such as fluconazole 400 mg daily, as an effective means of reducing Candida infection. Aciclovir (200–400 mg four times daily) is usually administered to prevent herpes simplex virus (HSV) reactivation. Quinolone antibiotics (e.g. ciprofloxacin 500 mg twice daily) are used by some units to reduce the risk of severe Gramnegative infections, although the evidence supporting this measure is inconclusive and practice should be guided by advice from local microbiologists concerning the prevalence and sensitivity of drug-resistant organisms. Patients should receive cotrimoxazole (480 mg twice daily three times per week) at the time of neutrophil engraftment (neutrophils >1.0 × 109/L) to prevent Pneumocystis jirovecii infection. If allergic to cotrimoxazole, nebulized pentamidine (300 mg monthly) can be substituted, although it should be remembered that this provides incomplete protection from Pneumocystis pneumonia and for this reason some units prefer to use dapsone. Allogeneic transplants, particularly recipients of TBIcontaining regimens, continue to be at long-term risk from infections caused by encapsulated bacteria such as Streptococcus pneumoniae and Haemophilus influenzae and require lifelong prophylaxis with penicillin (250 mg twice daily) or erythromycin (250 mg twice daily) if allergic to penicillin. Antibody titres to diseases for which childhood vaccination is performed decline after SCT. Revaccination is therefore recommended, particularly in allograft recipients, and most centres commence such a programme 12 months after transplantation. 736

Herpesvirus infection: CMV, HSV and varicella zoster Human CMV is a ubiquitous herpesvirus present in up to 60% of the general population, which reactivates after allogeneic SCT giving rise to either asymptomatic infection or, less commonly, end-organ damage (CMV disease) and death. Patients at the highest risk of CMV reactivation are seropositive recipients, especially those who receive TCD or unrelated donor grafts, and patients who develop GVHD requiring steroid therapy. CMV reactivation occurs in 40–80% of at-risk patients and until recently a substantial number of such patients developed CMV disease. CMV disease most commonly manifests as pneumonitis but can rarely cause gastrointestinal ulceration, hepatitis and retinitis. Until recently CMV was the commonest cause of infectious death after allogeneic transplantation. It is now possible to detect low levels of CMV infection after transplantation, using either polymerase chain reaction (PCR)-based detection of CMV or detection of pp65 antigen in peripheral blood leucocytes (CMV antigenaemia). The introduction of these sensitive diagnostic techniques coupled with the development of effective antiviral drugs has markedly reduced the incidence of CMV disease. Primary infection of seronegative patients may occur as a result of the infusion of stem cell or blood products from a CMV-positive donor but is rare. For this reason seronegative transplant recipients should receive CMVnegative or leucodepleted blood products to limit the possibility of primary infection. All patients at risk of CMV infection (all CMV-seropositive patients and any patient with a seropositive donor) should undergo weekly PCR or CMV antigenaemia testing from engraftment until 100 days after transplantation. Patients with CMV reactivation should be treated promptly with ganciclovir (5–10 mg/kg daily adjusted according to renal function), a nucleoside analogue that inhibits viral thymidine kinase. The major side-effect of ganciclovir is myelosuppression, which is especially problematic in patients transplanted using an unrelated or cord blood donor. Randomized studies have confirmed that this pre-emptive treatment strategy reduces the risk of CMV disease and death after sibling allogeneic transplantation. The use of prophylactic ganciclovir, which is administered regardless of whether there is evidence of CMV infection, does not improve outcome and is associated with significant bacterial and fungal infections consequent on high rates of myelotoxicity. For this reason a pre-emptive approach to prevention of CMV disease is generally preferred. Foscarnet, a DNA polymerase inhibitor, has less myelotoxicity than ganciclovir and is effective as part of a pre-emptive approach, although it is associated with significant nephrotoxicity. The incidence of CMV pneumonitis after allogeneic transplantation has substantially reduced since the advent of effective screening and pre-emptive treatment strategies. It occurs in patients with evidence of CMV reactivation within the first 100 days after transplantation and typically presents with dyspnoea, hypoxaemia and pulmonary infiltrates. Ganciclovir and foscar-

Stem cell transplantation

net are often ineffective in patients with established CMV pneumonitis. However, recent studies have demonstrated significant activity of cidofovir, which is considered in some units as firstline treatment in all patients with CMV pneumonitis. Cidofovir is nephrotoxic but can usually be safely administered if attention is paid to adequate hydration and other nephrotoxic drugs, particularly foscarnet, are discontinued. The role of high-titre CMV immunoglobulin in the treatment of CMV pneumonitis remains unclear, although it is still widely used, if available. The effective treatment of CMV infection delays the development of an immune response to CMV and as a result late (beyond 100 days post-transplant) CMV reactivation and disease is increasingly observed. Risk factors for late CMV infection include previous CMV reactivation, lymphopenia and the presence of active GVHD. Other members of the herpesvirus family have the potential to cause significant morbidity after allogeneic SCT. The incidence of HSV, which used to be very common in the first 30 days after SCT, has been sharply reduced by the use of prophylactic aciclovir. Reactivation of varicella zoster virus (VZV) occurs in up to 50% of at-risk patients after allogeneic SCT and typically presents as shingles with severe pain and a dermatomal vesicular eruption. Less commonly, VZV reactivation presents with atypical pain (headache or undiagnosed abdominal pain) in the absence of a rash. Prompt treatment of VZV infections with high-dose intravenous aciclovir is indicated after allogeneic SCT to prevent dissemination but also to reduce the severity of post-herpetic neuralgia. HHV-6 is also increasingly being reported in association with a syndrome variously associated with delayed engraftment, encephalitis and hepatitis. Fungal infections Fungal infections remain a major complication after SCT, reflecting the absence of accurate diagnostic tests and the inadequacy of current therapies. A high index of clinical suspicion is therefore required in transplant patients, particularly allografts, and most units administer systemic antifungal therapy early in the management of neutropenic fever. Risk factors for the development of fungal infection include prolonged neutropenia after SCT, the use of high-dose corticosteroids for treatment of GVHD and a history of prior fungal infection. Effective strategies exist for the prophylaxis and treatment of infection with yeasts (Candida spp.) but are lacking for infection with moulds such as Aspergillus spp. Candida infections typical manifest as oral thrush and less commonly as oesophageal candidiasis. Hepatosplenic candidiasis is seen occasionally, presenting with high spiking fevers at the time of engraftment in association with abnormal liver function tests. Ultrasound or computed tomography (CT) of the liver and spleen will confirm the diagnosis. Prophylactic use of fluconazole (400 mg daily) has proved effective in reducing the incidence of both superficial and invasive candidiasis. Patients who develop either hepatosplenic candidiasis or candidaemia should be treated with

systemic antifungals, usually liposomal amphotericin. All indwelling catheters must be removed. Emergence of fluconazoleresistant Candida species such as Candida kruseii or Candida glabrata is of concern and sensitivity data must be requested in any patient with Candida infection. Aspergillus infections usually present prior to or shortly after engraftment. The most common manifestation is as invasive pulmonary aspergillosis (IPA), which typically presents with an antibiotic-resistant fever, a significantly raised C-reactive protein, and abnormal chest radiography or high-resolution CT, often in the absence of respiratory symptoms. Rarely invasive Aspergillus infections can present with cerebral or hepatic disease. Accurate diagnosis of Aspergillus infections remains problematic since spores are only rarely cultured from lavage fluid or infected tissues and the sensitivity and specificity of other currently available diagnostic techniques is low. Contradictory results have been obtained using galactomannan detection assays and the initially encouraging results with PCR technology have not been confirmed by all groups. Operationally, the most helpful test in deciding whether IPA is a clinical possibility is high-resolution CT of the chest, which should be obtained in all patients with a neutropenic fever that has persisted for more than 72 hours. While the characteristic radiographic features of peripheral nodular shadows, with or without evidence of cavitation or a ‘halo’ sign, may take weeks to develop, the presence of any significant pulmonary infiltrate substantially increases the likelihood of Aspergillus infection and is an indication for the consideration of treatment doses of liposomal amphotericin or voriconazole. Importantly, the liklihood of IPA in a patient with a normal chest on high-resolution CT is low. Improved antifungal drugs now make it possible to contemplate allogeneic transplantation in patients with a previous proven or suspected invasive fungal infection. Risk factors for recrudescence of fungal disease in this setting include a short period (34 µmol/L), weight gain (>5% over baseline), and painful hepatomegaly. It is associated with evidence of damage to sinusoidal endothelial cells and hepatocytes and subsequent damage to the central veins in zone 3 of the hepatic acinus. In severe cases hepatic venular occlusion and widespread zonal disruption may lead to portal hypertension, hepatorenal syndrome, multiorgan failure and death. Risk factors for the development of VOD include the use of conditioning regimens containing busulfan or higher doses of TBI, pretransplant abnormalities of liver function tests, previous abdominal irradiation and recent exposure to the antiCD33 antibody gemtuzumab ozogamicin (Mylotarg). A diagnosis of VOD is most commonly made on clinical criteria.

Table 38.4 Causes of abnormal liver function tests after allogeneic SCT. Precipitant

Clinical presentation

Veno-occlusive disease Drugs

Hyperbilirubinaemia associated with weight gain, ascites and painful hepatomegaly Ciclosporin (hyperbilirubinaemia), azole antifungals (raised AST) Fall in Hb associated with unconjugated hyperbilirubinaemia and positive Coombs test Dilated biliary tree associated with gallbladder ‘sludge’ or cholelithiasis Viral hepatitis, fungal infection (disseminated aspergillosis or candidiasis), cholangitis lenta (progressive development of obstructive picture occurring days after an episode of sepsis)

Haemolysis

Biliary obstruction Infection

738

Hepatic Doppler studies demonstrating evidence of reversal of portal flow support the diagnosis. Definitive diagnosis requires transjugular venous liver biopsy, which has significant morbidity in the early post-transplant period and is therefore often avoided. It is important to exclude other causes of hyperbilirubinaemia, particularly ciclosporin toxicity (which can present with a very similar clinical picture), haemolysis (typically consequent on donor–recipient ABO mismatch or transplantation-associated microangiopathic haemolytic anaemia) and the hepatitis of sepsis (cholangitis lenta). Management is supportive, consisting of careful fluid balance, the judicious use of diuretics and, where necessary, haemofiltration. There is now convincing evidence that defibrotide can effectively treat severe VOD. Because of the clinical similarities between VOD and ciclosporin toxicity, it is wise to discontinue ciclosporin for at least 48 hours in any patient in whom a diagnosis of VOD is suspected. Renal toxicity The importance of the daily monitoring of weight, fluid balance and renal function in the effective management of patients after SCT cannot be overestimated. Impairment of renal function is frequently observed after allogeneic SCT unless careful attention is paid to fluid balance and the nephrotoxic potential of drugs commonly used during SCT, especially ciclosporin, amphotericin, aminoglycosides and loop diuretics. Ciclosporinrelated renal toxicity is usually easily reversible by temporary omission and dose reduction. Occasionally, ciclosporin toxicity manifests itself as a microangiopathic haemolytic anaemia with features of thrombotic thrombocytopenic purpura/haemolytic– uraemic syndrome. Withdrawal of the drug is mandatory. There is no convincing evidence that plasmapheresis is beneficial. Introduction of an alternative immunosuppressant (e.g. mycophenolate mofetil) should be considered. Late renal toxicity is observed in patients on long-term ciclosporin and patients who have undergone a TBI-based allograft. Pulmonary infections and non-infectious complications A range of pulmonary infections occurs after allogeneic SCT. In the first month after transplantation bacterial and fungal pneumonias are common. CMV, respiratory syncytial virus, influenza and parainfluenza are important causes of pneumonitis and typically occur in the first 90 days after transplantation. Pneumocystis pneumonia and Toxoplasma infection are still seen occasionally in the first few months after transplantation in patients who do not receive, or who are not compliant with, co-trimoxazole prophylaxis. Non-infectious pulmonary complications occurring after allogeneic transplantation can be reversible but demand prompt diagnosis if treatment is to be effective. Pulmonary oedema consequent on either increased capillary hydrostatic pressure caused by fluid overload or increased capillary permeability due to irradiation or sepsis is frequently seen in the post-transplant

Stem cell transplantation

period. Idiopathic pneumonia syndrome, defined as diffuse lung injury occurring after SCT for which no infectious or noninfectious aetiology can be identified, typically occurs 30–50 days after transplantation. The classic presentation includes dyspnoea, non-productive cough, hypoxaemia and non-lobar infiltrates on chest radiography and can progress rapidly to acute respiratory distress syndrome. Treatment is supportive but frequently unsatisfactory, and steroids have little effect on outcome. Diffuse alveolar haemorrhage is seen predominantly in patients undergoing autologous SCT but can also be seen in allogeneic recipients. This complication usually occurs within the first 2–3 weeks of transplantation and presents with dyspnoea, non-productive cough and hypoxaemia. Radiographic changes include interstitial or alveolar shadowing. Definitive diagnosis requires bronchoscopy, which shows fresh blood on repeated lavage. Early recognition of this disorder is essential since early intervention with high-dose steroids may significantly improve survival. Post-transplant lymphoproliferative disease Post-transplant lymphoproliferative disease (PTLD) includes a spectrum of EBV-driven B-cell hyperproliferative states that range from polyclonal benign proliferations to life-threatening neoplastic disease. Most cases of PTLD involve EBV-seropositive donors and present with lymphadenopathy and fever. In contrast to the PTLDs that develop following solid organ transplantation, the majority of cases after SCT are of donor origin and arise as a result of inadequate T-cell control of proliferation of EBV-infected B cells. Risk factors for the development of PTLD include TCD, particularly the use of ATG, and increased HLA disparity. Recent reports suggest that the use of alemtuzumab for TCD may now also be associated with an increased risk of PTLD. Weekly PCR quantitation of EBV viraemia is indicated in patients undergoing allogeneic transplantation that incorporates ATG or alemtuzumab and in all CBT procedures. Patients with a rising EBV load may be managed by a reduction in immunosuppression or treated with pre-emptive anti-CD20 monoclonal antibody (rituximab), although the triggers for intervention have not been precisely defined. In patients with a documented PTLD, early treatment with rituximab and, where possible, cessation of immunosuppressive therapy are the key to treatment. DLI using EBV-specific cytotoxic T lymphocytes has also been used with effect.

Intensive care support The likelihood of developing organ failure requiring admission to the intensive therapy unit (ITU) after SCT varies widely according to stem cell source (autologous vs. allogeneic), the intensity of the conditioning regimen and the presence of pretransplant comorbidities. While recent studies suggest a reduction in the proportion of transplant recipients requiring ITU admission, it remains the case that at least 10% of allografted patients, and in some institutions significantly more,

will require ITU admission at some stage during their treatment. Analysis of outcome in patients admitted to ITU has identified factors determining survival at varying stages after transplantation, allowing rational decisions about the likelihood of benefit of sustained intensive care in patients with multiorgan failure. Further sophistication in defining who will benefit from admission to ITU has been provided by the application of scoring systems. These include the Acute Physiology and Chronic Health Evaluation (APACHE) II and III mortality prediction models and Simplified Acute Physiology Score (SAPS) prognostic systems. Application of these scores prior to admission to ITU and, where appropriate, after a 72-hour period on ITU is helpful in identifying patients who will benefit from institution or continuation of intensive support. A number of studies have indicated that the development of progressive organ failure or the presence of multiorgan failure 72 hours after admission to ITU predicts an extremely poor outcome. The careful integration of these data into discussion with relatives and, where possible, patients can spare many the indignity of prolonged and futile intervention. Late complications As the results of allogeneic SCT have improved, its long-term complications have come to be better recognized. Chronic GVHD, secondary malignancies, and growth and fertility disorders represent an increasingly important cause of morbidity in patients who have been cured of their underlying disease.

Chronic GVHD Chronic GVHD refers to a complex syndrome occurring more than 3 months following allogeneic transplantation and is its commonest long-term complication. There is increasing recognition that its onset can be variable and that patients may present features of both acute and chronic GVHD (often referred to as overlap syndrome). Registry data show that chronic GVHD occurs in approximately one-third of patients undergoing T-replete transplants from HLA-identical siblings, rising to two-thirds of patients receiving T-replete grafts from unrelated donors. Risk factors for the development of chronic GVHD include increased recipient age, the use of PBSCs as opposed to bone marrow as stem cell source, transplantation from an unrelated or HLA-mismatched donor, the use of a T-replete stem cell inoculum and the presence of prior acute GVHD. Chronic GVHD may develop directly from acute GVHD (progressive), after the resolution of an episode of acute GVHD, or de novo in patients with no history of chronic GVHD. The clinical manifestations are characterized by features of both immunodeficiency and ‘autoimmunity’ (Figure 38.7). Previous grading systems for chronic GVHD have classified its severity as limited or extensive (Table 38.5), although the utility of this staging system has been questioned. Patients with progressive-type onset, extensive skin involvement, throm739

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Xerophthalmia Immunodeficiency Bronchiolitis obliterans Sinopulmonary infections Cholestasis

Xerostomia Oral ulceration Oesophageal web Immune cytopenias Autoantibodies Vulval/vaginal lichenoid changes

Lichenoid or sclerodermatous skin Fasciitis

Myositis Sensory or motor neuropathy

Figure 38.7 Clinical manifestations of chronic GVHD

Table 38.5 Classification of chronic GVHD. Limited chronic GvHD Either or both Localized skin involvement Hepatic dysfunction as a result of chronic GVHD Extensive chronic GvHD Either Generalized skin involvement or Localized skin involvement and/or hepatic dysfunction as a result of chronic GVHD plus Liver histology showing chronic aggressive hepatitis bridging necrosis or cirrhosis or Involvement of eye (Schirmer test with 50 bp) Total

25

3.0

Splice-site mutations Intronic Cryptic Total

20 2 22

2.4 0.2 2.6

Source: Graw et al. (2005) with permission.

Haemophilia B Inheritance and diagnosis Haemophilia B is an X-linked deficiency of FIX and the clinical manifestations are essentially the same as for haemophilia A. It is less common than haemophilia A and has an incidence of approximately 1 in 50 000 live births. FIX is responsible for the activation of FX in the presence of activated FVIII, calcium and phospholipid. FIX is synthesized in the liver and is a vitamin K-dependent protease that is similar to prothrombin, FVII, FX and protein C. The FIX gene is located on the long arm of the X chromosome and contains eight exons encoding a protein of 454 amino acids. The complete sequence of the gene has been determined and in virtually all cases of haemophilia B genetic mutations have been identified, the vast majority of mutations being missense. A haemophilia B database is also available at http://www.kcl.ac.uk/ip/petergreen/haemBdatabase.html. 802

q28 tel

38.2 9.3 47.5

Small deletions or insertions Small deletions Small insertions Combination of insertions and deletions Total

FVIII

cen

323 79 402

3' F8B F8A 5'

tel

F8A F8A

tel FVIII

3'

mRNA3' F8B F8A F8A

5' mRNA5' F8A tel

FVIII

Figure 41.4 How the tip flips: the mechanism of inversion through intron 22. The mutation is responsible for up to half of all cases of severe haemophilia A. During spermatogenesis, at meiosis the single X pairs with the Y chromosome in the homologous regions, but there is nothing to pair with most of the long arm of X. Unfortunately, the possibility of intrachromosomal pairing and crossover exists because there are three copies of a gene designated F8A, one lying within intron 22 and two in the opposite orientation situated 400 kb telomeric to the FVIII gene. Crossover with either the distal or the proximal F8A copy divides the FVIII gene in two, such that separately transcribed mRNAs are produced, neither of which encodes functional FVIII.

Isolated FIX deficiency is always hereditary and the clinical severity of haemophilia B generally shows the same relationship with the residual factor level as for FVIII in haemophilia A, although recent data suggest that on average the phenotype might be slightly less severe.

Treatment The mainstay of treatment is FIX concentrate. Previously, intermediate-purity prothrombin complex concentrates containing all the vitamin K-dependent factors were used. However, these concentrates were associated with a risk of thrombosis, particularly when used repeatedly, for example in surgery. This was probably due to accumulation of high levels of FX and FII, which have longer half-lives than FIX. Subsequently, high-purity FIX concentrates have been developed produced by either monoclonal antibody or affinity chromatography methods. FIX concentrates are prepared from the same screened plasma pools as FVIII and undergo similar viral inactivation procedures Although the FIX gene was cloned in 1982, the development of recombinant FIX was hampered by the posttranslational modification that it requires. Recombinant FIX is now widely used and although it is clinically effective, the recovery is only about 80% of that observed with plasmaderived FIX.

Inherited bleeding disorders

Dosage calculation in the treatment of haemophilia B follows the same principles as set out for FVIII deficiency, except that a higher initial dosage is required because of lower recovery: Dose to be infused (units ) = [ weight ( kg ) × increment needed ( units dL )] 0.9 (or 0.8 for recombinant FIX ) Also, the longer half-life (18 hours) means that daily infusions often suffice to maintain good levels after surgery, but as with FVIII the half-life may be much shorter in children and more frequent dosing required. Severely affected patients are usually maintained on twice-weekly prophylaxis to prevent spontaneous bleeds. This is practicable because of the longer half-life of the factor.

Complications of therapy Inhibitors Inhibitors in haemophilia B are much less common than in haemophilia A. Only about 2–3% of patients with severe haemophilia B will develop inhibitors, but interestingly many of these inhibitors present with anaphylaxis to infusions of FIX concentrate. Unlike FVIII inhibitors, FIX inhibitors are not time dependent, but can be similarly quantitated by a modified Bethesda assay. The majority of patients who develop inhibitors have large gene deletions or nonsense mutations occurring in the first 20% of the FIX gene. Acute management of bleeding can be achieved with recombinant FVIIa (as with haemophilia A). However, immune tolerance is frequently far less successful and can be complicated by the development of nephrotic syndrome.

Gene therapy for haemophilia Haemophilia is an excellent model for gene therapy because the clinical manifestation is the result of a deficiency of the single gene product and only a small amount of protein is required to ameliorate symptoms. Although preliminary data from Phase I trials were encouraging, the plasma levels of FVIII or FIX attained were notably less than in the animal models and certainly insufficient to free patients from treatment with clotting factor concentrates. Also, there has been a universal short duration of transgene activity. Although no inhibitors have been documented, this remains a theoretical risk. Furthermore, the detection of small amounts of viral vector genome in the semen continues to raise the possibility of the risk of germline integration. Insertional mutagenesis has occurred in a Phase I trial of Moloney retrovirus carrying the interleukin receptor, and this has been a cause for concern but was partly the result of the selection pressures applied to the transduced cells. New vectors, particularly modified serotypes and engineered self-complementary adenoassociated virus, which rarely integrates into the host genome,

will form the next generation of gene therapeutics and are about to enter clinical trials. The ethics of gene therapy for haemophilia are complex: it could be argued that for young patients with haemophilia, regular prophylactic factor infusions already deliver a nearnormal life. Gene transfer in haemophilia has played an integral role in furthering the science of gene therapy in general but it is still likely to be a long time before it becomes routine therapy for children with haemophilia.

General organization of haemophilia care As these are relatively uncommon disorders, with many and varied effects on patients and families at all stages of life who require care and support services across the whole field of medicine and social services, it is now accepted that this care can best be delivered comprehensively by referral centres. The staff of a major comprehensive care centre will include physicians, nurses, social workers, laboratory scientists and physiotherapists, devoting all or a substantial part of their time to haemophilia care. An orthopaedic surgeon or musculosketal rheumatologist prepared to see haemophilic patients regularly in a clinic set aside for their problems is a valuable addition to this team. In the UK, there is now a national service specification produced by the Haemophilia Alliance for Haemophilia and related conditions, which sets out the standards of care to be provided through the haemophilia centre network originally set up in the 1960s and early 1970s. It is recognized that not every centre can provide every facility and that there should be a fairly wide distribution according to population density, which determines numbers of patients. As defined by the national specification, the functions of a centre are to provide 24-hour emergency treatment for haemophilic patients and their families and a full range of diagnostic tests for identifying new patients and monitoring treatment. Full records should be kept of all treatments, whether given in hospital or as home therapy. Progress should be monitored through regular followup, with paediatric, dental and orthopaedic referrals being organized by the centre as necessary. (Many centres are indeed in paediatric departments or run by paediatricians.) Genetic counselling, including carrier detection and antenatal diagnosis, must be available for families of patients with haemophilia. Large centres providing all these facilities and treating at least 40 patients with severe haemophilia will be designated comprehensive care centres. It is envisaged that all patients will have access to a comprehensive care centre, either directly or via their smaller local centre. A part-time or full-time social worker should be part of the team, able to review the wider problems of living that affect the haemophilic patient at school, home and work. Centres provide access to specialized care for monitoring patients with HIV and HCV infection. On diagnosis, all patients are issued with a special medical card indicating 803

Postgraduate Haematology

96% 89%

80 Per cent alive

in inherited haemophilia, with a much greater incidence of soft tissue and mucosal bleeding, often out of proportion to the FVIII level. Treatment for bleeding is the same as for inhibitors in hereditary haemophilia and suppression of the inhibitor requires immunosuppressive therapy (e.g. corticosteroids, cyclophosphamide and anti-CD20 antibody).

97%

100

92% 88%

68%

60

59%

49%

40 20

Severe Mod/mild All UK males

von Willebrand disease

23%

0 0

15

25

35

45 55 65 Age (years)

75

85

Figure 41.5 Survival in men in the UK with haemophilia who were not infected with HIV and in the general male population of the UK in 1999. Note that these data still include deaths related to hepatitis C infection. (From Darby et al. 2007 with permission.)

laboratory test results, inhibitor status, main centre and local centre for treatment. Caring for haemophilic patients and their families is demanding but rewarding. Despite the setbacks in the 1980s due to virus transmission, the trend continues to be an ever-improving life expectancy and social participation, based on continuing medical progress and multiprofessional input into patient care. It is both tragic and ironic that the main foundation on which this progress rested (factor concentrate) has also been the route by which life-threatening infections have been introduced to about half of the most severely affected patients with haemophilia A. However, there are now effective therapies for both HIV and HCV infection. The younger generation of haemophiliacs is now treated with virus-safe or recombinant concentrates and has escaped both HIV and liver disease, and lead virtually normal lives (Figure 41.5).

Acquired haemophilia This chapter is devoted primarily to the inherited form of haemophilia arising from mutations in the FVIII and FIX genes but haemophilia can also arise as a result of autoantibodies that neutralize FVIII cofactor activity. The incidence is approximately 1.5 per million per year and it tends to occur predominantly in older patients, although there is also an increased frequency after pregnancy and in association with other autoimmune disorders. The antibodies usually show time-dependent (type 2) neutralization of FVIII and a residual FVIII level is often detectable. The pattern of bleeding is distinct from that 804

Although described in 1926, our understanding of this complex and variable bleeding disorder remains far from complete. The basic defect common to all variants is a deficiency of one or more aspects of VWF functional activity. The abnormality may be quantitative and/or qualitative.

von Willebrand factor VWF is encoded by a gene on chromosome 12, which was cloned simultaneously by several groups in 1986. The prepro-VWF primary translation product comprises a 22-aminoacid signal peptide, a 741-amino-acid propeptide and the 2050-amino-acid mature subunit. The pro-VWF monomer is composed of four types of domains (A–D) arranged as follows: NH 2– D1 – D2 – D ′ – D3 – A1 – A2 – A3 – D4 – B1 – B2 – B3 – C1 – C2 – CK–COOH. VWF is produced predominantly in vascular endothelial cells and also in megakaryocytes, although plasma VWF derives entirely from endothelial cell production. After translation the monomer enters the endoplasmic reticulum and the signal peptide is removed. The monomers then dimerize via the C-terminal cysteine knot domain before leaving the endoplasmic reticulum to the Golgi, where the dimers are joined via their N-termini to form a series of multimers with molecular masses ranging from 1000 to 20 000 kDa. During this process the molecule is extensively glycosylated with N- and O-linked glycans that form 20% of its final molecular weight. Importantly, approximately 13% of the N-linked glycans terminate in an ABO blood group antigen. In endothelial cells, multimerized VWF is then transferred into Weibel–Palade bodies, some of which pass directly to the cell surface to release their contents (constitutive pathway) while others remain in the cell for release when needed (regulated pathway). Platelet VWF does not contribute significantly to plasma VWF and is stored in the αgranules of platelets prior to release after platelet activation. During synthesis, the VWF propeptide, which is essential for multimerization, is cleaved from the mature molecule, stored with VWF in the Weibel–Palade bodies and released with it into plasma but is not known to have any further function thereafter. Some VWF is also secreted ablumenally from the base of the endothelial cell where it binds directly to collagen in the subendothelial matrix. Rapid release of VWF from the endothelial Weibel–Palade storage granules can be induced via the regulated pathway by a

Inherited bleeding disorders

number of agonists including thrombin, adrenaline, histamine and vasopressin. This can be used to advantage by using desmopressin to treat mild forms of VWD. Elevation of plasma VWF due to increased synthesis and secretion occurs as part of the acute-phase response to injury, inflammation, infection and neoplasia, and in pregnancy and hyperthyroidism. These responses are presumed to be physiological in promoting enhanced haemostasis but could reach pathological expression in being associated with an increased risk of thrombosis. The confounding effects of stress and inflammation need to be taken into account when measuring VWF levels in attempting to diagnose VWD. An appreciation of the complex structure–function relationship of VWF is essential in order to understand the classification of VWD. The two principal functions of VWF are (i) binding to matrix molecules, particularly collagen, at sites of vascular injury and subsequent capture of platelets to form the primary haemostatic plug and (ii) the stabilization of FVIII in the circulation. The adhesive function of FVIII requires functional binding sites for collagen and the platelet glycoproteins GPIb

and GPIIb/IIIa (also known as integrin αIIbβ3). The VWF released from endothelial cells into plasma is in the form of ultra-large multimers, which are subsequently cleaved into smaller forms by the action of the plasma metalloprotease ADAMTS-13. This is of critical importance because it is the high-molecular-weight multimers that have the greatest collagen- and platelet-binding activity: absent cleavage results in formation of platelet microthrombi and vascular occlusion and excessive cleavage results in increased bleeding. Although FVIII and VWF are entirely distinct entities with separate functions (Figure 41.6), they circulate together as a complex in which VWF protects FVIII from degradation, so that a deficiency of VWF or a reduction in its ability to bind FVIII may also result in a low plasma level of FVIII. Therefore, deficiency of VWF can give rise to a dual haemostatic defect: reduced plasma levels of FVIII (due to its shorter half-life in the absence of VWF) and a defect in primary haemostasis because of the failure in assisting platelets to adhere to the cut edges of small blood vessels (see Chapters 39 and 40). Clearly, the multistep synthesis, assembly and secretion of VWF and its multiple

Hepatocyte

Endothelial cell, megakaryocyte

X chromosome

q28

Chromosomes 12p113 Human von Willebrand factor gene

100 kb

qter

kb Exons EcoRI

G6PD

Factor VIII

0

10

20

30

40

5' 1

2 3 4 56

7 8 9 10 1112 13

14

23 24 25 26

3'

60

70

90

N VW AgII sp

VWF

B

a2

a1

Phospholipid binding

C N

C N

ss

C N

C N

200 kDa VW AgII dimer

VWF multimer: Signal A2 A3 peptide A1 cleavage

8.5 kb

N-terminal multimerization Carbohydrate processing

N

Propeptide cleavage: furin (PACE)

VWF binding

190

800 kDa

Pro-VWF multimer:

factor VIII

160 170 180

C-terminal dimerization

C

C

A3 C1 C2 COOH Single-chain

140 150

ss

N

A2

130

3'

Pre pro-VWF

Gene

9– kb poly(A)

2352aa NH2 A1

100 110 120

Translation 2813 aa Glycosylation

Pro-VWF dimer: mRNA

80

5' mRNA

10 kb 15 16 17 18 19 20 21 22

50

N

C

N

C

0.6–20 000 kDa

Folded factor VIII Weibel–Palade body Secretion

Constitutive

FVIII

Figure 41.6 Assembly of FVIII–VWF complex. FVIII synthesized by hepatocytes as a single chain is partially proteolysed and (possibly) complexed with VWF prior to release. VWF is synthesized as a single-chain precursor, which dimerizes with

FVIII Plasma

Regulated

FVIII

disulphide bond formation, then multimerizes with further disulphide exchange, with concomitant loss of a large propeptide segment. The propeptide is essential for multimer formation and probably functions as an acidic disulphide isomerase. 805

Postgraduate Haematology

binding interactions provide many ways in which mutations of the VWF gene can give rise to varying types of disease. In particular, the multimeric structure is susceptible to dominantnegative effects of mutant alleles. The diagnosis of VWD is based on the recognition that for normal VWF function it must be (i) present in adequate amounts, (ii) have a normal multimeric structure, and (iii) have intact functional domains (binding sites). When any of these properties is only slightly reduced, the presence or absence of a bleeding tendency may also depend on the quality and quantity of the other components, particularly platelets and collagen, with which VWF must interact.

Clinical features The clinical features vary according to the severity of the deficiency. The most common clinical picture is of an autosomal dominant, mild to moderately severe bleeding tendency. However, we now understand that the inheritance of VWD may be more complex than a simple autosomal dominant pattern (see below). Patients suffer from bruising, epistaxes, prolonged bleeding from minor cuts, menorrhagia and excessive, but not often life-threatening, bleeding after trauma or surgery. Patients often present for investigation in the second or third decade after prolonged bleeding from dental extraction or surgery has aroused clinical suspicion. Menorrhagia, unexplained by local or hormonal factors, can also be the presenting symptom: an estimated 20% of women with menorrhagia have VWD. The distribution of bleeding in VWD can be explained on the basis that VWF is required for platelet adhesion at high shear rate, which is the condition of flow found in the smallest blood vessels exposed to trauma in skin and mucous membranes. Haemarthroses do not occur in typical mild dominant VWD. Much less common is autosomal recessive (type 3) VWD, where VWF is completely absent and FVIII:C levels are usually around 1 or 2 units/dL. These patients have a bleeding tendency that clinically resembles severe haemophilia A, with haemarthroses, muscle bleeds and life-threatening haemorrhage after trauma, as well as proneness to small-vessel bleeding, which is not a feature of haemophilia A. Mild bleeding symptoms of the type typical of VWD are very common in the general population and thus are not specific for the presence of a bleeding disorder. It is now recommended that a simplified bleeding score (e.g. Bowman) is used to assess the likelihood of a significant defect in haemostasis and that the result is borne in mind when interpreting the results of laboratory investigations.

Laboratory diagnosis Preliminary diagnosis After obtaining a suggestive personal history and a family history, the preliminary tests required are a full blood count,

806

coagulation screen, FVIII assay, VWF:Ag and a measure of VWF activity, usually ristocetin cofactor activity (VWF:RCo). The laboratory diagnosis of VWD rests on assessing both the amount of VWF present (VWF:Ag) and its functional capacity. At present, it is generally possible to assess three important functions. 1 FVIII binding. Assessed first by a FVIII assay and then if this is reduced, by an assay of VWF FVIII-binding capacity using a modified ELISA-based technique. 2 Platelet-dependent function. The standard assessment of VWF adhesive functional activity remains the ristocetin cofactor assay (VWF:RCo). In this assay, dilutions of patient plasma are tested for their ability to promote platelet agglutination in the presence of the antibiotic ristocetin. 3 Collagen-binding function (VWF:CB). This recently introduced measurement is performed in an ELISA-based assay in which a well coated with collagen is used to capture VWF. Measures of VWF:RCo and VWF:CB are both sensitive to the loss of high-molecular-weight multimers, but measure different binding properties of VWF. Thus they should be seen as complementary rather than alternative assays. In addition, a global measure of primary haemostasis is also useful. Traditionally this was provided by the template bleeding time but this has a poor sensitivity for VWD and has largely been replaced by devices such as the PFA-100. The PFA-100 has very good sensitivity for VWD, although obviously does not distinguish it from platelet disorders and will not detect collagen abnormalities. Repeatedly normal PFA-100 results make a disorder of VWF function unlikely. Secondary tests for classification of VWD If a deficiency suggestive of VWD is detected then further tests, in particular multimeric analysis and ristocetin-induced platelet aggregation (RIPA), are recommended to allow accurate subtyping of the VWD. The most important of these is VWF multimer size analysis, which will demonstrate the distribution of VWF multimers and the pattern of flanking bands (Figure 41.7) adjacent to the main multimer bands. Using the tests described above, VWD can be classified into a limited number of types (Tables 41.4 and 41.5). The classification of VWD has been examined many times and extensive guidelines are available, detailed in the bibliography. What follows is necessarily a limited summary of much work. Type 1 VWD is defined as a simple quantitative deficiency of VWF and the VWF present should be functionally normal. This means that the VWF:Ag and VWF:RCo should be concordant and the VWF should have an essentially normal multimeric pattern. In practice it is acceptable that the ratio of antigen to function should be in excess of 0.7 and there should be no significant loss of high-molecular-weight multimers, although on close inspection many subtle abnormalities of multimer pattern can be discerned.

Inherited bleeding disorders

Figure 41.7 Multimer analysis of VWF from patients with VWD and normal control subjects. Note that many cases classified at present as type 2A were previously subdivided according to the details of the abnormality of triplet pattern.

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The characteristic of the 2A, 2B and 2M variants is a functional deficiency of VWF activity, which is reduced to below 0.7 of the antigenic measure. In type 2A, this results from a lack of high- and intermediate-size VWF multimers, whereas in type 2M a similar loss of platelet-binding activity is seen despite the presence of normal VWF multimeric composition and is caused by mutations in the VWF GPIb-binding site. Loss of highmolecular-weight multimers in type 2A may arise from failures of intracellular processing or from accelerated proteolysis in the circulation due to mutations close to the ADAMTS-13 cleavage site. VWD due to an isolated collagen-binding defect without loss of high-molecular-weight multimers is rare, although a handful of cases have been reported. The associated bleeding disorder is mild, perhaps because VWF contains two collagenbinding sites. In type 2B, VWF binds prematurely with platelets in the circulation and in the laboratory will agglutinate platelets at a low concentration of ristocetin (0.5 mg/mL) that has no effect with normal VWF (i.e. positive RIPA). If normal washed platelets are resuspended in the patient’s plasma, the phenomenon is reproduced, demonstrating that the abnormality is in the plasma. Studies with purified type 2B VWF show that, unlike normal VWF, it binds directly to platelets without prior activation. In vivo this results in the loss of high-molecular-weight multimers and platelets from the circulation due to in vivo formation of platelet aggregates and also to increased cleavage of VWF by ADAMTS-13. Thus routine platelet aggregation studies with a range of ristocetin dosages should be performed

Table 41.4 Primary classification of von Willebrand disease. Subclassification

Type of VWF deficiency

VWF protein function

Type 1

Quantitative partial deficiency Qualitative functional deficiency Quantitative complete deficiency

Normal

Type 2 Type 3

Abnormal Undetectable

as part of the diagnostic work-up, as it is important to detect this variant (see below), which does not always show up in the other tests. Indeed a recent analysis showed that many patients with type 2B VWD have normal platelet counts and VWF multimer patterns. A fourth type 2 variant, called type 2N (for Normandy), has been described. This is characterized by reduced affinity for FVIII but it is normal in other respects. Thus, laboratory investigations reveal only a reduced FVIII level (5–35%), which is easily mistaken for mild haemophilia. A clue to the correct diagnosis may come from the family history, and confirmation requires an assay of VWF FVIII-binding capacity or genetic analysis of FVIII and the VWF FVIII-binding site in the D′ domain. The majority of patients are found to be type 1, which accounts for 75% of kindreds. Types 2A and 2B are fairly common, together accounting for about 15% of kindreds. Although classification has proved useful in understanding VWD and can help in planning treatment, some forms remain difficult to classify and may show features of more than one type. Problems in diagnosis of type 1 VWD The diagnosis of type 1 VWD implies that the patient has a significantly low level of VWF, which is responsible for an increased tendency to bleed. It is often not easy to be certain of this conclusion for the following reasons. 1 Slightly low levels of VWF are common in the population. Although the lower end of the normal range for VWF in the general population is approximately 50 units/dL, most of the 2.5% of the population who have levels below this have normal haemostasis. In particular, individuals with blood group O have VWF levels on average 30% lower than those with non-O blood groups and the lower end of a ‘blood group O normal range’ is approximately 35 units/dL. Thus the lower limit of the normal range is not the minimum level required for normal haemostasis (Table 41.6). Nonetheless, individuals with blood group O are over-represented in the group with type 1 VWD and there is a continuous relationship between bleeding tendency and VWF level that extends into the normal range without a clear cut-off between normal and abnormal. 2 A history of minor bleeding episodes (e.g. easy bruising, epistaxes) is very common in the population and is not a good

Table 41.5 Secondary classification of type 2 von Willebrand disease. Subtype

Platelet-associated function

FVIII-binding capacity

High-molecular-weight VWF multimers

2A 2B 2M 2N

Decreased Increased affinity for GPIb Decreased Normal

Normal Normal Normal Markedly reduced

Absent Usually reduced/absent Normal and occasionally ultra-large forms Normal

808

Inherited bleeding disorders

Table 41.6 Influence of ABO blood group on VWF:Ag values (u/dl) in volunteer blood donors. ABO type

N

VWF:Ag geometric mean

VWF:Ag geometric mean ± 2SD

O A B AB

456 340 196 109

74.8 105.9 116.9 123.3

35.6–157.0 48.0–233.9 56.8–241.0 63.8–238.2

Note the significantly lower levels in donors of blood group O compared with non-O donors. Source: Gill et al. (1987) with permission.

predictor of bleeding in other circumstances such as operations. Equally, the patient may not yet have been exposed to a significant test of haemostasis. 3 As a result of the two points above, a history of, say, easy bruising and slightly low VWF levels will often be found together. However, this does not mean that the patient has VWD and caution should be exercised in drawing this conclusion as it has many consequences for the patient. 4 Intercurrent events such as stress, exercise, illness and pregnancy may all elevate the VWF level, making it difficult to be certain that a representative measure of VWF levels has been obtained. It is therefore necessary to perform carefully standardized sets of assays with concordant results on at least two occasions to be sure of the diagnosis and its severity. 5 The family history is often unhelpful, particularly in mild cases where penetrance is weak or variable. This is probably the result of the modifying effects alluded to in (1) above. It is now evident that in some families, VWD does not segregate with the VWF gene. By inference, therefore, some of the mutations responsible for the type 1 phenotype will occur at loci other than the VWF gene, although what these are remains to be determined. 6 Finally, review of many cases previously diagnosed as type 1 VWD has concluded that many are in fact better categorized as type 2M after better assessment of the VWF antigen-activity ratio. When there is a clear bleeding history and FVIII, VWF:Ag and VWF:RCo are all clearly reduced (e.g. < 25 units/dL) and the platelet count is normal, the diagnosis is easily made (see Tables 41.4 and 41.5). However, in the absence of a clear bleeding history, individuals with VWF activity in the range 25–50 units/dL are best regarded as simply being at slightly increased risk of bleeding. When VWF levels in the range 25–50 units/dL are associated with bleeding, it is very likely that other factors such as mildly impaired platelet function play a contributory role. This is especially true of the milder cases of type 1 VWD (see below).

Treatment Patients with mild or moderate VWD attend infrequently for treatment. The first-line treatment for minor bleeding after local measures have failed in type 1 VWD is desmopressin. This will produce a brisk rise in VWF and FVIII levels (30 min after intravenous infusion) and a shortening of the bleeding time (for details of therapy see discussion of mild haemophilia A; see p. 800). Desmopressin is much less effective in types 2A and 2M, presumably because the patient’s released VWF is highly abnormal and still unable to promote platelet adhesion. Desmopressin is generally regarded as contraindicated in type 2B VWD, as the released abnormal VWF will cause circulatory platelet aggregates to form, with a further fall in the platelet count. Before relying on desmopressin for therapy, patients should be given a test dose of DDAVP in order to determine the response at 30–60 min because it varies considerably. In addition, the duration of the response is also variable and further samples at 6–8 hours should be taken: some forms of type 1 VWD (e.g. VWF Vicenza) are characterized by a much shortened survival of VWF. A therapeutic trial may also be worthwhile in type 2A as some families do respond. In type 2N, the FVIII response is of normal magnitude but is of limited efficacy due to its short duration, emphasizing the importance of making the correct diagnosis. It may still be useful for minor procedures. Desmopressin is best avoided in small children (< 2 years) as there is a risk of hyponatraemia and consequent seizures. It is also contraindicated in elderly patients or those with arterial disease as there is a risk of arterial thrombosis. Desmopressin therapy is often combined with tranexamic acid. However, tranexamic acid may be effective on its own in some circumstances such as menorrhagia or as a mouthwash for oral cavity bleeding. In patients in whom desmopressin is ineffective or contraindicated, the next line of treatment is a concentrate containing adequate amounts of functionally active VWF with preservation of the high-molecular-weight multimers. As with all concentrates, the source of plasma and viral inactivation are also important. Recombinant VWF concentrate is not yet licensed but is in early clinical trials. Cryoprecipitate has been used in these circumstances but cannot be subjected to viral inactivation procedures and is therefore not recommended as first-line therapy. Factor concentrates will always be required for treatment of type 3 VWD. Depending on the responses obtained with desmopressin, the duration of treatment and the presence of other contraindications, they may also be required in other type 2 and type 1 variants. FVIII concentrates vary considerably in the amount of VWF they contain and in the extent to which this is degraded or remains in the form of functionally active high-molecular-weight multimers. Those that contain significant amounts of VWF that were effective in treating VWD were initially of intermediate purity, but high-purity more specifically designed VWF concentrates are now available that contain 809

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well-preserved VWF protein. The composition and VWF/FVIII ratio varies considerably between products. It is important to remember that following infusion of some high-purity VWF with low FVIII content there is a delay of approximately 12 hours before the level of FVIII rises substantially and, if rapid correction is required, FVIII concentrate should also be given. In general, none of these replacement treatments is reliable in correcting the bleeding time, but this is not necessarily a bar to effective haemostasis. This seemingly paradoxical result may be because they do not correct the deficiency of intraplatelet VWF or because the largest high-molecular-weight multimers are not present. In situations when concentrates fail to stop bleeding, cryoprecipitate and platelet concentrates may prove effective.

Clinical course and complications Patients with types 1 and 2 VWD lead relatively normal lives, with normal life expectancy. Menstruation is seldom a cause of severe blood loss, although menorrhagia is common. This can usually be managed satisfactorily with antifibrinolytics or by oral contraceptive estrogen/progesterone combinations. The Mirena coil is also effective. If these are not effective, then selfadministration at home of desmopressin by intranasal or subcutaneous routes can be useful. In later years, some patients require hysterectomy. During pregnancy, the VWF levels rise spontaneously to the normal or low-normal range in most patients with type 1 VWD. In type 2, the response is unpredictable and should be closely monitored: because of increased production of abnormal VWF, VWF:Ag may rise to normal levels but with VWF:RCo still significantly low. Type 2B is particularly complicated in pregnancy because the rise in endogenous VWF production exacerbates the thrombocytopenia. Patients with severe type 3 disease have a clinical course resembling severe or moderately severe haemophilia A, including the development of joint damage. A small number of patients with type 3 disease develop antibodies to VWF, which inhibit its platelet adhesion-promoting property and cause rapid removal from the circulation of infused material. Unlike anti-FVIII antibodies, some anti-VWF antibodies may mediate anaphylactic shock.

Molecular genetics The cloning of VWF cDNA and its gene has led to progress in identifying the underlying mutations responsible for the various phenotypes (Figure 41.8). As with the FVIII gene, the sheer size of the DNA region involved presents some problems of localization, but these are being overcome by powerful screening methods. A database of mutations responsible for VWD has been established at http://www.shef.ac.uk/vwf/vwd. html. 810

Type 1 In the last few years there have been extensive studies to determine the molecular basis of type 1 VWD and a number of important points have emerged. Firstly, in about 35% of cases, the disorder does not segregate with the VWF locus and is therefore presumably the result of the combination of other modifying genes, in particular the ABO locus. This phenomenon is most common in individuals diagnosed with VWD and VWF levels above 30 units/dL and involves a disproportionate number of blood group O individuals. The likelihood of the disorder segregating with the VWF gene and of a causative mutation being identified increases significantly as the VWF level falls and a mutation is found in all cases where the VWF multimer pattern is abnormal. Among those with type 1 VWD and an identified mutation, mutations are widely scattered throughout the molecule. Similarly, in the more severe cases a typical autosomal dominant pattern of inheritance with strong penetrance is seen, whereas in milder cases this is often not apparent. Type 2 Mutations responsible for type 2 VWD affect the relevant functional site in the molecule and the corresponding portion of the gene. Mutations causing types 2M and 2B both affect the binding site for platelet GPIb on VWF and are found within the A1 domain of the molecule. They are inherited dominantly. As discussed above, type 2A may arise from increased susceptibility to proteolysis by ADAMTS-13 and this type is associated with mutations in the A2 domain. Otherwise, type 2A mutations are scattered throughout the gene but particularly in the propeptide, D′–D3 and the cysteine knot. In general, type 2A is dominant but occasional recessive forms occur. Mutations causing type 2N are found in the FVIII-binding site in the D′ domain but because the binding capacity of VWF greatly exceeds the FVIII concentration, the inheritance is recessive. Type 3 Type 3 is characterized by the complete absence of VWF and so arises from inactivating or null mutations in both VWF alleles and is inherited in an autosomal recessive manner. The rare patients who develop antibodies to VWF nearly all have large deletions of their VWF gene.

Pseudo von Willebrand disease (platelet-type) Several families have been described with a disorder closely resembling type 2B VWD, but in whom mixing experiments show the defect to be in their platelets rather than their plasma. Patients with pseudo VWD have moderately reduced levels of VWF:Ag and platelets, with an enhanced response of their platelet-rich plasma to low levels of ristocetin (0.5 mg/mL). The addition of normal cryoprecipitate to their washed platelets

Inherited bleeding disorders

(a)

Propeptide sequence D3, A1–A3 Mutations Mutations 1405–1408delK V1279I L129M 1546–1548del R1315C M576I C1229G W542X P1413L N1231T Y1584C P1266L R1568S

B2, B3, C1, C2 Mutations C2340R S2497P G2343V T2647M R2379C nt8412insTCCC R2464C B1–3

COOH

NH2 D1 1

Table 41.7 Disorders associated with acquired von Willebrand syndrome and mechanism responsible.

D’

D3

preproVWF

22aa

Figure 41.8 (a) Mutations found in patients diagnosed with type 1 VWD in the Canadian and European studies. Note that not all these mutations have been shown to be causal and that classification as type 1 may sometimes be contentious. (From James et al. 2007 with permission.) (b) Distribution of mutations responsible for type 2 VWD. The domain structure of the monomer is shown, as well as the regions in which the mutations cluster in relation to the functional domains of the molecule. (From Goodeve et al. 2007 with permission.)

D2

A1

A2

2N

D1

D2

C1 C2 CK 2813

2050aa

5’ regulatory sequence Mutations D’–D3 Mutations –1665G>C K762E –1873A>G nt2435delC –1886A>C R854Q –2485G>A R924Q R924W –2520C>T C996E –2613A>G –2730C>T C1111Y Del–1522 Y1146C C1190R R1205H

SP

D4

VWF monomer

741aa

(b)

A3

D'

2M 2B D3

A1

A3–D4 Mutations nt5180insTT V1260I N1818S P2063S R2185Q T2104I E2233G

2A

2A

A2

A3

D4

B1 2 3 C1

C2

Propeptide FVIII Heparin

GPIb Heparin Collagen

Collagen

GPIIb/IIIa

Multimer

Dimer

Disorder

Mechanism

Treatment

Hypothyroidism Aortic stenosis Myeloproliferative

Correct thyroid status Replace aortic valve Restore platelet count to normal

IgG paraprotein

Reduced synthesis Shear stress Adherence to platelets and increased cleavage Immune

IgM paraprotein Wilms tumour

Immune Adsorption

IVIG, chemotherapy to reduce paraproteins Chemotherapy (IVIG ineffective) Tumour removal if possible

IVIG, intravenous immunoglobulin.

811

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causes spontaneous aggregation, whereas the reverse experiment is without effect (compare with type 2B). Missense mutations in platelet membrane GPIb, such that it spontaneously binds higher multimers of VWF, have been shown to be the underlying cause of this autosomally dominant mild bleeding syndrome. Treatment has not been extensively evaluated, but it should probably be with normal platelet concentrates, rather than desmopressin or cryoprecipitate. Thus this syndrome should be excluded by mixing tests as above or by genetic analysis before diagnosing type 2B VWD.

Acquired von Willebrand syndrome In a number of circumstances patients may acquire a deficiency of VWF function that is not inherited. The commonest association is with a paraprotein that binds to VWF and accelerates clearance, producing a type 2A or type 3 pattern. It is also associated with myeloproliferative disease when thrombocytosis is present and in hypothyroidism (Table 41.7). Severe aortic stenosis can cause sufficient shear damage to VWF to cause an acquired von Willebrand syndrome that resolves after valve replacement. In the long term, treatment of the underlying disorder will improve the syndrome. If urgent treatment is required, then replacement therapy may be necessary (taking into account accelerated clearance) or IgG paraproteins will usually respond to intravenous immunoglobulin.

Selected bibliography Darby SC, Kan SW, Spooner RJ et al. (2007) Mortality rates, life expectancy, and causes of death in people with hemophilia A or B in the United Kingdom who were not infected with HIV. Blood 110: 815–25. Escobar MA (2003) Treatment on demand: in vivo dose finding studies. Haemophilia 9: 360–7. Gill J, Endres-Brooks PJ, Bauer WJ et al. (1987) The effect of ABO blood group on the diagnosis of von Willebrand disease. Blood 69: 1691–5. Goodeve A, Eikenboom J, Castaman G et al. (2007) Phenotype and genotype of a cohort of families historically diagnosed with type 1 von Willebrand disease in the European study, Molecular and Clinical Markers for the Diagnosis and Management of Type 1 von Willebrand Disease (MCMDM-1VWD). Blood 109: 112–21. Erratum appears in Blood (2008) 111: 3299–300. Graw J, Brackmann H-H, Oldenburg J, Schneppenheim R, Spannagl M, Schwaab R (2005) Haemophilia A: from mutation analysis to new therapies. Nature Reviews. Genetics 6: 488–501. Haemophilia Alliance. A national service specification for haemophilia and related conditions. Available at www.haemophiliaalliance.org.uk

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Hay CR, Brown SA, Collins PW et al. (2006) The diagnosis and management of factor VIII and IX inhibitors: a guideline from the UK Haemophilia Centre Directors’ Organization. British Journal of Haematology 133: 591–605. James PD, Notley C, Hegadorn C et al. (2007) The mutational spectrum of type 1 von Willebrand disease: results from a Canadian cohort study. Blood 109: 145–54. Keeling DM, Tait C, Makris M (2008) Guideline on the selection and use of therapeutic products to treat haemophilia and other hereditary disorders. Haemophilia 14: 671–84. Laffan MA, Brown SA, Collins PW et al. (2004) The diagnosis of von Willebrand disease. A guideline from the UK Haemophilia Centre Doctors’ Organization. Haemophilia 10: 199–217. Lillicrap D (2009) Genotype/phenotype association in von Willebrand disease: is the glass half full or empty? Journal of Thrombosis and Haemostasis 7 (Suppl. 1): 65–70. Manco-Johnson MJ, Abshire TC, Shapiro AD et al. (2007) Prophylaxis versus episodic treatment to prevent joint disease in boys with severe hemophilia. New England Journal of Medicine 357: 535–44. Mannucci PM (2003) Haemophilia: treatment options in the twenty-first century. Journal of Thrombosis and Haemostasis 1: 1349–55. Mannucci PM, Schutgens RE, Santagostino E, Mauser-Bunschoten EP (2009) How I treat age-related morbidities in elderly persons with hemophilia. Blood 114: 5256–63. Mikaelsson M, Oswaldsson U, Sandberg H (1998) Influence of phospholipids on the assessment of FVIII activity. Haemophilia 4: 646–50. Ngo JCK, Huang M, Roth DA, Furie BC, Furie B (2008) Crystal structure of human factor VIII: implications for the formation of the factor IXa–factor VIIIa complex. Structure 16: 597–606. NHS Management Executive (1993) Health Service Guidelines: Provision of Haemophilia Treatment and Care. BAPS, Heywood. Nichols WL, Hultin MB, James AH et al. (2008) von Willebrand disease (VWD): evidence-based diagnosis and management guidelines. The National Heart, Lung, and Blood Institute (NHLBI) Expert Panel report (USA). Haemophilia 14: 171–232. Pasi KJ, Collins PW, Keeling D et al. (2004) Management of von Willebrand disease. A guideline from the UK Haemophilia Centre Doctor’s Organization. Haemophilia 10: 218–31. Rodriguez-Mercham EC, Lee CA (2003) Inhibitors in Patients with Haemophilia. Blackwell Science, Oxford. Sadler JE, Budde U, Eikenboom JC et al. (2006) Update on the pathophysiology and classification of von Willebrand disease: a report of the Subcommittee on von Willebrand Factor. Journal of Thrombosis and Haemostasis 4: 2103–14. United Kingdom Haemophilia Centre Doctors’ Organisation (2003) Guidelines on the selection and use of therapeutic products to treat haemophilia and other herditary bleeding disorders. Haemophilia 9: 1–23. von Willebrand Disease Database. Available at http://www.shef. ac.uk/vwf/vwd.html

CHAPTER 42

42

Rare bleeding disorders Flora Peyvandi and Marzia Menegatti A. Bianchi Bonomi Hemophilia and Thrombosis Center, IRCCS Cà Granada, Ospedale Maggiore, Milan, Italy

Introduction, 813 Fibrinogen deficiency, 815 Clinical manifestations and treatment, 815 Molecular defects, 816 Prothrombin deficiency, 816 Clinical manifestations and treatment, 816 Molecular defects, 816 Factor V deficiency, 818 Clinical manifestations and treatment, 818 Molecular defects, 818 Combined deficiency of factor V and factor VIII, 821

Clinical manifestations and treatment, 821 Molecular defects, 821 Factor VII deficiency, 821 Clinical manifestations and treatment, 822 Molecular defects, 823 Factor X deficiency, 825 Clinical manifestations and treatment, 825 Molecular defects, 825 Factor XI deficiency, 827 Clinical manifestations and treatment, 827 Molecular defects, 827

Introduction The rare bleeding disorders (RBDs), including the inherited deficiencies of such coagulation factors as fibrinogen, factor (F) II, FV, combined FV and FVIII, FVII, FX, FXI, FXIII and of multiple deficiency of vitamin K-dependent factors, are usually transmitted in an autosomal recessive manner. The prevalence of RBDs in the general population is low, and homozygous or double heterozygous deficiencies vary from 1 in 500 000 for FVII deficiency to 1 in 2 million for prothrombin and FXIII deficiencies (Table 42.1). The prevalence of RBDs is also strongly influenced by ethnicity and is significantly increased by a high rate of consanguinity in the population. Until 1980, only sparse information on RBDs was available, but in the last decade the number of reported studies has considerably increased, particularly pertaining to the molecular aspects of RBDs. At the same time, interest in the worldwide distribution of RBDs led to the creation of large databases aimed at collecting epidemiological data and at providing information to haemophilia organizations and treatment centres involved in reducing or preventing complications of bleeding. Two recent large data collections have helped to derive the worldwide distribution of RBDs (Figure 42.1), one by the World Federation of Haemophilia (WFH, http://www.wfh.

Postgraduate Haematology: 6th edition. Edited by A. Victor Hoffbrand, Daniel Catovsky, Edward G.D. Tuddenham, Anthony R. Green © 2011 Blackwell Publishing Ltd.

Factor XIII deficiency, 827 Clinical manifestations and treatment, 829 Molecular defects, 829 Vitamin K-dependent coagulation factors deficiency, 831 Clinical manifestations and treatment, 832 Molecular defects, 832 Concluding remarks, 832 Molecular defects, 832 Treatment, 832 Acknowledgements, 835 Selected bibliography, 835

org/) and the other by the International Rare Bleeding Disorders Database (RBDD, www.rbdd.org). The WFH started collecting information on haemophilia care throughout the world in 1998, but collection of data on RBDs only started in 2004. The current global survey includes basic demographic information, data on care resources and treatment products, and information on the prevalence of infectious complications such as HIV and hepatitis C in individuals with haemophilia, von Willebrand disease, platelet disorders and other RBDs. The RBDD, developed in 2004, has spearheaded the development of an international network of care providers to work together to discuss prevalence, clinical manifestations, and need for coordinated and consistent data collection. The goal of this international community effort is to better identify the number of affected individuals throughout the world, to define the clinical manifestations and sequelae associated with these disorders, and to share diagnostic and treatment expertise. Comparison of the data obtained by the two surveys shows similar results, confirming that FVII and FXI deficiencies are the most prevalent RBDs (representing about 29–33% of the total number of affected patients), followed by deficiencies of fibrinogen, FV and FX (8–9%), FXIII (∼ 6%) and combined FV and FVIII (∼ 5%), while the rarest disorder was FII deficiency with a prevalence of 2%. It is not possible to define a clear bleeding pattern among patients with RBDs, as symptoms are varied and heterogeneously distributed, but on the whole bleeding that endangers life, such as central nervous system (CNS) and musculoskeletal bleeding, appears to be less frequent than in haemophilia. 813

Postgraduate Haematology

Table 42.1 General features of autosomal recessive deficiency of coagulation factors. Deficiency

Estimated prevalence*

Gene (chromosome)

Fibrinogen

1 in 1 million

Prothrombin Factor V Combined factor V and VIII Factor VII Factor X Factor XI Factor XIII

1 in 2 million 1 in 1 million 1 in 1 million

FGA, FGB, FGG (all on 4q28) F2 (11p11–q12) F5 (1q24.2) LMAN1 (18q21.3–q22) MCFD2 (2p21–p16.3) F7 (13q34) F10 (13q34) F11 (4q35.2) F13A1 (6p24–p25) F13B (1q31–q32.1) GGCX (2p12) VKORC1 (16p11.2)

Vitamin-K dependent coagulation factors

1 in 500 000 1 in 1 million 1 in 1 million 1 in 2 million Reported in about 30 families

*Including dysfunctional proteins.

FXIII 6%

FXI 29%

FX 9%

Fibrinogen 8% FII 2% FV 8% FV+FVIII 5%

FVII 33%

Figure 42.1 Worldwide distribution of rare bleeding disorders derived from two main large data collections (WFH and RBDD).

The laboratory diagnosis of RBDs is currently carried out by means of coagulation screening tests, such as the activated partial thromboplastin time, the prothrombin time and the thrombin time, applied to subjects reporting a clinical and family history of bleeding. Abnormal results of screening tests should be followed by specific coagulation assays in order to make the specific diagnosis of an RBD. Molecular diagnosis is based on a search for mutation in the genes encoding the corresponding coagulation factors. Exceptions are combined deficiency of FV and FVIII, caused by mutations in genes encoding proteins involved in FV and FVIII intracellular transport

814

(MCFD2 and LMAN1), and combined deficiency of vitamin K-dependent proteins (FII, FVII, FIX and FX), caused by mutations in genes that encode enzymes involved in post-translational modification and in vitamin K metabolism (GGCX and VKOR). Genetic analysis is also important for prenatal and preimplantation genetic diagnosis, especially in those countries with a high rate of RBDs and consanguinity, particularly where genetic prevention by premature termination of pregnancy is not allowed. Since mutation detection became a relatively easy and available strategy in different laboratories worldwide, and it is not always clear whether the identified mutation could be the cause of the severe deficiency of a specific clotting factor, family history associated with allele frequency in the genetic population are important to determine if the identified mutations are common polymorphisms. Treatment of RBDs is a difficult task, since information on the clinical management of RBDs is often scarce and replacement therapy of coagulation factors may require the prescription of unlicensed products that are not readily available. A patient’s personal and family history of bleeding are important guides for management. Dosages and frequency of treatment depend on the minimal haemostatic level of the deficient factor, its plasma half-life and the type of bleeding episode. The main treatments in RBDs are represented by non-transfusional adjuvant therapies (antifibrinolytic amino acids, estrogen/progestogen) and replacement therapy of the deficient coagulation factor. Unfortunately, information on the safety and efficacy of the few available products is scarce and experience in their optimal use significantly more limited than that available for haemophilia. Particular care should be paid to women affected by RBDs. Since the latter are inherited in an autosomal recessive manner, roughly about 50% of patients are women. This implies that the global distribution of RBDs also reflects the global distribution of affected women, posing important social and medical problems, such as excessive menstrual bleeding (or menorrhagia) and bleeding during pregnancy and childbirth. Affected women also suffer the secondary consequences of such events, such as chronic iron-deficiency anaemia and a significantly reduced quality of life, due mainly to a complicated reproductive life but also to limitations in social activities and work. Postpartum bleeding often occurs if replacement therapy is not administered after delivery. Recurrent miscarriages are also described, but only in afibrinogenaemic and FXIII-deficient women. In the end, pregnancy is not contraindicated in patients with RBDs but requires a multidisciplinary approach: the best management of pregnancy should be decided through the coordinated action of a team composed of paediatricians, haematologists and obstetricians. In addition to female healthcare, the same team should ensure investigation and management of potentially affected newborns, particularly in families already having one affected child, because severe FVII, FX and FXIII deficien-

Rare bleeding disorders

cies present with a high risk of intracranial haemorrhage during the first week of life.

Fibrinogen deficiency Fibrinogen deficiency is heterogeneous and two main phenotypes can be distinguished: plasma and platelet levels of the protein are not measurable or very low, leading to afibrinogenaemia and hypofibrinogenaemia. In other cases, low clottable fibrinogen contrasts with normal or moderately reduced fibrinogen antigen leading to dysfibrinogenaemia and hypodysfibrinogenaemia. Fibrinogen is produced in the hepatocyte from three homologous polypeptide chains, Aα, Bβ and γ, which assemble to form a 340-kDa hexamer. The three genes encoding fibrinogen Bβ (FGB), Aα (FGA) and γ (FGG), ordered from centromere to telomere, are clustered in a region of approximately 50 kb on chromosome 4 in humans. Experimental disruption of the α-chain gene makes mice completely deficient in all the fibrinogen chains. There is no evidence of defective embryonic development, but the importance of fibrinogen in pregnancy is demonstrated in studies with fibrinogen knockout mice, which cannot maintain gestation to term. Both arterial and venous thromboembolic complications have been reported in afibrinogenaemic patients.

However, in the majority of patients no traditional thrombotic risk factors are present: one explanation for this predisposition is that even in the absence of fibrinogen, platelet aggregation is possible due to the action of von Willebrand factor and, in contrast to haemophiliacs, afibrinogenaemic patients are able to generate thrombin, both in the initial phase of limited production and also in the secondary burst of thrombin generation. In an in vivo thrombosis model, the number of embolized thrombi was sixfold higher in fibrinogen knockout mice than in wild-type mice, with large emboli very often leading to vessel occlusion (Table 42.2).

Clinical manifestations and treatment Dysfibrinogenaemic and hypofibrinogenaemic patients are usually asymptomatic, while those with afibrinogenaemia have a bleeding tendency that usually manifests in the neonatal period, with 85% of cases presenting umbilical cord bleeding. Bleeding may also occur in the skin, gastrointestinal tract, genitourinary tract or the CNS, while persistent damage to the musculoskeletal system and resulting handicap is less frequent. A milder symptom such as epistaxis is also frequent. Women may suffer from menometrorrhagia, but some have normal menses. First-trimester abortion is common in women with afibrinogenaemia but less common in those with dysfibrinogenaemia and

Table 42.2 Knockout mouse models of coagulation factors and vitamin K dependent proteins. Coagulation factor

Embryonic lethality

Compatible with survival to adulthood

Murine phenotype

Fibrinogen

No

Yes

FII

Partial

No

FV

Partial

No

FVII

No

No

FX

Partial

No

FXI FXIII

No No

Yes Yes

Vitamin K dependent (GGCX) Vitamin K dependent (VKORC1)

Partial

No

Normal embryonic development, bleeding events in one-third of newborns, females with deletion of both alleles do not support pregnancy due to vaginal bleeding Neonatal haemorrhage, no survival beyond several days, loss of vascular integrity Death caused by haemorrhage in neonates, no survival beyond 2 hours, vascular defects Normal embryonic development, death caused by haemorrhage in neonates, no survival beyond 24 days, no vascular defects Death caused by haemorrhage in neonates, no survival beyond 20 days, no vascular defects Normal life, no tendency to spontaneous bleeding Reproduction impaired and bleeding episodes associated with reduced survival Massive intra-abdominal haemorrhage

No

No

Death caused by predominantly intracerebral haemorrhage 2–20 days after birth

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Postgraduate Haematology

has not been reported in hypofibrinogenaemia. Postpartum bleeding is relatively frequent when no prophylactic replacement therapy is given. The conventional treatment is episodic and on-demand, in which fibrinogen is administered as soon as possible after onset of bleeding; however, effective long-term secondary prophylaxis with administration of fibrinogen every 7–14 days (particularly after CNS bleeds) has recently been described. Replacement therapy is effective in treating bleeding episodes in congenital fibrinogen disorders but, depending on the country of residence, patients receive fresh-frozen plasma (FFP), cryoprecipitate or fibrinogen concentrate. The latter is the treatment of choice because it is virally inactivated and thus safer than cryoprecipitate or FFP. In many countries fibrinogen concentrates remain inaccessible due to the cost of production.

Molecular defects In 1999, a homozygous deletion of approximately 11 kb in the FGA gene was first identified as a cause of inherited afibrinogenaemia in four members of a Swiss family by Neerman-Arbez and colleagues. Since this report many other mutations, the majority in FGA, have been identified in patients with afibrinogenaemia (in homozygosity or compound heterozygosity) or hypofibrinogenaemia. The relatively high number of mutations located in FGA led to the hypothesis that mutations tend to cluster in this gene, resulting in complete lack of production of fibrinogen Aα chain or of a severely truncated protein. Of particular interest are missense mutations that lead to complete fibrinogen deficiency. These are clustered in the highly conserved C-terminal globular domains of the Bβ and γ chains. Recent functional studies of these mutations in transfected cells have demonstrated either impaired assembly or impaired secretion of the fibrinogen hexamer, demonstrating the importance of these globular structures in quality control of fibrinogen biosynthesis. To date only two missense mutations, p.Cys64Phe and p.Met70Arg (Cys45Phe and Met51Arg, numbering without the signal peptide) located at the start of the coiled-coil in FGA, have been found in fibrinogen deficiency. Among the large number of mutations identified in patients with congenital afibrinogenaemia, two common mutations were found in individuals of European origin, both in FGA: the Asp153SerfsX4 mutation (or c.510+1G→T according to the nomenclature guidelines of the Human Genome Variation Society, www.hgvs.org) and the FGA 11-kb deletion, both found on multiple haplotypes. Accordingly, the Asp153SerfsX4 should be the first mutation to be screened in all new patients of European origin. Figure 42.2 shows the mutations accounting for afibrinogenaemia and hypofibrinogenaemia identified so far. Two further residues of prime interest in screening for dysfibrinogenaemia are residue Arg16 in exon 2 on FGA, which is part of the thrombin cleavage site in the fibrinogen Aα chain, 816

and residue Arg275 in exon 8 on FGG, which is important for fibrin polymerization.

Prothrombin deficiency Prothrombin deficiency is perhaps the rarest inherited coagulation disorder, with a prevalence of about 1 in 2 million. Based on the measurement of prothrombin (FII) activity and antigen level, two main phenotypes can be distinguished: hypoprothrombinaemia (both levels are concomitantly low) and dysprothrombinaemia (normal or near-normal synthesis of a dysfunctional protein); hypoprothrombinaemia associated with dysprothrombinaemia was also described in compound heterozygosity. FII, a vitamin-K dependent glycoprotein synthesized by liver, is the zymogen of the serine protease αthrombin and is encoded by a gene of approximately 21 kb located on chromosome 11. No living patient with undetectable plasma prothrombin has been reported so far, consistent with the demonstration that in mice complete prothrombin deficiency obtained by gene knockout is incompatible with life, resulting in embryonic or neonatal lethality (see Table 42.2).

Clinical manifestations and treatment Hypoprothrombinaemia, with plasma levels below 10%, is characterized by severe bleeding manifestations such as spontaneous haematomas and haemarthroses, intracerebral bleeding and gastrointestinal haemorrhages. Clinically, dysprothrombinaemia manifests as a variable bleeding tendency that is usually less severe than true deficiency, while heterozygote subjects are usually asymptomatic although, occasionally, excessive bleeding after surgical procedures can be observed. In homozygous women menorrhagia is frequent. Replacement therapy is needed only in homozygous patients, in case of bleeding or to ensure adequate prophylaxis before surgical procedures. Since no FII concentrate exists, FFP, prothrombin complex concentrate (PCC) or both are used for treating patients. In severe clinical settings, higher levels of prothrombin may be achieved with PCCs without the risk of potential volume overload induced by FFP. However, most PCCs contain other vitamin-K dependent coagulation factors, which could potentially induce thrombotic complications.

Molecular defects Genetic variants have been identified all along the prothrombin gene, although frequently they involve the catalytic area, consistent with the fact that they affect the enzymatic activity of the protein. Mutations in different exons may cause similar phenotypic expression of prothrombin. Most of the natural mutants derive from missense mutations (80%), although insertion/ deletions (10%), nonsense mutations (6%) and splice-site

Rare bleeding disorders

Exon 2

FGB IVS1+2076A>G p.Arg17X, p.Tyr41X, Lys83IlefsX3, Lysfs48X

1 2

p.Met118Lys

3

p.Asp134GlyfsX13

4

E domain Coiled-coil region

5 p.Arg255His, p.Arg264Gly, p.Trp226X, c.958+1G>A, p.Glu291AsnfsX24 IVS6-320A>T p.Asp316Tyr, p.Gly347Arg, p.Leu353Arg, p.Asn351X, c1245-1G>C

6

p.Gly400Asp, p.Trp402X, p.Gly414Ser, p.Gly19ValfsX17 p.Gly434Asp, p.Trp437Gly, p.Trp437X, p.Trp440X

8

7

D domain

FGA

Exon 4

Arg181X, Leu188PhefsX4, p.Leu185X, Leu212X, p.Lys219X, p.Trp229X, p.Ser293AlafsX109, Trp295X, p.Gly297GlugsX105 p.Gly297X, p.Trp315X, Gly342GlufsX79, Asn346ThrfsX75, p.Pro352LeyfsX69 p.Thr525LeufsX24, p.Tyr560PhefsX99, p.Pro343LeufsX59, p.Arg458Cys 5

Exon 4

Exons 5–6

Entire gene

6

p.Arg110X, p.Lys125SerX16, p.Gln131X, p.Arg149X, p.Asp153SerfsX p.Ser47PhefsX10, p.Cys45Phe, p.Met51Arg p.Val58delinsProLeuMetX, p.Tyr76X, p.Ser100X, c.180+2T>C p.Gly13X, p.Val21TrpfsX32, c.180+2T>C p.Phe21LeufsX21, c.54+1G>a, c.54+3A>G

4

Coiled-coil region

3 2 1

E domain

FGG p.Arg375Trp, p.Thr371Ile p.Gly284Arg, p.Ala289Val, p.His307Tyr, p.Ala341Val, p.Asn345Ser p.Arg197X, p.Gly200Val, p.Trp227Cys p.Asn230His, p.Asn230Asp, p.Glu231X p.Cys153Arg Leu150X p.Arg108X, p.Ala82Gly p.Gly161IlefsX1 p.Asn7ThrfsX4, c.123+1G>A, c.124–3C>G p.Tyr1ValfsX17

10 9 8 D domain 7 6 5 4 3 2 1

Coiled-coil region E domain

–1138C>T

Figure 42.2 Schematic representation of the localization of the main mutations along the human fibrinogen genes, projected onto the exons encoding the domains of the protein. Exons

(rectangles) are drawn to scale while introns (lines) are not to scale. Gross deletions are indicated by arrows.

817

Postgraduate Haematology

mutations (4%) have also been described. The abnormalities may be classified according to the site of the defect, i.e. in the activation mechanism or in the protease activity of thrombin. The latter group can be subdivided into two subtypes: one in which there is defective amidolytic activity and another where there is defective interaction of thrombin with substrates because of mutations in the molecular recognition domains. Other mutations indirectly affect the catalytic activity of thrombin, as they cause perturbation of the Na+-binding site of the enzyme. However, while in vitro Na+ binding seems to play a major role in thrombin activity, the natural prothrombin mutants with altered Na+ binding described so far (Arg517Gln and Lys556Thr, in exons 13 and 14, respectively) had only a mild haemorrhagic phenotype. To date several functional studies have been performed to elucidate the structure– function relationship. However, recent biochemical characterization of natural prothrombin mutants (Des-Lys9/10 and Phe299(7)Val) has revealed an important role of the A-chain for correct function of the catalytic B-chain of thrombin. These natural mutations could affect the interaction of divalent metals with thrombin, recently localized at the interface between the A and B chains, which seem to stabilize the active conformation of thrombin. Figure 42.3 shows the mutations identified so far.

Factor V deficiency FV has a double role in the coagulation process: it is a protein cofactor required by the prothrombinase complex for thrombin generation, but also contributes to the anticoagulant pathway by downregulating FVIII activity, so that FV deficiency may result in either haemorrhagic or thrombotic tendency. The majority of individuals with FV deficiency are characterized by concomitant deficiency of FV activity and antigen levels (type I deficiency), but approximately 25% have normal antigen levels, indicating the presence of a dysfunctional protein (type II deficiency). FV is mainly secreted by hepatocytes, but there is evidence that it can also be synthesized in vivo in the megakaryocyte/ platelet lineage. FV is encoded by a large (80 kb) and complex (25 exons) gene located on chromosome 1. Experimental deficiency of FV in gene knockout mice leads to defective embryonic development and early haemorrhagic death. However, mice expressing minimal FV activity below the sensitivity threshold of the detection assay (< 0.1%) differ from the complete knockout mice because they do survive (see Table 42.2).

Clinical manifestations and treatment Symptomatic patients usually present with skin and mucous membrane bleeding; epistaxis and menorrhagia are relatively frequent, even in patients with measurable FV levels. Typically, FV-deficient patients are difficult to diagnose and often come 818

to medical attention because of a positive family history or abnormal preoperative laboratory screen. On the whole, it appears that the clinical phenotype of patients with FV deficiency is completely different from that seen in the mouse knockout model. Haemarthroses and haematomas occur in only 25% of patients, and life-threatening bleeding episodes in the gastrointestinal tract and the CNS are rare. There are no published data on management of pregnancy in women with FV deficiency. Postoperative and oral cavity haemorrhages are common, but not fully predictable, as these symptoms also occur with plasma levels as high as 5–10%. This discrepancy is likely to be explained by the relatively poor sensitivity of FV bioassays, which do not measure those small amounts of the factor that are probably sufficient to make the deficiency compatible with life and a mild clinical phenotype. Replacement therapy with FV can be administered only through FFP, preferably virus inactivated, because no FV concentrate is available and FV is not contained in significant amount in cryoprecipitate or PCCs.

Molecular defects Among rare coagulation disorders, FV deficiency is one of the least characterized from the molecular point of view, with only 56 genetic defects hitherto described, the majority of them identified in the last 4 years (Figure 42.4). A total of 48 mutations seem to be responsible for the type I deficiency, only one being recurrent (Tyr1702Cys, repeatedly found in Italian individuals), confirming the remarkable allelic heterogeneity of the disease. A mild type I deficiency (decrease in FV levels) has also been associated with two FV variants: the functional Met2120Thr polymorphism, demonstrated to cause about 25% reduction in FV levels, and the HR2 haplotype defined by a group of more than 10 polymorphisms; among these, the dominant contribution of the Asp2194Gly variant was demonstrated by expression experiments in eukaryotic cells. Only one genetic defect, Ala221Val, associated with type II deficiency has so far been reported. Mutations causing FV deficiency do not cover the full spectrum of possible genetic lesions, since mutations located in the promoter as well as large deletions are absent. Missense mutations are completely absent in the large exon 13, coding for the whole B domain. This can probably be explained by an increased tolerance of this domain to variations, as suggested by the fact that it is highly polymorphic and not evolutionarily conserved. Finally, no clear genotype–phenotype correlation emerges between the clinical phenotype, plasma FV levels and the corresponding associated mutations in FV deficiency. In fact, despite carriership of the same mutations, some patients often present with a more severe bleeding pattern than others. Recently, the possibility that the severe FV-deficient phenotype may be modulated by a concomitant procoagulant defect was investigated by Duckers and colleagues, who showed that congenital FV deficiency associated with reduced plasma levels of

Rare bleeding disorders

Arg-39Gln

1

Arg-4Gln, Arg-2Trp, Arg-1Gln, Gln7Lys, Glu16Gly, Glu29Gly

2

Prepro region

GLA domain Tyr44Cys

3 4

Asp118Tyr, Thr122Met, Cys138Tyr

5 6

Glu157Lys, Arg220Cys

7

Kringle 1 region

Kringle 2 region Phe299Val,Glu300Lys, delLys301/302, Glu309Lys, Gly319Arg, Arg320His, Gly330Ser

Arg271Cys, Arg271His

Met337Thr, Arg340Trp, Ser354Arg, Trp357Cys, Arg382Cys, Arg382His, Arg388His

8 9

A chain

10 Arg418Trp

11

Arg457Gln, Glu466Ala

12

B chain

Arg538Cys, Lys556Thr, Gly558Val, Gln541stop, Glu548Ala, His562Arg, Trp569stop

Arg517Gln

13 14

Figure 42.3 Schematic representation of the localization of the main mutations along the human prothrombin gene, projected onto the exons encoding the domains of the protein. Exons (rectangles) are drawn to scale while introns (lines) are not to scale.

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Postgraduate Haematology

1 2 3 Glu119stop, FS150stop

4

A1 domain

5 Lys310stop, Phe325Leu, FS351stop

Ala221Val, Ser234Trp

6 7

IVS8+6T>C, IVS8-2A>G

Gly329Cys

Ile417Tyr

8 9

Cys472Gly, 478del Tyr, 1701G>T Tyr530Ser, FS545stop

10 11

Cys585Arg, FS592stop

FS689stop, Arg712stop, Gln773stop, FS836stop, FS900stop, FS930stop, Arg1002stop, Arg1133stop, FS1142stop, FS1259stop, FS1303stop, FS1381stop, FS1560stop

12

13

FS1590stop, Arg1606stop, Glu1608Lys

14

FS1659stop, Tyr1702Cys

15

FS1776stop

16

Val1813Met, FS1829stop

17

Gly1852Val, Trp1854stop

18 19 20 21

IVS16-3G>T

A2 domain

IVS18-12T>A IVS19+3A>T delN1982-S1983, FS1989stop IVS21+1G>A Pro2070Leu, Arg2074Cys, Arg2074His Gly2079Val

A3 domain

C1 domain

22 23 24

IVS24+1delGTAG Arg2174Leu

B domain

C2 domain

25

Figure 42.4 Schematic representation of the localization of the main mutations along the human FV gene, projected onto the exons encoding the domains of the protein. Exons (rectangles) are drawn to scale while introns (lines) are not to scale.

820

Rare bleeding disorders

tissue factor pathway inhibitor, which decreases the FV requirement for minimal thrombin generation to less than 1%, probably protects patients from severe life-threatening bleeding.

Combined deficiency of factor V and factor VIII For many years the molecular mechanism for combined deficiency of these two factors, each transmitted with different patterns of inheritance (autosomal recessive for FV, X-linked for FVIII) and involving proteins encoded by different genes, was not understood. Finally, in 1998 combined FV and FVIII deficiency (F5F8D) was causally associated with mutations in the LMAN1 gene, encoding lectin mannose-binding protein (previously named ERGIC-53), a 53-kDa type 1 transmembrane protein that acts as a chaperone in the intracellular transport of both factors. More recently, in 2003, another locus associated with the deficiency was identified in about 15% of affected families with no mutations in LMAN1; this was named the MCFD2 gene, encoding multiple coagulation factor deficiency (MCFD)2 protein, which acts as a cofactor for LMAN1, specifically recruiting correctly folded FV and FVIII in the endoplasmatic reticulum. Recent studies have failed to identify additional components of the LMAN1–MCFD2 receptor complex, supporting the idea that F5F8D might be limited to LMAN1 and MCFD2.

Clinical manifestations and treatment F5F8D is characterized by concomitantly low levels (usually 5–20%) of the two coagulation factors, both as coagulant activity and as antigen. It appears that the concomitant presence of two coagulation defects does not enhance the haemorrhagic tendency observed in each defect separately (see section on FV deficiency). The phenotypes associated with mutations in MCFD2 and LMAN1 are indistinguishable and manifested only by deficiencies of FV and FVIII, although a selective delay in secretion of the cargo protein procathepsin C has been observed in HeLa cells overexpressing a dominant-negative form of LMAN1. Symptoms are usually mild, with a predominance of easy bruising, epistaxis, and bleeding after dental extractions. Menorrhagia and postpartum bleeding have also been reported in affected women. More severe symptoms, such as haemarthroses and umbilical cord bleeding, are observed very seldom and gastrointestinal and CNS bleeding have been reported in only a few patients. Soft-tissue haematomas are unusual. Because of the mild clinical pattern, bleeding episodes are usually treated on demand and do not require regular prophylaxis. Sources of both FV and FVIII are needed and their differential plasma half-lives (FV 36 hours, FVIII 10–14 hours) have to be taken into consideration.

As there are no FV concentrates available and there is little or no FV present in cryoprecipitate or PCCs, replacement of FV can currently be achieved only through the use of FFP, preferably with virus-inactivated plasma. For FVIII replacement, a large number of products are available: the synthetic hormone desmopressin (DDAVP) can be successfully used for minor bleeding episodes to raise FVIII. However, the chemical efficacy of DDAVP needs to be tested in each patient and more than three to four consecutive treatments should be avoided. For severe bleeding, plasma-derived FVIII or recombinant FVIII concentrates are the treatments of choice and include FFP, plasma-derived concentrates and recombinant FVIII.

Molecular defects F5F8D is caused by mutations in LMAN1 or MCFD2 proteins. The first is encoded by a gene of approximately 29 kb located on chromosome 18 and containing 13 exons, while the latter is encoded by a shorter gene of approximately 19 kb located on chromosome 2 and containing four exons. To date, a total of 48 mutations (Figures 42.5 and 42.6) have been described (32 in LMAN1 and 16 in MCFD2; http://www.med.unc.edu/isth/ mutations-databases/FVandVIII_2006numbers.htm). All the LMAN1 mutations reported so far are null mutations, with the exception of a Cys→Arg mutation that disrupts a disulphide bond required for oligomerization and which also destabilizes the protein. In contrast, both null and missense mutations have been identified in MCFD2; in particular, five of six missense mutations reported (Asp81Tyr, Asp89Ala, Asp122Val, Asp129Glu and Ile136Thr) disrupt LMAN1 binding, indicating that LMAN1 and MCFD2 must function as a unit to transport FV and FVIII. Recently, Zhang and colleagues have performed a genotype–phenotype analysis to evaluate if mutations in the two different genes are associated with differences in FV and FVIII plasma levels. Mean plasma levels of FV and FVIII in patients with MCFD2 mutations were significantly lower than those in patients with LMAN1 mutations. These data suggest that MCFD2 may play a primary role in the export of FV and FVIII from the endoplasmatic reticulum. Some mutations in both LMAN1 and MCFD2 recurred in more than one family, for example Met1Thr, 89insG and Lys302stop on LMAN1 or IVS2+5G→A and Ile136Thr mutations on MCFD2. In particular, the MCFD2 IVS2+5G→A appears to be one of the most common mutations causing F5F8D and it has now been identified in at least 13 unrelated families from different geographic regions (India, Italy, USA, Serbia and Germany).

Factor VII deficiency FVII deficiency is the most common autosomal recessive coagulation disorder (1 in 500 000). A typical feature of this disease 821

Postgraduate Haematology

Met1Thr, 23delG, 31delG, 89insG

1

Gly114stop

2

422delC

3

Signal peptide

Lectin leg-like domain

4 Arg202stop

5

IVS5+1G>T 720del16bp 780delT IVS7-1G>A, IVS7-1G>C, IVS7+1G>A IVS7+62del10bp, IVS7+33insGGTT Lys302stop, Gln317stop, 795delC, 841delA, 912insA

6 7 8

Glu321stop, 1109delTC, Gln280stop

9

IVS9+2T>G, IVS9+2T>C 1208insT, 1214delAAATG

10

Arg456stop, 1270delG, 1356delC

11

Cys475Arg

12

Stalk domain

1519delA Transmembrane domain 13

is its clinical heterogeneity, which ranges in severity from lethal to mild, or even asymptomatic forms. FVII is synthesized in the liver and is encoded by the FVII gene (F7) located on chromosome 13, 2.8 kb upstream of the FX gene. Plasma levels of FVII coagulant activity (FVII:C) and FVII antigen (FVII:Ag) are influenced by a number of genetic and environmental factors (sex, age, cholesterol and triglyceride levels) and it is also well known that FVII levels are modulated by F7 polymorphisms. The majority of patients have concomitantly low levels of FVII:C and FVII:Ag, but several cases are characterized by normal or low borderline levels of FVII:Ag contrasting with lower levels of FVII:C. Most gene knockout mice made experimentally deficient in FVII develop normally, which suggests that complete lack of FVII is compatible with life (see Table 42.2).

822

Figure 42.5 Schematic representation of the localization of the main mutations along the human LMAN1 gene, projected onto the exons encoding the domains of the protein. Exons (rectangles) are drawn to scale while introns (lines) are not to scale.

Clinical manifestations and treatment The severity of symptoms of FVII deficiency is variable and generally reported to be poorly correlated with plasma levels. Some patients do not bleed at all after major episodes of haemostasis. Life- or limb-endangering bleeding manifestations are relatively rare, the most frequent symptoms being epistaxis and menorrhagia. CNS bleeding was also reported to have high incidence (16%) in a series of 75 patients affected by FVII deficiency, the study concluding that the greatest risk factor for development of bleeding was trauma related to the birth process. However, these data were not confirmed by two subsequent large series of Italian and Iranian patients. Thrombotic episodes (particularly deep vein thrombosis) have also been reported in 3–4% of patients with FVII deficiency, particularly

Rare bleeding disorders

N-term

1

–1G>C 103delC

2

Signal

3

EF-1

IVS2+5G>A 210del35bp, 249delT, 263del8bp, Asp81Tyr, Asp89Ala,374insGA

IVS3+1G>A EF-2

Asp122Val, Asp129Glu, Tyr135Asn, Ile136Thr, Ser144stop

4

Figure 42.6 Schematic representation of the localization of the main mutations along the human MCFD2 gene, projected onto the exons encoding the domains of the protein. Exons (rectangles) are drawn to scale while introns (lines) are not to scale.

in the presence of surgery and replacement treatment, but spontaneous thrombosis may also occur. Lack of correlation between measured FVII:C in vitro and the clinical phenotype is well known and is probably due to the fact that only trace amounts of FVIIa are required to initiate coagulation in vivo. Empirical studies and mathematical modelling suggest that as little as 5 pmol/L (0.05% of normal FVII concentration) is sufficient to induce clot formation. None of the in vitro tests could differentiate between null and extremely low levels of FVII:C capable of initiating coagulation in vivo. A number of therapeutic options can be offered to patients with FVII deficiency, including FFP, still used in developing countries, PCCs, plasma-derived FVII concentrates and recombinant FVIIa, which has to be considered the optimal replacement therapy because it can be used at very low dose (10–20 µg/ kg). Prophylaxis has been a debated issue in FVII deficiency, especially because of the very short half-life of infused FVII in blood. Ex vivo studies have shown that infused recombinant FVIIa disappears quickly from the circulation but persists extravascularly bound to pericytes. These observations would support the feasibility of prophylaxis in FVII deficiency, but larger trials are needed to assess the optimal schedule. The occurrence of frequent menorrhagic menses is almost invariably associated with iron deficiency and anaemia in

women with the severe form of FVII deficiency. Pregnancy itself does not require special precautions and uncomplicated delivery is possible without prophylaxis. However, all cases of postpartum bleeding reported so far occurred in women with low FVII coagulant activity (< 15 units/dL) not receiving prophylaxis. Therefore, delivery should occur under the coverage of a short-term replacement.

Molecular defects The molecular bases of FVII deficiency are more extensively characterized than those of other defects, perhaps due to the relatively high frequency of this defect and small size of the gene. Mutations are very heterogeneous: missense variants are the most frequent lesions (70–80%), splice-site changes are also well represented, while nonsense mutations and small deletions are rare, particularly in the homozygous condition. Mutations are located throughout the gene, suggesting that all domains are important in maintaining the overall structure and function of FVII. The previously available online database http://europium. csc.mrc.ac.uk/ allowed rapid access for researchers to the entire listing of F7 mutations but is currently under revision; however, an updated overview of mutations is shown in Figure 42.7. The severe cases are all either homozygous or doubly heterozygous

823

Postgraduate Haematology

–96C>T, –94C>G(sp1), –79C>T, –65G>C, –62CT, –61T>G(HNF4), –60T>C(HNF4), –60T>G, –59T>G, –55C>G, –55C>T(HNF4), –44T>C, –39A>G, –32A>C, –30A>C Met–60Ile, Met–60Val, Gln–57stop, –55delC, –52delCTstop Leu–48Pro, Leu–42Pro, Gly/Val–39Ser/Ile, –26delCT, Gln–35stop Leu–20Pro, Val–17Ile, Val–15Ala, Alla-10Asp, Val–7Ile IVS1a+5G>A, IVS1-11G>A, IVS1-8del4bp Met1Val, Phe4Leu, 3892delTTC, Pro10Gln, Leu13Gln, Glu14delA, Glu14insA, Glu16Lys, Cys22Phe, Cys22Arg, Ser23Pro, Glu23Lys, delPhe24, Glu26stop, Arg28Gly, Arg28Pro, Glu29Lys, Lys32Gln, Lys32stop

IVS2+5G>T, IVS2+1G>C, IVS2+1G>A, IVS2+1delG, IVS2-3C>G

Entire gene

Gln49stop, Ser52stop, Asn57Asp, Asn57Ile, Ser60Pro, Cys61Phe, Cys61stop, Leu65Pro, Tyr68Cys, Gly78Asp, Arg79Trp, Arg79Gln

IVS3-1G>A IVS3+1G>A IVS3+5G>A

1a Prepro region 1b Gla domain 2 3 4

IVS4+1G>A

Cys91Ser, Glu94Lys, Gly96Ser, Gly97Ser, Gly97Cys, Gly97Val, Gln100Arg Cys102Tyr, Ser103Gly, Thr106delC, Arg110Cys, Ser111Phe, Gly117Arg Leu121Pro, Aps123Asn, Asp123Tyr, Ser126Phe, Pro129Ser

EGF1 and EGF2 region

5

IVS5-12T>A IVS5-2A>G Pro134Thr, Pro134Leu, Cys135Arg, Lys137Glu, Ile140Ser Arg152stop, Arg152Gln, 8968delG, Val154Gly, Gly156Asp, 156delG

6

IVS6-1G>T, IVS6+1G>A, IVS6+3A>G, IVS6+1G>T Cys178Tyr, Gly179Arg, Gly180Arg, Thr181Asn, 188delC, Ala191Thr, Ala191Glu Ala191Val, Cys194Arg, Cys194Tyr, Leu204Pro, 9711delC, Ala206Thr

Protease 7

IVS7+3del4, IVS7+5G>A, IVS7+7A>G, IVS7+2T>G, IVS7+1G>A, IVS7+3delGGGT 10554del15bp, 10554ins15bp, Asp212Asn, Gln211stop, Asp217Asn, Arg223Trp, Arg224Gln, Gln227stop, Thr238Ile, Thr239Pro, 10586del16bp, His241Gln, Asp242Asn, Asp242His, Ala244Val, Ala244Thr, Arg247Cys, Arg247His, Val252Met, 10696delC, Leu261Phe, Glu265Lys, Arg266Gln, Arg271insGG, Thr272Met,10743delG, Arg277Cys, Arg277His, Val281Phe, Val281Pro, Ser282Arg, Trp284stop, Gly283Ser, Gly285Ser, 10785delC, Arg290Cys, Ala294Val, Met298Val, Met298Ile, Leu300Pro, Pro303Thr, Pro303His, Pro303delCC, Pro303Arg, Arg304Trp, Arg304Gln, Leu305Val, Met306Asp, Met306Gln, Asp309Ser, Cys310Phe, Arg315Trp, Thr324Met, Met327Ile, Met327Thr, Met328Val, Phe328Ser, Cys329Gly, Cys329Arg, Gly331Cys, Gly331Ser, Gly331Asp, Ser339Phe, Ser339Cys, Lys341Glu, Gly342Arg, Gly342Glu, Asp343Asn, Asp343His, Ser344Asn, His348Arg, His348Gln, 10983delT, 10896_10913del18bp, Gly354Cys, Trp356stop, Gly357Glu, Thr359Met, Val362Phe, Ser363Ile, Trp364Cys, Trp364Phe, Trp364stop, Gly365Ala, Gly365Cys, Ala369Thr, Gly372Asp, Gly375Glu, Tyr377Phe, Arg379Gly, Gln382stop, Gly385Lys, Arg402stop, 404delC

Figure 42.7 Schematic representation of the localization of the main mutations along the human FVII gene, projected onto the exons encoding the domains of the protein. Exons (rectangles)

824

8

are drawn to scale while introns (lines) are not to scale. Gross deletion is indicated by the arrow.

Rare bleeding disorders

for mutations that disrupt expression of the protein (e.g. deletions, insertions, splice junctions and promoter mutations), resulting in FVII:C levels typically less than 2% of normal. Individuals with a mild/moderate clinical phenotype are homozygous or doubly heterozygous for missense mutations; however, some missense mutations are also associated with a severe phenotype. Frequent mutations (Ala294Val, 11125delC, Arg304Gln, Cys310Phe) identified among hundreds of patients with FVII deficiency by different groups are associated with a range of clinical severity and neither FVII:C nor gender provide an explanation for this heterogeneity, suggesting that other modifying factors may influence the different clinical manifestations in subjects carrying the same mutation. It is known that FVII levels are modulated by F7 polymorphisms: different genotypes demonstrated up to fivefold differences in mean FVII levels. Tightly linked polymorphisms in the 5′ regulatory region regulate FVII levels and differential promoter activity in vitro; the p.Arg353Gln substitution in the catalytic domain, tightly associated with the decanucleotide insertion (g.–323 to −324insCCTATATCCT) and with the −401T allele in the promoter region, decreases FVII secretion, therefore reducing its plasma levels. The same effect is shown by the g.11293– 11294insAA polymorphism in the 3′ untranslated region of F7 that negatively affects the steady state of mRNA levels, leading to a reduction in FVII mRNA by 40%, which subsequently reduces FVII protein levels. An additional polymorphism, located in intron 7, is characterized by the presence of a variable number of 37-bp repeats. This polymorphism is reported to be associated with variable rates of FVII transcriptional activity according to the number of repeats present in the genotype. The peculiar presence of such repeated regions, and of polymorphic variation of repeats within F7, requires careful design of PCR primers to detect gene rearrangements when molecular diagnosis is performed.

Factor X deficiency FX is a glycoprotein that plays a pivotal role in the coagulation cascade, being the first enzyme in the common pathway of thrombin formation. FX is mainly synthesized by the liver and is encoded by the FX gene, comprising 22 kb and located on chromosome 13, a few kilobases downstream of the F7. The clinical phenotypes of FX deficiency are characterized by concomitantly low levels of coagulant activity and antigen, or low coagulant activity contrasting with normal or low borderline antigen values. Mice rendered experimentally deficient in FX by targeted inactivation of the FX gene showed frequent embryonic lethality. Those who survived bled to death intra-abdominally at birth and in the CNS within the first 3–4 weeks of life (see Table 42.2).

Clinical manifestations and treatment In FX deficiency the bleeding tendency may appear at any age, although the more severely affected patients (FX coagulant activity < 1%) present early in life with, for instance, umbilicalstump, CNS or gastrointestinal bleeding. Patients with severe deficiencies also commonly experience haemarthroses and haematomas. The most common bleeding symptoms reported at all levels of severity are epistaxis and menorrhagia. In FX deficiency there is reasonable correlation between the severity of clinical manifestations and residual FX activity, confirming animal study results. Data from the United Kingdom Haemophilia Centre Doctors’ Organisation (UKHCDO) registry shows that the proportion of patients with this deficiency who require treatment is much higher than that of other rare coagulation deficiencies. There is no specific FX product, plasma derived or recombinant, available as yet for treatment of patients with FX deficiency; hence current treatment is based on the administration of FFP and PCCs. However, these treatments could be associated with some complications stemming from the use of large amounts of plasma, particularly in children and elderly patients with cardiac disease, or because of the high concentrations of FII, FVII and FIX in PCCs that could be associated with a risk of thromboembolic events. To overcome this problem, a freeze-dried human coagulation FIX and FX concentrate with specified amounts of FX (and FIX) has been developed recently. Therapeutic options for the control of menorrhagia include both medical (e.g. antifibrinolytics, oral contraceptives, levonorgestrel intrauterine device and clotting factor replacement) and surgical (e.g. endometrial ablation and hysterectomy). Apart from menorrhagia, women with FX deficiency, as well as women with other RBDs, are likely to develop other gynaecological problems, such as corpus luteum haemorrhage or haemoperitoneum associated with ovulation, that may warrant the adoption of prophylactic treatment. Even if pregnancy is accompanied by increased concentrations of FX, women with severe FX deficiency and a history of adverse pregnancy outcomes, such as abortion, placental abruption or premature birth, may benefit from continuous replacement therapy. Finally, heterozygotes have also been reported to have bleeding after delivery that required treatment with FFP.

Molecular defects The gene structure and organization is homologous to that of the other vitamin-K dependent proteins, with the exception of prothrombin, suggesting evolution from a common ancestor by a process of duplication and divergence. To date, 105 mutations comprising 85 missense, 14 insertions/deletions (three gross deletions plus 11 micro insertions/deletions), six splice site, two nonsense, and one in the 5′ flanking region have been reported 825

Postgraduate Haematology

(Figure 42.8). Although most mutations are located in exon 8, the number of mutations located in each exon is proportional to the length of the exon itself, indicating the absence of a hotspot region on FX gene. Among the 105 mutations so far reported, only two were nonsense, and only one (C61X) was identified in the homozygous state in three unrelated patients. Moreover, only a few of the entire group of missense mutations so far reported have been recurrent and found in more than one family from the same geographical area. In particular, Arg–1Thr, associated with a severe phenotype (FX coagulant activity < 1%), was identified in the homozygous state in four unrelated patients from Iran, while Gly380Arg, associated with intracranial haemorrhages, was identified in six

homozygous patients from Costa Rica; Pro343Ser (FX Friuli), associated with reduced FX coagulant activity (4–9%), was identified in more than 10 patients from northern Italy. Other mutations, such as Gly–20Arg, Gly94Arg and Gly222Asp, all associated with a severe laboratory phenotype, have been reported from different groups worldwide. To assess whether genotypic variations could have an influence on FX plasma level, four different polymorphisms located in the promoter region (a TTGTGA insertion between position −343A and −342G, a C/T polymorphism at position −222, a C/A polymorphism at position −220 and a C/T polymorphism at position −40) have been investigated but no association was found.

–40C>T Met–40Val, Leu–34Ile, Leu–32Pro, Ser–30Arg Gly–20Arg, 33delGC IVS1-1G>C, IVS1+1G>A IVS1+3A>T Thr–2Met, Arg–1Thr, Ser3Cys, Glu7Gly, Gly7Lys Gly11Val, Glu14Lys, Glu14Gly, Glu16Lys Glu19Ala, Glu25Lys, Glu26Asp, Phe31Ser Ser30Arg, Glu32Gln, p.Glu69Lys IVS2-3T>G

1 Prepro region 2

Glu51Lys, Asn57Thr, Cys61stop Gly78Asp, Cys81Tyr, p.Asp103His

Linking region

4 EGF1–2 domains

IVS4–8delCTT Exon 7 + part od exon 8

Entire gene + part of exon 7

3

Gla domain

Gly94Arg, Asp95Glu, Glu102Lys, Cys109Tyr Cys111Tyr, Gly114Arg

5

Tyr30stop, Gly133Arg, Arg139Cys, Arg139Ser Arg142Met, Gly152Arg, Val196Met, Gly204Glu Gly204Arg, 163delAT, 514delT, 556delC

6

Gly222Asp, Gly223Val, Gly223Arg Thr233Met, Ala234Ser, Val246Met Gly249Arg, 838delC

7

Connection and activation region

IVS7-1G>A Catalytic domain

Arg251Trp, Glu264Lys, Phe281Leu, Asp282Asn, Arg287Trp Val298Met, Pro304Ser, Arg306Cys, Trp308Leu, Glu310Lys Thr318Met, Gly323Ser, Arg326Cys, Glu329Gly, Ser334Pro Val342Ala, Pro343Ser, Tyr344Cys, Arg347Cys, Arg347His Cys350Phe, Ser353Tyr, Ser354Arg, Phe356Cys, Phe363Ile Cys364Arg, Gly366Ser, Asp368Asn, Ser379Lys, Gly380Arg Gly381Asp, Phe382Leu, His383Gln, Ala404Thr, Arg405Gly Lys408Asn, Ile411Phe, Trp421Arg, 882insC, Ile269delTCA 28299delATGCC, 28376insTGCCGCCA, 1151del17bp

Figure 42.8 Schematic representation of the localization of the main mutations along the human FX gene, projected onto the exons encoding the domains of the protein. Exons (rectangles) 826

8

are drawn to scale while introns (lines) are not to scale. Gross deletions are indicated by the arrows.

Rare bleeding disorders

Factor XI deficiency FXI deficiency is characterized by a decrease in the functional activity of this plasma protein, usually accompanied by correspondingly low FXI antigen levels. More rarely, normal or borderline levels of plasma FXI antigen are associated with a dysfunctional form of the protein. The estimated prevalence of the severe deficiency in most populations is about 1 in 1 million, but is reported to be much higher in Ashkenazi Jews, heterozygosity for FXI deficiency being as high as 8%. FXI is mainly synthesized in the liver, although tiny amounts of transcript can also be detected in megakaryocytes and platelets. The protein is encoded by the FXI gene, comprising 23 kb and located on chromosome 4. The existence of a platelet FXI transcript, originating from the skipping of exon 5, was hypothesized for a long time but never confirmed by subsequent work. Nonetheless, recent data support the view that FXI transcripts undergo alternative splicing, leading to the synthesis of FXI isoforms whose physiological role and importance still need to be demonstrated. In knockout mice the loss of the gene coding for this factor is compatible with life, with no tendency for spontaneous bleeding (see Table 42.2).

Clinical manifestations and treatment The relationship between FXI levels in plasma and the bleeding tendency is not as clear-cut as for other coagulation factor deficiencies. The phenotype of bleeding is not correlated with the genotype but with the site of injury. When a site of injury with local high fibrinolytic activity is involved (e.g. urogenital tract, oral cavity after dental extraction or tonsillectomy), the risk of bleeding is increased (49–67%) in comparison to sites with less local fibrinolytic activity (1.5–40%). Usually, patients with severe FXI deficiency (1% or less) are mildly affected and most bleeding manifestations are injury-related. Surprisingly, patients with low but detectable levels of FXI are also mild bleeders, so that clinical phenotypes are not strikingly different in these two groups. This observation, already made for Jewish patients, was recently confirmed in a series of Iranian non-Jewish patients with severe or moderate deficiency (FXI coagulant activity < 1 to 5%) and in patients with mild deficiency (6–30%). All patients were mild bleeders, but those symptoms that define the severity of the bleeding tendency, such as muscle haematomas and haemarthrosis, showed a similar frequency in the two groups of factor-deficient patients (about 25%). The most frequent symptoms were oral and postoperative bleeding, which occurred in more than 50% of patients. Women with FXI deficiency are prone to excessive bleeding during menstruation, but case series of women affected by severe FXI deficiency showed that 70% of pregnancies were uneventful, despite no prophylactic treatment. Also, patients with alloantibodies that inhibit FXI do not bleed spontaneously but only during surgery or

trauma. Patients with inhibitors treated with FFP or FXI concentrates can suffer from prolonged bleeding if the presence of inhibitors is not diagnosed prior to surgery. After being exposed to plasma, 7 of 21 FXI-deficient Israeli patients homozygous for the Glu117stop mutation developed inhibitors to FXI. Recently, Zucker and colleagues showed that this also happened in an Italian woman homozygous for the same mutation after three injections of Rh immunoglobulin and without previous exposure to blood products. Treatment is based on the use of antifibrinolytic agents, FFP, FXI concentrate and recombinant FVIIa, but care should be taken to reduce the risk of complications such as thrombotic events (especially when FXI concentrate or recombinant FVIIa is planned), volume overload, allergic reactions and development of inhibitors. Patients with partial deficiency of FXI who have no bleeding history do not require prophylactic treatment.

Molecular defects To date, more than 180 mutations affecting FXI gene have been identified (Figure 42.9). These can be accessed on three different online databases: • http://www.med.unc.edu/isth/mutations - databases/ FactorXI_2007.html • http://www.wienkav.at/kav/kar/texte_anzeigen.asp?ID=7137 • http:www.factorxi.com. The high allelic heterogeneity of FXI deficiency is highlighted by the distribution of genetic defects throughout the whole gene. Missense mutations represent more than 60% of all variants, only seven of them being reported to lead to the dysfunctional form of the deficiency. Two mutations are responsible for most cases of FXI deficiency in Ashkenazi Jews, while mutations are more varied in non-Jews. The so-called type II Jewish mutation is a stop codon in exon 5, compatible with the observation that patients homozygous for this defect usually have undetectable levels of FXI. The so-called type III Jewish mutation is a missense mutation in exon 9, leading to the Phe283Leu substitution. The mutation causes defective secretion of the protein from cells, but some FXI is ultimately produced so that these patients have measurable levels of FXI (about 10%). Type II/III compound heterozygosity is the commonest cause of severe to moderate FXI deficiency in Ashkenazi Jews.

Factor XIII deficiency FXIII is a transglutaminase, is the last enzyme to be activated in the blood coagulation pathway and functions to cross-link the α and γ fibrin chains, resulting in a stronger clot with an increased resistance to fibrinolysis. The plasma factor consists of two catalytic A subunits (FXIII-A) and two carrier B subunits 827

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–17G>T 1 Met–18Ile, Tyr–14stop, Ser–4Leu, Gly–1Arg, Glu1stop Gln5stop, Asp16His, Val20Ala, Pro23ln, Pro23Leu, Ser24Arg Cys28Phe, Gln29His, Thr33Ile, Thr33Pro, Hys35Tyr Cys38Arg, Cys38Trp, Cys38stop, 192insG, Pro48Leu, Pro52Leu, Arg54stop

IVS2+6T>G

3 Tandem repeat 1 4 IVS4+1G>A

Cys92Gly, Met102Thr, Gly104Asp, Gln116stop, Glu117stop Cys118stop, Cys122Tyr, Tyr123Met, His127Arg, Cys128stop Thr132Met, Tyr133Cys, Tyr133Ser, Ala134Pro, Arg144Cys

5 IVS5+5G>C, IVS5–2A>G

Gly155Glu, Leu172Pro Ala181Val, Cys182Tyr, Arg184Gly, Pro188Ser, 644delTCGACA Asp198Asn, Arg210stop, Cys212Arg, Cys212Ser, Phe221Ser 717insT, Ser225Phe, Gln226Arg, Gln226stop, Trp228Cys Trp228stop, Gln233stop, Arg234Ile, Arg234Lys, Arg234Ser

Phe283Leu, 952delG, 961delTG, 962delG, Ile290Phe, Ile290Thr Glu296Lys, Glu297Lys, Glu279stop, Leu302Pro, Thr304Ile, Val307Phe Arg308Cys, Cys309stop, Thr313Ile, Cys321Phe, Glu323Lys, 324insG

Tandem repeat 2

6 IVS6+3A>G 7

Cys237Tyr, Glu243Asp, Gly245Glu, Ser248Asn, Arg250Cys Arg250His, Lys252Ile, Gln263stop, Val271Leu

Tandem repeat 3 8

IVS8del11bp, IVS8G>C

Gly336Arg, 1072delA, 1118delA, Ile341Met, Leu342Pro Gly344Arg, Gly350Ala, Gly350Arg, Gly350Glu Tyr351Ser, Tyr351stop, Leu355Ser, Cys356Arg

Exons 11–15

2

IVS3+2T>A

Thr57Ile, Cys58Arg, Cys58Phe, Cys58Tyr, Pro69Thr, Gly79Ala Ser81Tyr, 307ins7bp, Lys83Arg, Gln88stop, Ala91Tyr

Entire gene

Leader region

9 Tandem repeat 4

IVS9 –2A>G

IVS10–1G>A, IVS10–4delGTTG, IVS10+1G>A, IVS10+5G>A Val371Ile< Gly372Ala, Ala375Val< Arg378Cys, Try381Leu, Pro382Leu, Trp383stop Thr386Asn, His388Pro, Thr389Pro, Cys398Tyr, Gly400Ser, Gyl400Val Ser401Ala, Gln406stop, Try407Cys, Tyr410Ile, Ala412Ser, Ala412Thr, Ala412Val IVS11–10T>A, IVS11+12G>A Arg425Cys, Arg426Cys, Tyr427Cys Gln433Glu, Phe442Val, Glu447stop Gly460Arg, Thr475Ile IVS12 +2T>G Arg476stop, Arg479stop, Cys482Arg, Cys482Try Ser485Pro, Tyr493His, Trp497Cys Val498Met, Try501Cys, Trp501stop, 501insGly, Tyr503dupG IVS13 +2T>G Lys518Asn, Pro520Leu, Cys527Tyr, Gly544Ser, Glu547Lys, 1757del14bp

10

11

12

13

Catalytic domain

14

IVS14–2A>G, IVS14+1G>A Gly555Glu, Asp556Gly, Cys563Phe, Tp569Ser Thr575Met, Ser576Arg, Gly578Cys, Glu579Lys Cys581stop, Tyr590His, Tyr590stop, Trp599Arg, Ile600Ser

15

Figure 42.9 Schematic representation of the localization of the main mutations along the human FXI gene, projected onto the exons encoding the domains of the protein. Exons (rectangles)

are drawn to scale while introns (lines) are not to scale. Gross deletions are indicated by arrows.

(FXIII-B). FXIII-A is synthesized in cells of bone marrow origin, while FXIII-B is produced in the liver. The corresponding genes are located on chromosomes 6 and 1. FXIII deficiency is probably, together with prothrombin deficiency, the rarest of the

recessively transmitted coagulation factor deficiencies (1 in 2 million). In inherited FXIII deficiency, plasma levels of FXIII-A measured as functional activity or immunoreactive protein are usually extremely reduced, whereas the FXIII-B subunit is

828

Rare bleeding disorders

Table 42.3 Classification of FXIII deficiency according to activity and antigen determinations of the FXIII subunits.

FXIII-A deficiency

Plasma activity Plasma antigen A2B2 Plasma antigen A Platelet antigen A Platelet antigen B

reduced but always at measurable levels. Table 42.3 shows the classification of FXIII deficiency according to activity and antigen determinations of the FXIII subunits. Studies on homozygous null mice generated by deletion of exon 7 of the FXIII gene showed that these mice were fertile, although reproduction was impaired and bleeding episodes were associated with reduced survival (see Table 42.2). A new aspect of the relationship between FXIII and wound healing has recently been reported. Following an acute myocardial infarction in an experimental model, infarct expansion, heart failure and cardiac rupture were observed in FXIII-deficient mice, probably due to inadequate healing of the myocardial tissue. Spontaneous haemorrhagic events with subsequent inflammation and fibrosis were also reported in the heart tissue of FXIIIdeficient mice, suggesting that low FXIII levels may impair cardiac healing and lead to complications in patients with acute myocardial infarction.

Clinical manifestations and treatment Patients with FXIII-A deficiency have a bleeding tendency that is usually severe, particularly because of the early onset of lifethreatening symptoms such as umbilical-cord and CNS bleeding. Bleeding from the umbilical stump in the first few days of life occurs in approximately 80% of patients, and CNS bleeding in up to 30%. Ecchymoses, haematomas and prolonged bleeding following trauma are also typical. Haemarthroses and intramuscular haematomas may appear unexpectedly, although less frequently. In a recent study made on the largest group of patients with severe FXIII deficiency (93 Iranians), the most frequent mucosal tract bleeding symptom was bleeding in the oral cavity (lips, tongue, gum) followed by menorrhagia and epistaxis. Gastrointestinal bleeding is not unusual. Soft-tissue bleeding such as spontaneous haematoma and haemarthrosis occur in a large proportion of patients. In women of reproductive age, 20% had intraperitoneal bleeding that occurred at the time of ovulation, in some cases leading to hysterectomy; 50% of women had at least one miscarriage during pregnancy. The same pattern of symptoms was also recently shown by Ivaskevicius and colleagues in an international registry formed mostly of European data, where joint bleeding is reported to be more commonly periarticular than intra-articular.

FXIII-B deficiency

Quantitative defect

Qualitative defect

Reduced Reduced Reduced Reduced > 30%

Reduced Normal/near normal Normal/near normal Normal/near normal > 30%

Moderately reduced Reduced Moderately reduced Normal Reduced

On the whole, the clinical impact of FXIII deficiency can shift from that of a very severe disease to that of a mild one, depending on the adoption of prophylactic treatment. All these symptoms usually lead to an early diagnosis, so that patients who survive are often treated prophylactically, starting early in life. Monthly prophylactic infusions of FXIII concentrate are also recommended in FXIII-A-deficient women who are pregnant in order to prevent bleeding and fetal loss. In fact, it was recently reported that among these women there were seven successful pregnancies and five had received FXIII replacement. Moreover, it was found that all women with deficiency of the FXIII-A subunit miscarried, with one exception, if replacement therapy was not given during pregnancy. These data suggest that implementation of replacement therapy is crucial for pregnancy and should commence as early as possible. This approach to treatment is rendered simple and feasible by the fact that plasma levels of FXIII of 2–5% are sufficient to prevent bleeding, and that the long in vivo half-life of the factor (11–14 days) makes it possible to infuse cryoprecipitate or concentrates at intervals of 1 month or longer. Because of the risk of blood-borne diseases, FFP and cryoprecipitate are less satisfactory for treatment or prophylaxis, and FXIII concentrate is recommended whenever available. A new recombinant FXIII-A2 concentrate (rFXIII-A2) has recently been manufactured in Saccharomyces cerevesiae. The rFXIII-A2 homodimers associate in plasma with the endogenous FXIII-B to form the stable heterotetramer FXIII-A2B2. In a Phase I clinical trial, rFXIII-A2, administered to 11 patients with FXIII deficiency, seemed to have a similar half-life and pharmacokinetics to those of the native protein and appeared to be safe. Only four cases of FXIII-B deficiency have been reported to date and the bleeding symptoms appear milder than in FXIII-A-deficient patients.

Molecular defects More than 70 mutations within F13A have been published (Figure 42.10) and most of them are missense/nonsense point mutations. Multiple missense/nonsense mutations have also been reported for a number of codons (77, 260, 326, 413 and 541) with codon changes AAC/AAA and AAC/AAG. In the review by Ivaskevicius and colleagues, the two most frequent 829

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IVS1-7to-20insTT, E1/IVS1del20bp

1

5’-UTR

27delT

2

Activation peptide

3

β-sandwich domain

131delAG, Asn60Lys, Tyr69stop, Arg77His, Arg77Cys 249del13bp, 291delGG and ins6bp, Glu102Lys IVS3-1G>T, IVS3+5G>A, IVS3+6T>C Met159Arg, Arg171stop, Pro186Leu, 189insGly 599delAA, 604delA, 607delG Gly210Arg, Gly215Arg Leu235Arg, 709delG, Met242Thr Arg252Ile, Arg260His, Arg260Cys Arg260Leu, Gly262Glu,Ser263Phe

4 IVS4+2insT

IVS5-1G>A

6

Tyr283Cys, 869insC, 873dupG, Ser295Arg, Val316Phe, Ala318Val Exons 4–11

5 Core 1 domain

7

IVS7+1G>A Arg326stop, Arg326Gln, 1033delAAT, Leu354Pro

8

IVS8-2T>G Trp375Cys, Ala378Pro, Arg382Gln, Arg382Ser Ala394Val, Thr398Asn, Gln400stop, 1286insC

9

Arg408Gln, Ser413Leu, Ser413Trp, Val414Phe, Gly420Ser

10

IVS10+1G>A Tyr441stop, 1392delAATT, 1405delCAAA

Core 2 domain 11

IVS11+1G>T Leu498Pro, Gly501Arg, Asn541Lys 1642dupA, 1652del10bp, Gly562Arg

12

13 1981delC, Leu660Pro, Arg661stop, Leu667Pro 2035delAAG

Barrel 1 domain

14

IVS14-1G>A Barrel 2 domain Trp691stop, Arg703Trp, Ser708Asn, His716Arg

15 3’-UTR

Figure 42.10 Schematic representation of the localization of the main mutations along the human FXIII-A gene, projected onto the exons encoding the domains of the protein. Exons

(rectangles) are drawn to scale while introns (lines) are not to scale. Gross deletions are indicated by arrows.

mutations affecting F13A were found to be the IVS5–1G→A splice-site defect and the Arg661stop mutation. Haplotype analysis in four patients with the IVS5–1G→A mutation suggested a founder effect, while the concentration

of Arg661stop mutation in Finland, Arg77Cys in Switzerland and Arg77His in Iran might also be due to founder effects, although the occurrence of these mutations at CpG dinucleotides raises the alternative possibility of independent origin.

830

Rare bleeding disorders

1

Signal sequence

IVS1-2delA 2

c.300insAAC

3 4 5

6

and Glu651Gln. The Val34Leu variant is the most studied polymorphism, with the amino acid substitution occurring in the activation peptide sequence, three amino acids upstream from the thrombin-cleavage site. Studies have shown that the Leu34 allele variant promotes accelerated cleavage of the activation peptide once activated by thrombin and has been associated with a decreased risk of myocardial infarction. However, controversy has surrounded the role of Val34Leu in coronary artery disease, with the literature supporting both sides of the debate. Only four mutations have been identified in F13B (Figure 42.11), a single-nucleotide deletion at the acceptor splice junction of intron A/exon 2 (IVS1(−2)delA), a Cys430Phe missense mutation, a G deletion in exon 9 and an AAC triplet insertion within codon Tyr80 in exon 3.

7 Sushi domain Cys430Phe

1498delG

8

9

10

11

12

Figure 42.11 Schematic representation of the localization of the main mutations along the human FXIII-B gene, projected onto the exons encoding the domains of the protein. Exons (rectangles) are drawn to scale while introns (lines) are not to scale.

Mutations are scattered throughout the F13A gene and in a few studies the mutant proteins were expressed in cultured mammalian cell lines, showing that introduction of the mutation caused destabilization of protein structure and intracellular degradation. Several polymorphisms in F13A have been identified. The most common are Val34Leu, Tyr204Phe, Pro564Leu, Val650Ile

Vitamin K-dependent coagulation factors deficiency The first case of vitamin K-dependent coagulation factors deficiency (VKCFD) was reported more than 40 years ago in a 3-month-old girl with multiple bruises and haemorrhages. The girl was studied further when she was 15 years old and was found to have immunologically measurable FII, FVII, FIX and FX that were lacking γ-carboxyglutamic acid residues. Vitamin K-dependent coagulation factors such as FII, FVII, FIX and FX require γ-carboxylation of glutamic acid residues at their Gla domains to enable binding of calcium and attachment to phospholipid membranes. The γ-carboxylation is catalysed by hepatic γ-glutamyl carboxylase (GGCX) and its cofactor reduced vitamin K (KH2). During the reaction, KH2 is converted to vitamin K epoxide (KO), which is recycled to KH2 by the vitamin K epoxide reductase (VKOR) enzyme complex. Heritable dysfunction of GGCX or the VKOR complex results in the secretion of poorly carboxylated vitamin K-dependent coagulation factors, leading to combined deficiency of the vitamin K-dependent clotting factors. The γ-carboxylation of glutamic acid residues is also required for activity of the anticoagulant factors protein C, protein S and protein Z, and although protein S and protein C levels are low in VKCFD, there are no reports of venous or arterial thrombosis. Thus, the effect of VKCFD is clearly in the bleeding area. The GGCX and VKOR proteins are encoded by two corresponding genes: GGCX (13 kb, 15 exons) located on chromosome 2 and the unusually small VKORC1 (5126 bp, three exons) located on chromosome 16; the latter gene was so named because of evidence suggesting that VKOR is a multisubunit complex. Studies on heterozygous mice carrying a null mutation for GGCX showed normal development and normal activity of the vitamin K-dependent coagulation factors, but only half of the γcarboxylase-deficient mice developed to term and those 831

Postgraduate Haematology

delivered died due to massive intra-abdominal haemorrhages (see Table 42.2).

Concluding remarks Molecular defects

Clinical manifestations and treatment VKCFD is a rare autosomal recessive bleeding disorder that often presents during infancy, although the routine administration of vitamin K may delay the diagnosis of VKCFD in neonates. Severe symptoms such as intracranial haemorrhage or umbilical stump bleeding are usually associated with factors levels below 5 units/dL. Severely affected children may also present with skeletal abnormalities such as nasal hypoplasia, distal digital hypoplasia, epiphyseal stippling and mild conductive hearing loss. Older patients can present with easy bruising and mucocutaneous or postsurgical bleeding. Treatment with oral or parenteral vitamin K1 should be started as soon as possible in all patients at diagnosis. However, some patients show insufficient response, and there are limited data on the effectiveness of prophylaxis with weekly administration of vitamin K1 10 mg. In fact, massive parenteral doses of vitamin K do not always correct FII, FVII, FIX and FX activities, and there is clear biochemical evidence that the molecules are not fully carboxylated by such treatment. In this group of patients, factor replacement by virally inactivated FFP could also be used in cases of acute bleeding episodes or before surgery. PCC is the alternative choice when a rapid increase in clotting factor levels is necessary. However, PCCs have been associated with thrombosis and disseminated intravascular coagulation, and therefore should be used with caution in some patients.

Molecular defects The rare VKCFD autosomal disorder arises from point mutations in either the GGCX (type I) or VKOR (type II) genes, the only exception being a kindred with a 14-bp deletion in intron 1 of GGCX. To date, few mutations have been described for the type I disorder: Asp31Asn, Trp157Arg, Leu394Arg, His404Pro, Arg485Pro, Trp501Ser, Thr591Lys and two recently identified splice-site mutations: a G→T transversion of the first nucleotide of intron 2 (c.1358+1G→T) and an A→G transversion of the third nucleotide of intron 11 (c.10363+3A→G). With regard to the Arg485Pro mutation, Rost and colleagues identified it in two patients originating from the same region in western Germany. Since VKCFD represents a rare entity, they hypothesized a founder effect that was strongly supported by further examination of three intragenic polymorphisms of GGCX (c.44–140_–127dup14 and c.44–32G→A in intron 1, and c.215–385G→C in intron 2) and five microsatellite markers surrounding the GGCX gene (D2S417, D2S2232, D2S388, D2S237 and D2S440). The only mutation identified so far leading to type II is the homozygous Arg98Trp mutation. 832

In general, in these patients a defect in the DNA can be identified in the genes encoding the different coagulation factors. Combined FV and FVIII deficiency, explained by defects located in genes encoding transport proteins, is a typical exception. In each coagulation defect the mutations are multiple and the majority of them are private mutations, unique for any given patient. The unique nature of the mutations complicates the approach to the control of these diseases through prenatal diagnosis in families with affected members, because it renders necessary the actual identification of the underlying mutation in each kindred. Prevention of rare coagulation disorders through prenatal diagnosis of the underlying mutations is feasible in couples who have already had affected children. Primary prevention might be achieved by discouraging consanguineous marriages. Even though the cultural, religious and economic roots of this practice are deep in Muslim communities, consanguineous marriages are becoming much less frequent in large cities and among younger generations. The relation between genotype and phenotype is not always clear-cut. Even though in general severe mutations predicting no protein production (stop codons, deletions, insertions, splicing abnormalities) are associated with severe factor deficiencies and severe clinical phenotypes, there are a number of cases with severe deficiencies and phenotypes associated with missense mutations. Usually, missense mutations are associated with milder phenotypes despite unmeasurable factor levels, probably because some protein is produced in these cases that cannot be detected with the currently available assays. Expression of the mutations in cultured cell lines and characterization of the recombinant proteins has been useful for understanding how well-defined molecular lesions lead to structural abnormalities and to functional defects of the protein. In some cases expression studies have documented the mechanism whereby a mutant protein is synthesized normally but is not ultimately secreted in plasma from cells. In most cases impaired folding and/or conformational changes of the mutant protein leads to both intracellular and extracellular instability, which in turn causes factor deficiency in plasma. Expression work is still in its infancy and needs to be expanded, mainly in cases of missense mutations that appear of special functional interest when projected onto the crystal structure of the coagulation factors.

Treatment Treatment of rare coagulation disorders is with the most purified blood product available that contains the missing factor. Because of the rarity of each factor deficiency, purified factor

Rare bleeding disorders

concentrates are not as freely available as they are for haemophilia A and B. Dosages and frequency of treatment depend on minimal haemostatic levels of the deficient factor, plasma halflife and type of bleeding episode. General recommendations for treatment on-demand, prophylaxis, pregnancy and delivery,

and for surgery of patients with rare coagulation disorders are summarized in Tables 42.4–42.6. The avoidance of transmission of blood borne infectious agents is the primary requisite in the choice of replacement material. Solvent/detergent-treated plasma is an important

Table 42.4 Treatment of recessive coagulation disorders. Deficient factor

Recommended trough levels

Plasma half-life

On demand

Fibrinogen

50–100 mg/dL

2–4 days

Cryoprecipitate (5–10 bags) SD-treated plasma (15–30 mL/kg) Fibrinogen concentrate (50–100 mg/kg)

Prothrombin

20–30%

3–4 days

SD-treated plasma (15–20 mL/kg) FIX concentrate and PCC (20–30 units/kg)

Factor V

10–20%

36 hours

SD-treated plasma (15–20 mL/kg)

Factor V and factor VIII

10–15%

FV 36 hours FVIII 10–14 hours

As for FV

Factor VII

10–15%

4–6 hours

FVII concentrate (30–40 mL/kg) PCC (20–30 units/kg) rFVIIa (15–30 µg/kg every 4–6 hours)

Factor X

10–20%

40–60 hours

SD-treated plasma (10–20 mL/kg) PCC (20–30 units/kg) FX/FIX concentrate (10–20 units/kg)

Factor XI

15–20%

50 hours

SD-treated plasma (15–20 mL/kg) FXI concentrate (15–20 units/kg)

2–5%

9–12 days

Cryoprecipitate (2–3 bags) SD-treated plasma (3 mL/kg) FXIII concentrate (50 units/kg for high haemorrhagic events)

Factor XIII

Vitamin K dependent

Vitamin K (10 mg i.v. or s.c. for minor bleeding)

PCC, prothrombin complex concentrate; rFVIIa, recombinant activated FVII; SD, solvent/detergent. Table 42.5 General recommendations for long-term prophylaxis. Deficient factor

Fibrinogen

Recommended trough levels

Plasma half-life

50–100 mg/dL

2–4 days

Reported dose schedule for successful long-term prophylaxis Products

Dose

Frequency

Cryoprecipitate

1 unit 3 units 30–100 mg/kg

Three times per week Every 7–10 days Every week

Fibrinogen concentrate Prothrombin

20–30%

3–4 days

PCC

25–40 units/kg

Once per week

Factor V

10–20%

36 hours

SD-treated plasma

30 mL/kg

Twice per week

Factor V and factor VIII

10–15%

FV 36 hours FVIII 10–14 hours

No data

No data

No data

833

Table 42.5 Continued Deficient factor

Recommended trough levels

Plasma half-life

Factor VII

10–15%

Factor X

10–20%

Factor XI Factor XIII

Reported dose schedule for successful long-term prophylaxis Products

Dose

Frequency

4–6 hours

FFP pdFVII rFVIIa

10–15 mL/kg 10–50 units/kg 15–30 µg/kg

Twice per week Three times per week Two to three times per week

40–60 hours

PCC

30–40 units/kg

FX/FIX concentrate

20–40 units/kg

Two to three times per week One to two times per week in children

15–20%

50 hours

No data

No data

No data

2–5%

9–12 days

Cryoprecipitate SD-treated plasma FXIII concentrate

2 units 15–20 mL/kg 10 units/kg

Every 3 weeks Every 4–6 weeks Every 4–6 weeks

Vitamin K

10 mg

Once per week orally dependent

Vitamin K

PCC, prothrombin complex concentrate; pdFVII, plasma-derived FVII; rFVIIa, recombinant activated FVII; SD, solvent/detergent.

Table 42.6 General recommendations for treatment during pregnancy and delivery and during surgery. Deficient factor

Pregnancy/delivery

Surgery

Minimum level

Treatment

Minimum level

Treatment

Fibrinogen

> 150 mg/dL

Successful pregnancy in afibrinogenaemia is difficult without fibrinogen replacement therapy No strong data to support routine postpartum fibrinogen prophylaxis beyond the first 1–2 days

100 mg/dL

Fibrinogen concentrate (50– 100 mg/kg) Antifibrinolytic agents (dental extraction)

Prothrombin

> 25%

No data

20–40%

SD-treated plasma (15–20 mL/kg) PCC (20–30 units/kg, higher doses for major surgery)

Factor V

15–25%

SD-treated plasma (15–20 mL/kg, until after delivery)

25%

SD-treated plasma (15–20 mL/kg)

Factor V and factor VIII

As for FV

As for FV

Factor VII

> 15–20%

Not required during preganacy, unless there is a bleeding history with previous pregnancies If FVII < 20%, peripartum prophylaxis should be considered

10–15%

FVII concentrate (30–40 mL/kg) PCC (20–30 units/kg) rFVIIa (15–30 µg/kg every 4–6 hours)

Factor X

10–20%

In severe FX deficiency and history of adverse outcome, replacement therapy to cover delivery (up to 3 days) is indicated

10–20%

SD-treated plasma (10–20 mL/kg) PCC (20–30 units/kg) FX(FIX) concentrate (10–20 units/kg)

834

Rare bleeding disorders

Table 42.6 Continued Deficient factor

Pregnancy/delivery

Surgery

Minimum level

Treatment

Minimum level

Treatment

Factor XI

15–20%

FXI levels 15–70 units/dL, with no bleeding history: give tranexamic acid for up to 3 days (first dose during labour) FXI levels < 15 units/dL: replacement therapy should be considered at the onset of labour

15–20%

Antifibrinolytic agents (250 mg/ day)

Factor XIII

10%

FXIII concentrate: 250 units per 7 days in early period, 500 units per 7 days after 23 weeks Factor replacement should be given at delivery to maintain FXIII levels > 20%

5–10%

Cryoprecipitate (2–3 bags) SD-treated plasma (3 mL/kg) FXIII concentrate (10–20 units/kg)

PCC, prothrombin complex concentrate; rFVIIa, recombinant activated FVII; SD, solvent/detergent.

source of replacement that is recommended in the majority of these disorders; virus-inactivated concentrates, when commercially available, are also safe but expensive especially for developing countries. Non-virus-inactivated plasma and cryoprecipitate should be avoided if possible. Of course the treatment of choice may change depending on the facilities of the country where the patient is resident. Cost is the next most important determinant. Virally inactivated factor concentrates are available for several deficiencies and should be preferred when virally inactivated plasma is not available or repeated infusions causing fluid overload are needed, as may occur during surgery or in cases of bleeding in the CNS. An updated registry of the available clotting factor concentrates is published by the WFH (available at http://www.wfh.org/index. asp?lang=EN).

Acknowledgements We would like to thank Professor P.M. Mannucci for his great supervision and all our colleagues who have helped us recently to develop a chapter on rare bleeding disorders in Seminars of Thrombosis and Hemostasis. Their revision of specific types of deficiency helped us to more accurately revise our present chapter. Thanks, therefore, to Philippe de Moerloose and Marguerite Neerman-Arbez, Stefano Lancellotti and Raimondo De Cristofaro, Rosanna Asselta and Marta Spreafico, Guglielmo Mariani and Francesco Bernardi, Stefano Duga and Ophira Salomon, Mehran Karimi, Zsuzsanna Bereczky, Nader Cohan and László Muszbek, and Benjamin Brenner, Amir A. Kuperman,

Matthias Watzka and Johannes Oldenburg. We would also like to thank Isabella Garagiola and Roberta Palla for their help in writing the manuscript.

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CHAPTER 43

Acquired coagulation disorders Peter W Collins1, Jecko Thachil2 and Cheng-Hock Toh2

43

1

Cardiff University School of Medicine, University Hospital of Wales, Cardiff, UK School of Clinical Sciences, University of Liverpool, Liverpool, UK

2

Introduction, 839 Tests of coagulation and point-of-care testing, 840 Routine tests of haemostasis, 840 Disseminated intravascular coagulation, 841 Pathophysiology, 841 Clinical features, 843 Diagnosis, 843 Treatment, 844 Haemostatic dysfunction in acute promyelocytic leukaemia, 845 Vitamin K and related disorders, 845 Vitamin K metabolism, 845 Vitamin K deficiency, 846 Haemostatic disturbance in liver disease, 847 Acute hepatitis, 848 Chronic liver disease, 848 Liver transplantation, 849 Hypercoagulability in liver disease, 850

Haemostatic disturbance in renal disease, 850 Pregnancy-related haemostatic dysfunction, 851 Haemostatic dysfunction associated with cardiopulmonary bypass surgery, 851 Haemostatic dysfunction associated with trauma, 852 Coagulopathy in massive blood loss, 853 Bruising, 853 Purpura simplex (normal/easy bruising), 853 Non-accidental bruising, 853 Senile purpura (atrophic or actinic purpura) and steroid-related purpura, 853 Psychogenic purpura, 853 Scurvy, 853 Inherited disorders of collagen and elastic fibres, 854 Haemostatic dysfunction associated with vasculitis, 854

Introduction Acquired disorders of haemostasis are a heterogeneous group of conditions with varied and often complex aetiologies. Patients may have multiple and overlapping causes for haemorrhagic symptoms and distinct conditions often have similar pathophysiologies. In order to manage acquired haemostatic failure it is important to understand the mechanisms by which haemostatic disturbance occurs and how these apply in different ways to a variety of conditions. Systemic disease may present to haematologists with symptoms of bleeding or bruising or abnormalities of coagulation tests. Alternatively, patients with known disorders may need haematological input to manage symptoms or at the time of

Postgraduate Haematology: 6th edition. Edited by A. Victor Hoffbrand, Daniel Catovsky, Edward G.D. Tuddenham, Anthony R. Green © 2011 Blackwell Publishing Ltd.

Arteriovenous malformations, 854 Hereditary haemorrhagic telangiectasia, 854 Kasabach–Merritt syndrome, 855 Microthromboembolic disease, 855 Cholesterol embolism, 855 Fat embolism syndrome, 855 Warfarin-induced skin necrosis, 855 Haemostatic dysfunction associated with paraproteinaemia and amyloidosis, 856 Paraproteinaemia, 856 Amyloidosis, 856 Acquired inhibitors of coagulation factors, 856 Acquired haemophilia A, 856 Acquired von Willebrand disease, 858 Acquired factor V deficiency, 858 Acquired protein S deficiency, 858 Prothrombin deficiency associated with lupus anticoagulant, 858 Selected bibliography, 858

invasive procedures. Assessment of patients with symptoms of bleeding or bruising requires clinical review and laboratory investigation. Serial laboratory testing is often required because the haemostatic disturbance may evolve rapidly. The possibility of a congenital disorder should also be considered. Haematologists are also often involved in the management of acutely bleeding patients who may have complex medical conditions and in whom bleeding is usually multifactorial. Critical to optimizing the management of these patients is a well-organized multidisciplinary team approach. Hospitals should have systems in place to respond rapidly to massive haemorrhage and hospital transfusion committees should establish protocols for these medical emergencies. Laboratories should be geared to producing accelerated full blood count (FBC) and coagulation results. The rapid supply of blood products and efficient transfer of these to the patient is vital to success. ‘Fire drills’ to test proficiency of response to emergencies are good practice in areas where massive haemorrhage often occurs, such as on labour wards and in casualty departments. 839

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Tests of coagulation and point-of-care testing Routine tests of haemostasis To investigate a patient suspected of an acquired haemostatic defect, a platelet count will be required and examination of the blood film is often useful. Routine coagulation tests include the prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and Clauss fibrinogen level. It is important that derived fibrinogen assays are not used because results are likely to be misleading in the context of acquired haemostatic defects. If screening tests are abnormal, correction studies help to distinguish factor deficiencies from inhibitors. Comparison of TT with the reptilase time will establish whether abnormal results are due to heparin. Measurement of fibrin breakdown products, such as D-dimers, is required for investigation of possible disseminated intravascular coagulation (DIC). Further measurement of individual coagulation factor and von Willebrand factor (VWF) levels may be required. Assessment of platelet function may be useful in some circumstances through platelet aggregation studies, platelet nucleotides or a bleeding time, although these results may be difficult to interpret in the context of an acquired haemostatic defect. The role of global platelet function analysers remains to be defined but these are available in many hospitals. Thromboelastography This is a point-of-care test that assesses the viscoelastic properties of whole blood samples under low-shear conditions (Figure 43.1). Its main advantage is the global assessment of the different components of clot formation including platelets and

coagulation factors. It has been used to guide blood product administration in patients undergoing liver transplantation and cardiopulmonary bypass. Other reported uses include the assessment of coagulation in liver disease, neonates, obstetrics and trauma as well as monitoring the effects of bypassing agents in haemophilia patients with inhibitors, although more studies are required to validate its role in these areas. The two main types of instruments are TEG and ROTEM, which measure the viscoelastic properties of an evolving clot in similar but distinct ways and then graphically display the changes to show all stages of the developing and dissolving clot. Different activators are available and heparinase cups can be used to investigate heparinized samples. Thrombin generation assays These tests measure the amount of thrombin generated over time following activation of a sample and may reflect an individual’s coagulation potential. They are not available in routine practice. Assays are initiated by the addition of a trigger, usually tissue factor (TF), to recalcified plasma in the presence of phospholipids and thrombin is detected via cleavage of a chromogenic or fluorogenic substrate. When low concentrations of TF are used, contact activation must be inhibited by maize trypsin inhibitor. Several variations of these assays have been developed by varying the concentrations of TF, maize trypsin inhibitor, phospholipid and protein C-sensitizing reagents such as soluble thrombomodulin. Thrombin generation tests have been studied in the context of haemophilia, including monitoring of inhibitor bypass agent therapy. Other studies have examined thrombophilia and liver disease and monitoring of anticoagulant therapy and warfarin reversal. The clinical utility of many of these potential applications remains to be demonstrated.

α-angle MA

A60

2 mm 20 mm 60 min

r time

k time Coagulation

Time

840

Fibrinolysis

Figure 43.1 Representative thromboelastographic trace.

Acquired coagulation disorders

80

80

Figure 43.2 Representative normal and biphasic waveforms. Activated partial thromboplastin time waveform patterns: (a) normal; (b) biphasic. Dashed lines denote transmittance level at 18 s (TL18) as the quantitative index: (a) TL18 = 100; (b) TL18 = 63.

Transmittance (%)

100

Transmittance (%)

100

60

40

20

60

40

20

0

0 12

18

24 30 36 42 Time (seconds)

APTT biphasic waveform The Multichannel Discrete Analyser is a coagulation analyser that generates an optical profile charting changes in light transmittance over time during the process of clot formation on the routine APTT. In contrast to the sigmoidal appearance of a normal APTT waveform, a ‘biphasic’ appearance correlates with acquired haemostatic dysfunction (Figure 43.2). The reliability of this method has been validated in several reports and correlates with the diagnosis of overt DIC according to the International Society for Thrombosis and Haemostasis (ISTH) criteria. This biphasic response is due to calcium-dependent complex formation between C-reactive protein and very low density lipoprotein, which has been shown to increase thrombin generation. Activated clotting time This test is predominantly used by anaesthetists to monitor anticoagulation with heparin during cardiopulmonary bypass (CPB) surgery. It uses whole rather than citrated blood and has a linear response at the high concentrations of heparin used during CPB. The APTT cannot be used because plasma is unclottable using this method at these heparin concentrations. Measured using a specialized analyser, the activated clotting time (ACT) reference range varies according to the method and usually falls within 70–180 s. During heparinization for CPB, the goal is to exceed 400–500 s. Off-pump cardiac surgery has been described using less anticoagulation and lower ACT reference ranges of 200–300 s. Measurement of activated coagulation factors Many publications have reported the measurement of activated coagulation factors and inhibitors during various clinical situations. Numerous assays and markers of coagulation are

48

12

18

24 30 36 42 Time (seconds)

48

available, of which the commonest are thrombin–antithrombin complexes, prothrombin fragment 1+2 and plasmin– antiplasmin complexes. These assays are useful in a research context for understanding the pathogenesis of abnormal haemostasis but have not been shown to be useful in clinical practice.

Disseminated intravascular coagulation DIC is characterized by the loss of localization or compensated control of intravascular activation of coagulation. Arising from diverse causes (Table 43.1), its pathology can manifest systemically and contribute to a worse prognosis for the patient. Uncoupling of the highly regulated balance between procoagulant, anticoagulant, profibrinolytic and antifibrinolytic processes can result in simultaneous bleeding and microvascular thrombosis at different vascular sites (Figure 43.3). Removal of the inciting cause is the best means of restoring haemostatic control but may not always be possible. Sepsis and malignancy account for more than half the cases of DIC.

Pathophysiology There are several important themes in the pathophysiology of DIC (Figure 43.3): first is the central role played by the generation of thrombin; second, mechanisms that fuel and perpetuate thrombin generation become pathogenic in its dissemination; third is the parallel and concomitant activation of the inflammatory cascade; and fourth is the importance of the endothelial microvasculature in this process. The excess bleeding in DIC is partly attributable to the depletion of coagulation factors and platelets. However, several other factors may contribute, including abnormal platelet function 841

Table 43.1 Clinical conditions associated with disseminated intravascular coagulation. Sepsis/severe infection Potentially any microorganism, including Gram-positive and Gram-negative bacteria, viruses, fungi and rickettsial infections Malaria and other protozoal infections Trauma Serious tissue injury Head injury Fat embolism Burns Malignancy Solid tumours Haematological malignancies (e.g. acute promyelocytic leukaemia) Obstetric complications Placental abruption Amniotic fluid embolism

Pre-eclampsia Intrauterine fetal demise Vascular abnormalities Giant haemangiomas (Kasabach–Merritt syndrome) Large vessel aneurysms (e.g. aortic) Severe toxic or immunological reactions Snake bites Recreational drugs Severe transfusion reactions Transplant rejection Miscellaneous Severe pancreatitis Heat stroke ABO mismatch transfusion

Procoagulant (factors VIII, V, XI)

Normal coagulation

Profibrinolytic (tPA)

Antifibrinolytic (TAFI)

Thrombin

Anticoagulant (APC) Disseminated intravascular coagulation Bleeding

Prolongation of PT and APTT Thrombocytopenia and hypofibrinogenaemia

FFP, platelets, cryoprecipitate or fibrinogen concentrate

Clotting factor and platelet consumption

Antifibrinolytics Hyperfibrinolysis Bleeding

Excess thrombin

Excess antifibrinolysis Thrombosis Increase in TAFI and PAI levels

Increased D-dimers Increased thrombin– antithrombin complexes

Anticoagulant consumption Activated protein C Antithrombin Heparin

Thrombosis

Figure 43.3 The changes in disseminated intravascular coagulation are compared with the normal coagulation. The excess thrombin generation in DIC leads to either bleeding or thrombosis (left) based on the predominant coagulation change 842

Increased thrombin– antithrombin complexes Decreased protein C and AT

(right) which has occurred. The therapeutic intervention in each setting is given by the arrow towards each double box. TAFI, thrombin activatable fibrinolysis inhibitor; PAI, plasminogen activator; AT, antithrombin.

Acquired coagulation disorders

due to renal dysfunction and decreased clotting factor synthesis due to liver impairment. Conversely, microvascular thrombosis can be precipitated by reduced levels of circulating anticoagulant proteins as well as loss of receptors, such as thrombomodulin, in the microvasculature. Endothelial dysfunction can also lead to the depletion of nitric oxide and result in uninhibited platelet activation. Thrombin generation in vivo The TF pathway plays a major role in initiating thrombin generation and different aetiologies promote this in different ways. In sepsis, the infecting microorganisms induce TF expression on monocytes and other inflammatory cells, while in trauma thromboplastin-like substances can be released from injured tissues like the brain. During obstetric complications, the placenta and amniotic fluid act as rich sources of TF and phospholipid while malignant cells release products with TF-like activity. Although the TF pathway is considered more important in thrombin generation, the contact pathway through factor (F)XII activation contributes to the pathological state by activation of the kallikrein–kinin system, causing vasodilatation and hypotension and activation of fibrinolysis. Mechanisms for disseminating and sustaining thrombin generation When the inciting insult is persistent or severe, the amount of thrombin generated becomes continuous and excessive. This can lead to consumption and depletion of both coagulation and anticoagulant factors. Increased exposure of negatively charged phospholipid surfaces facilitates the assembly and enhances the rate of coagulation reactions. Such surfaces, mainly rich in phosphatidylserine, are provided by externalization of the inner leaflet of cell membranes upon activation and apoptosis. Cell damage also leads to the generation of microparticles from platelets, monocytes and endothelial cells that increase the circulating surface area for coagulation reactions to occur. Phospholipid surfaces are also provided by very low density lipoprotein, which can increase several fold in severe sepsis to further enhance and sustain thrombin generation. Together, these mechanisms promote a spatially and temporally expanded response that is the hallmark of DIC. Links between inflammation and coagulation Once activated, the inflammatory and coagulation pathways interact to amplify the response. While cytokines and proinflammatory mediators such as tumour necrosis factor (TNF), interleukin (IL)-1 and high mobility group box protein (HMGB)-1 can induce activators of coagulation, thrombin and other serine proteases interact with protease-activated receptors (PARs) on cell surfaces to promote further activation and additional inflammation. When this process becomes generalized, it escapes the well-developed local checks and balances and results in a dysregulated undirected response that fuels a vicious cycle between inflammation and coagulation. Reactive oxygen species

generated during sepsis and trauma can lead to further recruitment of neutrophils, cause lipid peroxidation and stimulate nuclear factor (NF)-κB to initiate the transcription of cytokine genes. An important role for the complement system has also been increasingly identified. Thrombin can convert C5 to C5a and the mannan-binding lectin pathway triggers coagulation by converting prothrombin to thrombin. C4b-binding protein of the complement pathway also binds and thus depletes free protein S, the cofactor for activated protein C. Endothelial cell activation and dysfunction Dysfunction and failure of the endothelium beyond the host adaptive response can lead to the development of DIC. The degree to which this occurs and the dominance of thrombotic or bleeding sequelae depend on genetic and other host-related factors. Damage to and activation of the endothelium downregulates and depletes its anticoagulant receptors, such as thrombomodulin and endothelial protein C receptor, exposes subendothelial collagen that binds activated platelets, and releases plasminogen activator inhibitor (PAI)-1 that inhibits fibrinolysis and ultra-large VWF multimers to increase platelet aggregation. In vitro studies show a downregulation of endothelial TF expression.

Clinical features The perturbed coagulation associated with DIC can manifest clinically at any point in the spectrum from bleeding to thrombosis. Although bleeding, ranging from oozing at venepuncture sites to major gastrointestinal or intracranial haemorrhage, is the archetypal and most obvious manifestation of DIC, organ failure due to microvascular thrombosis is much more common and often unrecognized. For example, in meningococcal septicaemia and rarely pneumococcal infection, thrombosis of the adrenal vessels can lead to adrenal insufficiency and Waterhouse– Friderichsen syndrome.

Diagnosis DIC is a clinicopathological syndrome and, as such, there is no single laboratory test that can confirm or refute the diagnosis. In clinical practice, the diagnosis is usually made by a combination of routinely available coagulation tests in a clinical situation where DIC is suspected. DIC must be differentiated from other acquired disorders of haemostasis (Table 43.2). The typical findings are a prolonged PT and APTT, elevated products of fibrin breakdown (e.g. D-dimer), thrombocytopenia and reduced fibrinogen. However, results within the normal range for these tests do not exclude a significant consumptive coagulopathy, especially because the acute-phase response results in shortening of the APTT and increased fibrinogen. A fall in platelet number within the normal range may also be significant. 843

Postgraduate Haematology

Condition

Similarities

Differences

Liver disease

Bleeding common PT, APTT abnormal Platelet count low Fibrinogen low

D-dimer usually normal* FVIII levels not affected

Microangiopathic haemolytic anaemia (e.g. HELLP, TTP)

Microthrombi common Platelet count low

Bleeding uncommon Coagulation tests normal†

Hyperfibrinolysis

PT, APTT abnormal Fibrinogen low

Platelet count normal

Catastrophic antiphospholipid antibody syndrome

PT, APTT abnormal Platelet count low

Fibrinogen not low D-dimer normal‡

Massive transfusion

PT, APTT abnormal Fibrinogen low Platelet count low

D-dimer normal

Table 43.2 Differential diagnosis of disseminated intravascular coagulation (DIC).

*Unless additional disorders which increase the D-dimer coexist. † Unless coexisting DIC. ‡ Some antibodies can affect the D-dimer results. HELLP, haemolysis, elevated liver enzymes, low platelet count (syndrome); TTP, thrombotic thrombocytopenic purpura.

It is also important to recognize that DIC is a dynamic process and thus interpreting a series of laboratory tests over time is more relevant than looking at a single set of results. The ISTH Sub-Committee of the Scientific and Standardization Committee on DIC has recommended the use of a scoring system for overt DIC (Table 43.3). This has been prospectively validated in various studies, indicating a very high sensitivity and specificity. A strong correlation between an increasing DIC score and mortality has been demonstrated. The optical light transmittance profile of the APTT, referred to as the biphasic waveform, has also been shown to correlate well with the overt DIC score. The biphasic waveform occurs independently of prolongation in the clotting times in patients with DIC. Markers of increased thrombin generation (thrombin– antithrombin complexes) and increased fibrinolysis (plasmin– antiplasmin complexes) as well as endothelial markers have also been investigated as laboratory markers for DIC but single determinations are neither sensitive nor specific for DIC. More importantly, their general availability is limited due to practical considerations.

Table 43.3 ISTH Sub-Committee of the Scientific and Standardization Committee on DIC recommended scoring system for overt disseminated intravascular coagulation (DIC). 1 Risk assessment: does the patient have an underlying disorder known to be associated with overt DIC? If yes, proceed If no, do not use this algorithm 2 Order global coagulation tests (platelet count, PT, fibrinogen, soluble fibrin monomers or FDPs) 3 Score global coagulation test results Platelet count (×109/L): > 100, score 0; < 100, score 1; < 50, score 2 Elevated fibrin-related marker (e.g. soluble fibrin monomers or FDPs): no increase, score 0; moderate increase, score 2; strong increase, score 3 Prolonged PT (s): < 3, score 0; > 3 but < 6, score 1; > 6, score 2 Fibrinogen (g/L): > 1, score 0; < 1, score 1 4 Calculate score 5 Score ≥ 5 compatible with overt DIC. Repeat scoring daily

Treatment The mainstay of treatment of DIC is to remove the underlying cause. However, DIC often continues even after appropriate treatment for the underlying condition. Supportive therapy with blood products may be necessary. Although the efficacy of blood product replacement has not been proven in randomized 844

FDPs, fibrin degradation products; PT, prothrombin time.

controlled trials, it is a biologically rational option to replace both thrombin-promoting and thrombin-opposing proteins, particularly when there is significant depletion of these factors in a patient who is either bleeding or at risk of bleeding. In those

Acquired coagulation disorders

patients not bleeding, transfusion of platelets or plasma in patients with DIC should not be undertaken based on laboratory results. There are no clinical or experimental data to suggest that platelet or plasma transfusions worsen the thrombotic process. In patients with DIC and bleeding or at high risk of bleeding (e.g. after invasive procedures) and a platelet count less than 50 × 109/L, transfusion of platelets should be considered, especially because platelet function may be impaired. In bleeding patients with DIC and prolonged clotting times, fresh-frozen plasma (FFP) should be administered. If fluid overload is an issue, prothrombin complex concentrates (PCCs) could be considered but these will only correct vitamin K-dependent components. Activated PCCs should not be used because these can precipitate DIC. Severe hypofibrinogenaemia (< 1 g/L) requires the use of fibrinogen concentrate or cryoprecipitate to correct. A recent large trial in patients with severe sepsis showed a non-significant benefit of low-dose heparin on mortality and suggested that this is continued in patients with DIC and abnormal coagulation parameters, in the absence of overt bleeding. Notably, these patients are at highest risk of venous thromboembolism due to immobility, recent surgery and a proinflammatory state. However, the role of heparin remains controversial and unproven. Treatment with recombinant human activated protein C in adults with severe sepsis significantly reduced mortality at 28 days from 30.8% to 24.7% and should be considered in sepsisrelated DIC. It is used as a 96-hour infusion and caution is required in patients with a platelet count less than 50 × 109/L due to the increased incidence of intracerebral haemorrhage. Although not licensed, (unactivated) protein C concentrate has been reported as useful in meningococcal septicaemia. No evidence exists to support the use of antithrombin or tissue factor pathway inhibitor concentrates. The use of pro-haemostatic agents may be necessary in certain instances. There are some reports of the successful use of recombinant (r)FVIIa in patients with DIC and life-threatening bleeding. Antifibrinolytic agents, such as tranexamic acid and ε-aminocaproic acid, are not ideal treatments for DIC except if increased fibrinolysis with bleeding is observed (e.g. some cases of prostate cancer and giant haemangiomas).

Haemostatic dysfunction in acute promyelocytic leukaemia Acute promyelocytic leukaemia (APL), in particular the microgranular variant (AML-M3v), is associated with major coagulation disturbance including DIC in at least 80% of cases. Although the introduction of all-trans retinoic acid (ATRA) and arsenic trioxide as differentiation agents has markedly reduced the rate of early haemorrhagic death and almost 90% of patients are cured, the 10% mortality from bleeding complications has

not improved. Bleeding does not correlate with the clotting parameters but with a high white cell count. The cause remains poorly understood and probably relates to enhanced proteolysis, including fibrinolysis and disruption of endothelial barrier integrity. APL blast cells express TF and can also stimulate the production of inflammatory cytokines, which can further amplify TF levels and promote thrombosis. Cancer procoagulant, a cysteine proteinase detected on APL blast cells, can activate FX independent of FVII. Both these procoagulants are noted to be progressively reduced once patients have been commenced on ATRA. ATRA has also been demonstrated to inhibit vascular endothelial growth factor (VEGF) production, which indirectly limits TF production. Annexin II expression is enhanced on the surface of the blast cells and can act as a cell surface receptor for plasminogen and its activator, tissue plasminogen activator (tPA). Elastase, cathepsin-G and proteinase-3 are present in the granules of the APL blasts and can directly degrade fibrinogen, α1-antitrypsin, C1 esterase inhibitor and VWF, inducing the loss of highmolecular-weight multimers. Management of the haemostatic abnormalities revolves around supportive care, including platelet transfusions to maintain a count of at least 30 × 109/L (or 50 × 109/L if the patient is bleeding) and the adequate replacement of fibrinogen to at least 1.5 g/L. The use of antifibrinolytic agents is supported by some evidence but remains controversial. Although markers of coagulation activation and fibrinolysis fall rapidly and completely following the start of ATRA therapy, there appears to be a slower resolution of procoagulant markers (up to 30 days). This may partly explain the clinical observation of thromboembolic events occurring in patients on ATRA, and thus prophylactic use of low-molecular-weight heparin or other anticoagulants should be considered once bleeding manifestations have settled.

Vitamin K and related disorders Vitamin K metabolism Vitamin K (‘K’ denoting Koagulation) is a group of lipophilic and hydrophobic vitamins that are needed for the posttranslational modification of proteins, mostly required for blood coagulation. All forms of vitamin K share a methyl-naphthoquinone ring but differ in the structures of the side-chain. Phylloquinone (vitamin K1) is derived from plants and has a phytyl side-chain, while menaquinones (vitamin K2), which differ in the constituent of the side-chain, are synthesized by bacteria in human and animal intestine. Menadione (vitamin K3), a synthetic water-soluble derivative without a side-chain, has been withdrawn because its use often resulted in haemolytic anaemia and kernicterus, especially in patients with glucose 6-phosphate dehydrogenase deficiency. 845

Postgraduate Haematology

Vitamin K is a cofactor for vitamin K-dependent carboxylase, an enzyme that catalyses the carboxylation of glutamic acid (Glu) residues in several proteins (Figure 43.4). A free cysteine residue in the carboxylase converts reduced vitamin K (KH2) into a strong base that can extract hydrogen from the γ-carbon of glutamic acid. Subsequently, carbon dioxide is added to form γ-carboxyglutamic acid (or Gla). This reaction results in vitamin K 2,3-epoxide, which is recycled to reduced vitamin K by vitamin K epoxide reductase (VKOR), the enzyme inhibited by coumarin anticoagulants. The Gla domains of the vitamin K-dependent coagulant and anticoagulant proteins (FII, FVII, FIX, FX and proteins C and S) allow calcium-dependent binding to negatively charged phospholipids of activated cell membranes. This enhances the rate of the enzymatic reactions. In the bones, carboxylation of the Gla protein osteocalcin is essential for incorporation of calcium into hydroxyapatite crystals. Retrospective studies suggest that long-term therapy with coumarin-based anticoagulants can affect vertebral bone density and fracture risk.

Vitamin K deficiency Vitamin K deficiency can occur at any age but is more common in infants because vitamin K does not cross the pla-

Glutamic acid

Vitamin K-dependent carboxylase

γ-Carboxyglutamic acid

Vitamin KH2

Vitamin K

Vitamin K epoxide Vitamin K epoxide reductase

Figure 43.4 Vitamin K metabolism.

centa, breast milk has low levels and there is low colonic bacterial synthesis. The clinical presentation of vitamin K deficiency in infants is with bleeding. It was previously called ‘haemorrhagic disease of the newborn’ but is now termed vitamin K deficiency bleeding (VKDB). Supplementation of vitamin K is necessary to reduce the risk of bleeding, especially in exclusively breast-fed babies. A single intramuscular injection of vitamin K 1 mg prevents VKDB; however, if oral replacement is used, prolonged administration is required, although the exact dose is still controversial. In adults, vitamin K deficiency is uncommon due to recycling of the vitamin and an adequate gut flora. However, a poor dietary intake in combination with antibiotic therapy can cause deficiency. Other causes of vitamin K deficiency include malabsorption, cholestatic liver disease (poor enterohepatic circulation) and drugs such as anticonvulsants and warfarin. Vitamin K-dependent coagulation factors in healthy fullterm infants are about half of normal adult values. Adult values are reached by about 6 months except for protein C, which does not reach adult levels until adolescence. Coagulation tests should be compared with age-matched reference ranges to distinguish physiological and pathological deficiencies. VKDB in young children has been classified into early, classical and late types. The clinical features are shown in Table 43.4. The diagnostic criteria for VKDB includes a PT more than four times control in the presence of at least one of (i) a normal platelet count, normal fibrinogen level and absent fibrinogen degradation products, (ii) normalization of coagulation tests after parenteral vitamin K administration or (iii) the presence of proteins induced by vitamin K absence or antagonism (PIVKA) in plasma. The presence of PIVKA without a coagulation deficit is a marker of subclinical vitamin K deficiency. The treatment of a non-life-threatening bleed is with vitamin K1 (phytomenadione) given slowly intravenously. A dose of 1–2 mg is enough to fully correct the deficiency in infants aged up to 6 months, higher doses offering no advantage in efficacy or speed of reversal. There have been reports of anaphylactic reactions with the parenteral form although these are rare. The

Table 43.4 Features of vitamin K-deficiency bleeding (VKDB).

Presentation Cause Clinical presentation

Incidence of intracranial haemorrhage

846

Early VKDB

Classical VKDB

Late VKDB

First 24 hours of life Infants of mothers on vitamin K-inhibiting drugs Severe, with intracranial and intrathoracic bleeds

Days 2–7 of life Inadequate feeding

High

Rare

Day 8 to 6 months of life Cholestasis or malabsorption syndromes Severe, with high incidence of intracranial bleeds with ‘warning bleeds’ in up to one-third Very high

Milder, with gastrointestinal or umbilical bleeds being common

Acquired coagulation disorders

PT should improve within 48 hours and if this has not occurred after three doses, continuation of vitamin K in most cases is unlikely to help. Oral replacement may be used if there is no active bleeding and absorption is normal. Patients with severe bleeding should be treated with FFP or PCC. Poisoning with warfarin follows the accidental ingestion of large doses of warfarin or compounds with similar properties. Diagnosis is suggested by poor correction of PT or International Normalized Ratio (INR) with normal doses of vitamin K. Serum assays of warfarin concentration can be undertaken for confirmation.

(a)

Figure 43.5 Haemostatic disturbance in liver disease. The haemostatic system in liver disease (bottom) compared with normal haemostasis (top). In normal healthy adults, the procoagulant and anticoagulant systems and the profibrinolytic and antifibrinolytic systems form a balance that is not easily disturbed. In patients with liver impairment, all four components are variably reduced and additional stimuli like infections or renal failure can upset the balance very easily, leading to either bleeding or thrombosis.

Haemostatic disturbance in liver disease (Figure 43.5) Almost all procoagulant factors, natural anticoagulants and inhibitors of coagulation are synthesized in the liver. The liver is also involved in the clearance of activated clotting factors from the circulation. The effect of liver disease on haemostasis is therefore complex and a balance between procoagulant and antithrombotic changes (Figure 43.5; Table 43.5). Routine coagulation tests such as PT and APTT are commonly pro-

Procoagulant Antifibrinolytic

Profibrinolytic

Anticoagulant

(b) Thrombosis Procoagulant Antifibrinolytic

Profibrinolytic

Anticoagulant

Bleeding

Table 43.5 Causes of increased bleeding tendency or coagulation abnormalities in liver disease. Decreased coagulation factors Decreased synthesis Vitamin K deficiency Increased clearance Abnormal coagulation factors due to hypocarboxylation Decreased platelets Sequestration in spleen or liver Thrombopoietin deficiency Destruction due to toxins or antiplatelet antibodies or toxins Toxic effect of alcohol on marrow Dysfunctional platelets Altered platelet arachidonic acid metabolism Defective signal transduction Storage pool deficiency Abnormalities of the platelet glycoprotein Increased platelet inhibitors nitric oxide and prostacyclin

Hypofibrinogenaemia Impaired synthesis Loss into extravascular spaces (ascites) Increased catabolism Loss due to massive haemorrhage Dysfibrinogenaemia: abnormal sialic acid content Abnormal fibrinolysis Increased release and decreased clearance of tissue plasminogen activator Decreased synthesis of α2-antiplasmin, plasminogen and thrombin-activated fibrinolysis inhibitor Infections Endogenous heparinoids: anti-FXa activity Renal failure Sinusoidal endothelial cell dysfunction Hypothermia

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longed in liver disease but are only sensitive to decreases in procoagulant factors and hence are liable to overestimation of the haemostatic defect. Patients with liver disease have similar thrombin generation to healthy control individuals, when tests are performed in the presence of thrombomodulin to make them sensitive to protein C deficiency.

Acute hepatitis Patients with acute hepatitis are often thrombocytopenic (platelets 100–150 × 109/L). The mechanism for this may be immune, due to concurrent hypersplenism or DIC. Platelet function may be impaired in acute hepatitis, although this is unlikely to be of clinical significance. In acute liver disease, hepatic biosynthesis of clotting factors is impaired and this may be reflected in a prolongation of coagulation tests. The PT and FV, FVII, antithrombin and protein C levels are the most sensitive to hepatic biosynthetic dysfunction. The PT and FVII level predict survival in acute liver disease. The plasma fibrinogen may be raised in acute hepatic disturbance as part of an acute-phase response. A low fibrinogen level is associated with a poor prognosis in this context. A pattern of reduced fibrinogen and increased fibrin degradation products seen in acute liver disease may be due to hyperfibrinolysis.

Chronic liver disease Low platelet count and abnormal platelet function are also common in liver disease and have been thought to lead to bleeding. Mild to moderate thrombocytopenia is noted in up to 30% of patients with chronic liver disease and in up to 90% of patients with terminal liver disease. In patients with cirrhosis, this is frequently related to hypersplenism, secondary to portal hypertension. In addition, dietary problems, such as folate deficiency and the direct toxic effect of ethanol on megakaryocyte function, may contribute to thrombocytopenia. A more profound thrombocytopenia in patients with hepatitis C virus (HCV) infection may be autoimmune, although the response to steroids is poor; however, approximately half of adult such patients treated with interferon alfa responded with a rise in platelet count. Recently, a Phase II study using eltrombopag, an oral thrombopoietin receptor agonist, increased platelet counts in the majority of patients with cirrhosis associated with HCV infection to more than 100 × 109/L in a dose-dependent manner. Several platelet function abnormalities have also been demonstrated in liver disease. Despite this, recent evidence shows that VWF which binds to platelet glycoprotein is found to be increased tenfold and has been shown to support platelet adhesion despite reduced functional capacity. In addition, desmopressin did not show any efficacy in reducing blood loss in patients undergoing partial liver resection or liver transplantation. Coagulation factor and natural anticoagulant synthesis is affected by chronic liver disease. FIX synthesis is usually reduced 848

less than that of FII, FVII, FX and protein C. The levels of these factors, especially FVII, have been demonstrated to fall proportionately with increasing severity of disease, with levels of FVII shown to be an independent predictor of survival (level below 34% associated with 93% mortality). Chronic liver impairment also leads to vitamin K deficiency, mainly by decreased absorption from the gut as a result of reduced bile salt secretion in cholestasis (parenteral vitamin K responsive) and decreased synthesis in parenchymal disease (parenteral vitamin K nonresponsive). The PT, a test for the vitamin K-dependent factors, has been incorporated into prognostic indices of chronic liver disease, such as the Child–Pugh and Mayo End-Stage Liver Disease scores. Despite the prolongation of clotting tests such as PT and APTT, bleeding after liver biopsy or other potentially haemorrhagic procedure in patients with cirrhosis is rare, suggesting that these tests are not on their own indicative of the haemorrhagic tendency. Both these laboratory tests were developed to investigate coagulation factor deficiencies in patients with a bleeding history and as such they only measure the procoagulant factors. Overall, haemostasis is a complex interrelationship between endothelium, platelets, procoagulant proteins and inhibitors. The levels of such inhibitors (or anticoagulant proteins), antithrombin and proteins C and S are reduced in a similar manner to other clotting factors, thus potentially providing a net balance in haemostasis. Evidence for normal thrombin production in cirrhosis despite abnormal conventional coagulation tests has been recently demonstrated by thrombin generation tests, which include the other haemostatic parameters including platelets. It is common practice to use the INR instead of the PT to access patients with liver disease. However, the INR is standardized for patients taking vitamin K antagonists and its use in patients who have liver disease requires determination of an international sensitivity index (ISI) relevant to liver disease. Recently, attempts have been made to use modifications of the INR called the INRliver, which is free of the warfarin-based reference range. Most patients with stable chronic liver disease have normal or increased fibrinogen, although in advanced disease fibrinogen falls. This may be due to impaired synthesis, loss into extravascular spaces (ascites), increased catabolism or massive haemorrhage. Some patients develop dysfibrinogenaemia due to increased activity of sialyltransferase expressed by immature hepatocytes generated during hepatic injury, which leads to low-molecular-weight fibrinogen with abnormal α-chains and higher sialic acid content. Dysfibrinogenaemia is reflected by a prolonged TT and/or reptilase time, with the fibrinogen antigen level reduced less than the Clauss fibrinogen. Cirrhosis can also be associated with accelerated fibrinolysis due to decreased clearance of tPA, and decreased synthesis of α2-antiplasmin, plasminogen and thrombin-activatable fibrinolysis inhibitor (TAFI). However, the level of TAFI deficiency in cirrhosis is not associated with increased plasma fibrinolysis when a global test

Acquired coagulation disorders

of fibrinolysis is employed. Also, the levels of plasminogen (a fibrinolytic) are reduced and those of PAI-1 (an antifibrinolytic) are high in liver disease, ultimately providing a balance for the fibrinolytic system. However, hyperfibrinolysis has been found to be a predictor of the first episode of upper gastrointestinal bleeding in cirrhotic patients with portal hypertension. This is due to delayed clotting activation because of the consumption of clotting factors and inhibition of fibrin polymerization and also secondary to reduced platelet adhesion and induction of platelet disaggregation. Recent experiments have thrown light on non-haematological factors contributing to the bleeding diathesis in liver disease. These include bacterial infections, endogenous heparinoids, sinusoidal endothelial dysfunction and renal failure. Bacterial infections are a frequent coexisting problem in cirrhotic patients and are related to the degree of liver dysfunction. Infection is also an independent risk factor for variceal bleeding, with spontaneous bacterial peritonitis commonly preceding variceal bleeding and prophylactic antibiotic therapy shown to prevent early rebleeding. The endotoxins and inflammation due to infection can also release endogenous heparinoids from the endothelium and mast cells. These substances can have anti-FX activity that could in theory lead to bleeding. The dysfunction in sinusoidal endothelial cells makes them unable to respond to abrupt repeated increases in portal pressure (e.g. postprandial situations), which can lead to progressive dilatation of varices in cirrhosis. Life-threatening haemorrhage in patients with liver disease is often related more to portal hypertension (the localized coagulation problem is termed ‘accelerated intravascular coagulation and fibrinolysis’) than to net dysfunction of the clotting cascade, wherein the varices play a major part. Renal failure is also common in advanced liver disease (hepatorenal syndrome), which can impart a bleeding risk due to various reasons discussed separately. Hypothermia used as a therapeutic measure to prevent hepatic encephalopathy can reduce the enzymatic activity of plasma coagulation proteins and also has an effect on preventing the activation of platelets. Reduced hepatic clearance of activated clotting factors and lower synthesis of natural anticoagulants may predispose individuals with hepatic disease to DIC. This may occur following Gram-negative infection or in association with the insertion of a peritoneovenous shunt. Ascitic fluid should be discarded at the time of shunt insertion to prevent this complication. The management of a bleeding patient with hepatic dysfunction depends on the site of bleeding and the haemostatic dysfunction. Clotting function should be assessed by means of the PT, APTT, TT, and fibrinogen and D-dimer levels, although the results should be interpreted in the clinical context before transfusions are undertaken. Vitamin K should be administered intravenously (10 mg daily for 3 days) to aid biosynthesis of vitamin K-dependent factors. The widespread practice of managing the coagulopathy of liver disease with transfusions of FFP, cryoprecipitate or platelets should only be considered in the

presence of active bleeding. Large volumes of FFP may be required and this may present a management problem in patients with hepatic disease who are at risk of fluid overload. Platelet transfusion may be necessary, although platelet recovery may be reduced because of hypersplenism or immunemediated destruction. There are no evidence-based guidelines to establish safe coagulation levels for liver biopsy and similar procedures and as such it is important to allow these only when the benefits outweighs the risks (transjugular route safer than percutaneous for liver biopsy). However, correction of the coagulopathy is particularly important before the placement of an intracranial pressure transducer in patients with hepatic encephalopathy. Spontaneous intracranial bleeding has been reported in such patients, though rarely, and this remains one of the principal concerns regarding abnormal coagulation in liver disease. In patients with evidence of increased fibrinolysis, antifibrinolytic drugs such as tranexamic acid should be considered.

Liver transplantation The changes in the balance of haemostasis seen during liver transplantation are complex and multifactorial and can be due to surgical and non-surgical causes. Traditionally, the process of liver transplantation is split into three stages: stage I, the preanhepatic stage, which ends with the occlusion of the recipient’s hepatic blood flow; stage II, the anhepatic phase, which ends with the reperfusion of the donor liver; and stage III, the reperfusion and neohepatic period. The risk of bleeding in the pre-anhepatic stage is related directly to the preoperative haemorrhagic risk related to the underlying liver disease. A reduction in procoagulant factors may also be seen, especially if large blood losses necessitate transfusion, leading to the dilution of clotting factors. During the second anhepatic stage, many studies have reported enhanced fibrinolytic activity. The lack of tPA clearance and the reduction in α2-antiplasmin may be responsible for this enhanced primary fibrinolysis. The mechanism of the increase in tPA levels is most likely due to lack of hepatic clearance. Reperfusion of the liver during the postanhepatic phase is the crucial point of the intervention where once again fibrinolysis seems to play a pivotal role. There is a dramatic increase after reperfusion in almost three-quarters of patients who undergo liver transplantation. Usually, hyperfibrinolysis subsides within an hour, but in damaged donor liver sustained increased fibrinolytic activity may be observed. The endothelium of the donor liver is an important source of tPA; the ischaemic damage to the graft during preservation may explain the dramatic increase in plasminogen activators. Trapping of platelets in the graft may also play a role in the bleeding tendency. After reperfusion, the release of heparin-like compounds has also been shown. In the postoperative period, a reduction in platelet count is related to platelet activation and blood loss in addition to low thrombopoietin levels. The levels 849

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of this hormone increase significantly on the first day after liver synthetic function is restored, with the platelet count normalizing in 2 weeks. There is little evidence that one can predict the transfusion needs of a patient by examining the preoperative coagulation results. The profound coagulation abnormalities seen in fulminant hepatic failure should be corrected preoperatively. However, the correction of a mild coagulopathy in a patient with chronic liver disease prior to the operation is less likely to be of benefit. During surgery, the aim is to transfuse blood components to prevent the development of intractable coagulopathy. Whole-blood near-patient tests of haemostasis are often used in theatre to complement the tests performed in the coagulation laboratory. Many departments use algorithms based on coagulation and near-patient testing to guide FFP, red cell, cryoprecipitate and platelet transfusion during the procedure. Aprotinin can be administered to counteract the increase in fibrinolysis observed.

Hypercoagulability in liver disease Tipping of the balance in the complex interplay between endogenous procoagulants and the anticoagulant system in liver disease can also lead to a hypercoagulable state that negates the generally believed notion of an ‘autoanticoagulated state’. The hypercoagulable events may be clinically evident disease, as with portal vein thrombosis and venous thromboembolism, but in the microvasculature can be a major pathophysiological contributor to portopulmonary hypertension, liver fibrosis (termed ‘parenchymal extinction’ in this setting), thrombosis of extracorporeal circuits and progression of non-alcoholic steatohepatitis to cirrhosis. Hypercoagulation in liver disease may be related to poor flow, endothelial dysfunction and vasculopathy associated with a chronic inflammatory state, increased levels of FVIII and VWF, or decreased synthesis of the naturally occurring anticoagulant proteins. Hepatic fibrogenesis may be caused by tissue ischaemia and direct thrombin-mediated stellate cell activation by PAR-1 cleavage. FV Leiden mutation, protein C deficiency and increased expression of FVIII were associated with rapid progression to cirrhosis in chronic HCV. The association between hypercoagulation and increased fibrosis has led to experiments which have shown that interference with either the generation of thrombin or its downstream activity may reduce hepatic fibrosis.

Haemostatic disturbance in renal disease Bleeding was a frequent cause of morbidity and mortality in patients with renal failure before the advent of dialysis. Common manifestations are gastrointestinal bleeding from angiodysplasia and peptic ulcers as well as prolonged bleeding from skin puncture sites. Subdural haematomas and haemorrhagic peri850

carditis are also seen but bleeding at the time of renal biopsy is rare. Bleeding is seen despite normal or elevated circulating levels of coagulation factors, suggesting that platelet abnormalities are the likely cause. This is supported by the finding of a prolonged bleeding time and reduced platelet aggregation to various agonists. Severe thrombocytopenia (platelets < 50 × 109/L) secondary to renal failure is rare and its presence should suggest concomitant conditions such as HCV infection or vasculitis. Platelet dysfunction with renal failure is multifactorial and can be divided into (i) intrinsic platelet defects, (ii) abnormal interaction of platelets with the endothelium, (iii) effects of uraemic toxins, (iv) effects of anaemia on platelets and (v) dialysis-related. Intrinsic platelet abnormalities reported in association with uraemia are shown in Table 43.6. The interaction between uraemic platelets and the endothelium can be markedly reduced, due partly to impaired VWF binding to platelets. As a result of excess urea, l-arginine is shunted to form guanidinosuccinic acid, which can upregulate endothelial production of nitric oxide, an inhibitor of platelet aggregation. Uraemic patients with prolonged bleeding times also have raised prostaglandin (PG)I2 levels. Anaemia-related haemostatic dysfunction is primarily a result of reduced displacement of platelets to the vessel wall by red cells. Decreased red cell number also results in reduced ADP release and decreased platelet interaction with collagen. Dialysis can improve bleeding symptoms and a likely explanation is the chronic low-level platelet activation associated with the procedure. Antifibrinolytic agents can help because fibrinogen fragments of fibrinolysis can interfere with platelet receptors. Antiplatelet drugs, antibiotics (especially those which can accumulate in renal failure) and heparin may contribute to the bleeding risk. The bleeding time better correlates with clinical bleeding than tests of renal function, although measurement is rarely clinically useful. Adequate dialysis will improve symptoms and the bleeding time in most patients. Intravenous or intranasal Table 43.6 Platelet abnormalities reported in uraemia. Decreased GPIb complexes Reduced serotonin and ADP in the granules Increased levels of cyclic AMP Defective ristocetin-induced platelet aggregation Abnormal mobilization of free cytoplasmic calcium in response to agonists Reduced release of arachidonic acid from membrane phospholipids Decreased conversion of arachidonic acid to thromboxane A2 Abnormal dense-granule and α-granule secretion Abnormal cytoskeletal assembly Deficient tyrosine phosphorylation

Acquired coagulation disorders

administration of desmopressin in conjunction with antifibrinolytics has also been used successfully to control uraemic bleeding. Although the response is brief (4–8 hours), the rapid onset of action is beneficial. Care is required to avoid hyponatraemia and fluid retention. Desmopressin is also thought to improve platelet function by the enhanced release of endothelial VWF multimers and also by increasing the levels of platelet glycoprotein (GP)Ib/IX. Estrogen preparations have been used for a more prolonged effect, although the mechanism of action is not well understood. Correction of anaemia with recombinant human erythropoietin so that the haematocrit exceeds 30% improves platelet interaction with the vessel wall and has also been shown to increase the number of reticulated platelets. Other potentially beneficial effects of recombinant human erythropoietin include improved platelet intracellular calcium mobilization, increased expression of GPIb and improved platelet signal transduction. Cryoprecipitate transfusion for uraemic bleeding is going out of favour, although it may be tried if the patient is haemodynamically unstable.

Pregnancy-related haemostatic dysfunction Normal pregnancy is associated with physiological changes in haemostasis, with increased levels of procoagulant proteins such as fibrinogen, VWF and FVIII and a fall in anticoagulants such as protein S. These changes partly contribute to the increased risk of venous thromboembolism during pregnancy. Obstetric haemorrhage is the third most common cause of maternal death in the UK after venous thromboembolism and pre-eclampsia. The cause of bleeding is often multifactorial, with a combination of physical and acquired haemostatic defects. Consumption leads to depletion of platelets and coagulation factors, particularly fibrinogen. A particularly aggressive consumptive coagulopathy is seen with amniotic fluid embolus triggered by TF and phosphatidylserine, and is often fatal. Consumptive coagulopathy may also be triggered by placental abruption, intrauterine fetal death, pre-eclampsia and acute fatty liver of pregnancy. Once bleeding and resuscitation has started, a dilutional coagulopathy may exacerbate the haemostatic failure and contribute to the ongoing bleeding. Treatment requires rapid recognition of bleeding and a coordinated response from obstetricians, midwives, anaesthetists and haematologists. Physical methods to control bleeding include the use of oxytocins to contract the uterus, B Lynch sutures, intrauterine balloon tamponade and uterine artery embolization. Assessment of haemostatic failure requires an urgent FBC and a coagulation screen that includes a Clauss fibrinogen because the PT and APTT may be normal despite a low fibrinogen. Blood product replacement is often required before the results of the blood test are available and empirical

replacement therapy may be necessary. In patients with severe haemostatic failure, a standard dose of FFP (15 mL/kg) is unlikely to be adequate to correct the haemostatic defect and the use of larger volumes should be anticipated. Fibrinogen should be maintained above at least 1.5 g/L and platelets above 50 × 109/L. Some centres now routinely use fibrinogen concentrate in obstetric haemorrhage to rapidly correct this clotting factor, although this product is not licensed in the UK. rFVIIa may be used if haemostasis cannot be secured by standard methods but will not be useful in the presence of low fibrinogen or thrombocytopenia. As soon as bleeding has been controlled the patient is likely to be at high risk of venous thromboembolism; this should be assessed and appropriate prophylaxis started.

Haemostatic dysfunction associated with cardiopulmonary bypass surgery Coronary artery bypass graft (CABG) surgery is associated with excessive bleeding in about 5% of cases. Reoperation for bleeding is required in 3–10% of cases, with a mortality of about 30%. Bleeding is more common with revision procedures. A surgically correctable source of bleeding is found in over half of cases. Bleeding due to haemostatic disturbances can be patient related or CPB related. Antiplatelet, anticoagulant or fibrinolytic drugs, particularly at the time of emergency CABG after stenting procedures, commonly contribute to perioperative bleeding. Despite its antiplatelet activity, aspirin has not been demonstrated to cause increased blood loss after CABG, and has been shown to reduce the risk of death by one-third. Haemostatic abnormalities associated with CPB are related to the interaction of blood with the extensive non-endothelial bypass surfaces and retransfusion of pericardial blood. CPB decreases both the number and function of platelets. Platelet counts fall by 25–60% within 15 min of first passage of blood through the primed CPB circuit, in association with a prolonged bleeding time. This is due to the exposure of platelets to the high concentration of unfractionated heparin, the haemodilution caused by by the oxygenator primer and the non-biological extracorporeal surface. The platelet count rarely falls below 100 × 109/L and persistent or profound thrombocytopenia should prompt consideration of alternative causes including heparin-induced thrombocytopenia. The observed changes in platelet function in patients undergoing CPB are due to modification of membrane components with loss of receptors, decrease in granule contents, aggregation to fibrinogen adsorbed onto the bypass circuit, mechanical trauma, exposure to hypothermia and heparin. CPB also results in increased numbers of platelet fragments and microparticles, denoting platelet activation. Following CPB, platelet function returns to normal within 1 hour, although the platelet count 851

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may take several days to normalize. Coagulation factors fall because of haemodilution, which can be exacerbated by use of cell-salvage systems. Fibrinolysis is also enhanced by returned blood from the pericardiotomy suction. Blood component administration in patients with excessive bleeding related to CABG is generally empirical, with replacement of FPP and platelets. Near-patient whole-blood testing has been used to assess haemostasis and fibrinolysis in the context of cardiac surgery and has been shown to help rationalize blood product usage. The time delay in obtaining routine laboratory tests often limits their use in the acute management of bleeding patients. The off-licence use of PCCs and fibrinogen concentrates has also been reported. Case reports suggest that off-licence use of rFVIIa may be considered in patients who have failed standard treatment but randomized studies have not supported a role for its use as prophylactic treatment. Aprotinin has been used to reduce bleeding during CPB by inhibition of fibrinolysis. Double-blind studies have consistently shown its effectiveness in reducing blood loss in comparison with tranexamic acid, α-aminocaproic acid and desmopressin. However, recent reports have shown a doubling of the risk of renal failure requiring dialysis in patients undergoing complex coronary artery surgery and a 55% increase in cardiovascular and cerebrovascular events. The current use of aprotinin is contentious and under intense debate.

Haemostatic dysfunction associated with trauma Coagulopathy associated with trauma is multifactorial and the important initiators include tissue injury, loss of procoagulant factors and platelets, physiological and therapeutic haemodilution, inflammation, hypothermia and acidosis. The localization of coagulation to the sites of injury (in contrast to DIC of sepsis) has made some experts use the term ‘acute coagulopathy of trauma-shock’. Tissue damage in trauma initiates coagulation via TF, with head injury causing release of brain-specific thromboplastins into the circulation. Increased fibrinolysis due to the direct release of tPA from the endothelium is also seen, together with abnormal fibrin polymerization that increases susceptibility to cleavage by plasmin. Massive blood loss leads to deficiencies of haemostatic factors and up to half of the fibrinogen and one-third of platelets may be lost before treatment of the injury has begun. Tissue under-perfusion contributes to the coagulopathy by enhancing anticoagulation through activated protein C and fibrinolysis through reduced TAFI. Physiological filling of the vascular space with fluid from cellular and interstitial spaces, along with the administration of intravenous fluids and plasma-poor red cells, causes haemodilution to worsen the coagulopathy. Plasma expanders are also associated with anticoagulant effects such as reduced VWF levels and their use should be limited. 852

The presence of an abnormal coagulation test on arrival in the emergency department correlates with the severity of injury and the mortality rate. A raised PT is the most frequent abnormality (12.4% of patients) while a low platelet count is least frequent (5.6%). An abnormal PT and APTT increased the risk of mortality by 6.3-fold and 10.7-fold, respectively. A guide to blood product support, with transfusion triggers, that may be used in conjunction with repeated laboratory tests is given in Table 43.7. Delay in obtaining coagulation and platelet count results dictates that empirical blood product replacement is often required. Recent observations, predominantly based on experience of battlefield trauma, have prompted recommendations for aggressive and early plasma and platelet replacement. This has been termed ‘damage limitation resuscitation’ (ratio of 1 : 1 : 1 for red cells, plasma and platelets). This strategy has been reported in non-randomized studies to improve outcome but definitive evidence is lacking for this approach. Issues such as hypothermia and acidosis need to be addressed by using prewarmed fluids and extracorporeal warming devices and appropriate resuscitation. Table 43.7 Guide to blood product replacement in massive blood loss. 1 Control bleeding using surgical and/or radiological interventions 2 Restore an appropriate circulating blood volume 3 Control exacerbating factors: hypothermia and acidosis 4 Blood product support as detailed below Red cells Use O-negative red cells first If no record of red cell antibodies, ABO- and Rh-compatible cross-matched blood should be available within 30 min (maximum 45 min) Replace red cells as required to maintain circulating blood volume Use blood warmer to avoid hypothermia Fresh-frozen plasma Transfuse FFP early to prevent coagulopathy to maintain PT and APTT < 1.5 times normal If PT and APTT > 1.5 times normal FFP at doses > 15 mL/kg will be required Platelet transfusion One to two adult doses after 1.5–2 blood volume replacement (equivalent to 8–10 bags of red cells) Aim for platelet count > 50 × 109/L Fibrinogen Cryoprecipitate (dose: two donation pools) Fibrinogen concentrates (30–50 mg/kg) Aim for fibrinogen level > 1.5 g/L

Acquired coagulation disorders

A randomized controlled trial of rFVIIa versus placebo in 301 patients with blunt and penetrating injuries showed a reduction in 2.6 RBC units transfused for the blunt trauma subgroup and a similar though non-significant trend in the penetrating injury subgroup. The incidence of adult respiratory distress syndrome was decreased in patients with blunt injury who received rFVIIa, although there were no differences in survival or in the incidence of thromboembolic events and multiorgan failure. In view of these data, use of rFVIIa in the management of trauma has to be considered on an individual basis.

Coagulopathy in massive blood loss Massive blood loss is usually defined as 1 blood volume (about 5 L in an adult) in 24 hours or 50% blood volume in 3 hours or more than 150 mL/hour. The associated coagulopathy is complex and multifactorial. It is partly dilutional as blood is replaced with volume expanders and red cells but DIC, hypoxia, hypothermia and acidosis contribute in some clinical situations. Haemostatic management should be to maintain a platelet count of at least 75 × 109/L, a threshold that is likely to be reached after a 2-blood-volume transfusion. A higher platelet count of about 100 × 109/L will be needed in major trauma or central nervous system bleeding. Fibrinogen should be maintained above at least 1.5 g/L, a level that is likely to be reached after about a 1.5-blood-volume transfusion, assuming no significant additional consumptive coagulopathy. Haemostatic therapy is to infuse platelets and FFP in volumes likely to maintain a safety margin above these critical levels. Hypofibrinogenaemia unresponsive to FFP may require cryoprecipitate or fibrinogen concentrate. It is important to predict ongoing dilution and consumption of coagulation factors and to replace these expectantly if bleeding continues. Regular measurement of FBC and coagulation screens (including Clauss fibrinogen) are important for monitoring replacement treatment, although often these are not available rapidly enough to guide initial management and empirical treatment may be required in the early phases (see Table 43.7).

Bruising Purpura simplex (normal/easy bruising) Distinguishing normal from pathological bruises may be difficult and individuals have variable thresholds for presenting for medical review. If bruising is not associated with other symptoms suggestive of a bleeding disorder and routine tests of coagulation (including VWF levels and platelet number) are normal, the patient can be reassured. Drugs such as non-steroidal anti-inflammatory agents or selective serotonin reuptake inhibitors are sometimes implicated.

Non-accidental bruising Bruising is a common feature of non-accidental injury in both children and adults. Bruises that affect unusual sites, are in different stages of maturation or shaped like a hand or instrument should raise concern. In the case of children, appropriate liaison with child protection agencies should be undertaken, although the possibility of an underlying congenital or acquired bleeding disorder should also be thoroughly investigated. Selfharm may also present with bruising and should be suspected if the pattern of bruising is atypical. Deliberate ingestion of anticoagulants and long-acting vitamin K antagonists is also possible.

Senile purpura (atrophic or actinic purpura) and steroid-related purpura Bruising is more common in elderly people due to atrophy of subcutaneous tissues and loss of collagen and elastin fibres in subcutaneous tissues. Blood vessels in the skin can be broken by minor trauma or shearing forces and typically purpura are seen on hands and forearms. If routine tests of platelet number and coagulation are normal, no further investigation is required. Long-term steroid use is also associated with bruising secondary to atrophy of collagen fibres supporting blood vessels.

Psychogenic purpura Psychogenic purpura or Diamond–Gardner syndrome is a rare condition usually seen most commonly in women with psychological problems. It presents with painful ecchymotic lesions, mostly on the extremities and the face and rarely on less accessible parts of the body. The skin lesions can be preceded by paraesthesia or pain and are usually reported after surgery or minor trauma. The usual tests for haemostasis reveal no abnormalities. Diamond and Gardner suggested the possibility of a local reaction to the patient’s own red cell stroma and gave it the name ‘autoerythrocyte sensitization syndrome’.

Scurvy Scurvy is caused by a lack of vitamin C (ascorbic acid), which is required for the hydroxylation of prolyl and lysyl residues in the formation of mature collagen. Vitamin C deficiency renders the collagen unable to self-assemble into rigid triple helices, resulting in blood vessel fragility and poor wound healing. The characteristic clinical finding is perifollicular haemorrhage but large ecchymoses can occur on the legs and trunk. Follicular hyperkeratosis and corkscrew hairs are other cutaneous features. Bleeding gums are also common. Scurvy is more common in people with poor nutrition, especially alcoholics. The diagnosis is made clinically although levels of plasma or leucocyte 853

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vitamin C can be measured. A prompt response to vitamin C treatment is also diagnostic.

Inherited disorders of collagen and elastic fibres Ehlers–Danlos syndrome (EDS) is a group of dominantly inherited disorders of connective tissue characterized by lax hyperelastic skin, joint laxity and poor wound healing. There are six subtypes and subtype IV is associated with severe varicosities and spontaneous rupture of major blood vessels. Bruising is seen in 90% of patients with EDS and has also been reported in people with lax joints who do not fulfil the criteria of EDS. Although mild platelet and coagulation factor dysfunction has been reported in association with EDS, it is likely that these are coincidental. Some patients have a prolonged bleeding time and this can respond to DDAVP. Other disorders such as osteogenesis imperfecta and Marfan syndrome may also be associated with bruising. Recently, a pseudoxanthoma elasticum-like disorder has been described where deficiency of the vitamin K-dependent clotting factors was noted along with the typical excessive skin folding (or cutis laxa).

Haemostatic dysfunction associated with vasculitis The vasculitides are a heterogeneous group of disorders characterized by inflammation within blood vessel walls of different organs. It is classified by the size of the blood vessel involved and can be associated with both thrombosis, for example Behçet’s disease (see Chapter 46), and haemorrhage. The classic presentation of Henoch–Schönlein purpura is palpable purpura without thrombocytopenia or coagulopathy, arthritis/arthralgia, abdominal pain and renal disease. Henoch–Schönlein purpura is more commonly associated with bleeding than thrombosis.

Arteriovenous malformations Hereditary haemorrhagic telangiectasia Hereditary haemorrhagic telangiectasia (HHT) is a dominantly inherited, highly penetrant, familial disease in which telangiectasia develop on the skin and mucous membranes. Associated large arteriovenous malformations may occur in solid organs. International consensus diagnostic criteria have been developed based on epistaxis, mucocutaneous telangiectasia, visceral lesions and an affected first-degree relative. A patient is diagnosed as having ‘definite HHT’ when three crite854

ria are present, ‘suspected HHT’ when two criteria are present, or ‘HHT unlikely’ when only one criterion is present. All the offspring of an affected individual are at risk of having the disease since HHT may not manifest until late in life. Mutations in the ENG gene (encoding endoglin) and ALK1 gene (encoding activin receptor-like kinase 1) have been described. These genes encode proteins that are involved in signalling by the transforming growth factor (TGF)-β superfamily. In healthy endothelial cells, ALK-1 and endoglin cooperate in the TGF-β/Smad pathway. In HHT types 1 and 2, either endoglin or ALK-1 fail in cooperative signalling resulting in impaired TGF-β signalling. The latter can lead to a decreased capacity to form cord-like structures typical of the angiogenic process. This leads to dysregulation in the organization of the capillary network and arteriovenous malformation. Recently, it has also been shown that TGF-β1 has a biphasic effect on angiogenesis depending on its concentration. At high concentrations TGF-β1 inhibits VEGF-induced endothelial cell invasion and capillary lumen formation, whereas lower TGF-β1 concentrations potentiate VEGF activity. Increased levels of VEGF have been demonstrated in HHT (especially in patients who have frequent bleeds) and agents that inhibit VEGF, like thalidomide and bevacizumab, have been successful in treating severe HHT. Cerebral, pulmonary and neurological involvement is described more commonly with ENG mutations, whereas liver involvement has been associated with ALK1 mutations. Telangiectasia usually appears during the second and third decades of life but presentation at all ages has been described. Typical lesions are red 0.5–3 mm spots that blanche with pressure and are often noted on the skin of face and lips and mucosal surfaces of the tongue and mouth. Symptoms range from cosmetic and recurrent epistaxis and mouth bleeding to iron deficiency and intracranial haemorrhage. Epistaxis is seen in the majority of patients and is usually recurrent. Gastrointestinal bleeding from both upper and lower bowel and haematuria are also seen. Emboli, including septic emboli, may occur and antibiotic prophylaxis at the time of invasive procedures such as dental extraction is recommended. Occasionally bleeding can occur from internal organs without visible lesions. Virtually all internal organs can be affected. The lungs develop pulmonary arteriovenous fistulae in about 30% of patients leading to haemoptysis, sometimes catastrophic, hypoxia, dyspnoea, high-output cardiac failure and clubbing. Transient ischaemic attacks, cerebral infarction and systemic emboli due to right-to-left shunting occur in about half of the patients with pulmonary arteriovenous fistulae. Pulmonary arteriovenous fistulae may worsen during pregnancy. Liver involvement is present in the majority of patients and, though often asymptomatic, can lead to cirrhosis, portal hypertension and high-output cardiac failure. Conjunctival telangiectasia may cause benign bleeds while more serious retinal bleeds can occur. Intracranial haemorrhage is associated with arteriov-

Acquired coagulation disorders

enous fistulae but embolization including septic emboli also commonly cause cerebral symptoms. Screening tests in patients with mucocutaneous telangiectasia should include FBC, ferritin, liver function tests, chest radiography, liver ultrasound and contrast echocardiography (to identify pulmonary shunts). The role of gastrointestinal endoscopy and CT angiography or MRI of lung or brain in asymptomatic patients is unclear. Treatment options for symptomatic lesions include local options such as laser therapy of skin and mucosal telangiectasia and embolization of arteriovenous malformations in lung or nervous system. Systemic treatment with estrogens and tranexamic acid may reduce bleeding symptoms.

venous plexus becomes visible because of increased amounts of desaturated venous blood. Organ involvement from cholesterol emboli is dependent on the vascular supply, with renal ischaemia being the commonest. The diagnosis of this condition is often missed unless the occurrence of the clinical features is related to the triggering procedure. Fundoscopy can reveal the presence of retinal cholesterol crystals (Hollenhorst plaques) in about 25% of cases. Biopsy of the cutaneous lesion or the affected organ is required for definitive diagnosis. The prostacyclin analogue iloprost has been used successfully in painful cutaneous necrotic lesions and renal insufficiency in a single report.

Fat embolism syndrome Kasabach–Merritt syndrome Kasabach–Merritt syndrome is a rare condition in which a vascular tumour (Kaposiform haemangioendothelioma) is associated with thrombocytopenia, hypofibrinogenaemia and bleeding, which can be life-threatening. The thrombocytopenia in Kasabach–Merritt syndrome is presumed to be due to platelet adhesion to the abnormally proliferating endothelium. This results in the activation of platelets with secondary consumption of clotting factors, including VWF, leading to systemic haemostatic failure. Excessive flow and shear rates secondary to arteriovenous shunting also contribute. The platelet count is often less than 20 × 109/L and platelet half-life dramatically reduced. Intralesional bleeding can cause rapid enlargement of the haemangioma and can worsen the consumptive coagulopathy. Intralesional thrombosis may rarely occur, causing spontaneous resolution of some lesions. In addition to the thrombocytopenia and abnormal coagulation, some degree of microangiopathic haemolysis may be seen, suggesting intravascular coagulopathy. Management involves supportive care and, if possible, removal of the lesion. Steroids, interferon, chemotherapy with vincristine, radiotherapy and anti-angiogenic agents have been tried.

Fat embolization is characterized by release of fat droplets into the systemic circulation after a traumatic or iatrogenic event. Its incidence depends on the bone involved (fractures of the femoral shaft > tibia or fibula > neck of femur), isolated or multiple fractures, age (10–40 years) and gender (occurs more in males). Although more common after lower limb fractures, fat embolism syndrome can also occur after liposuction, bone marrow harvesting, total parenteral nutrition, sickle cell crisis and pancreatitis. Classically, it presents with the triad of respiratory distress, mental status changes and petechial rash 24–48 hours after pelvic or long-bone fracture. The rash is pathognomonic and is seen usually on the conjunctiva, oral mucous membranes and upper body possibly due to embolization of fat droplets accumulating in the aortic arch. The diagnosis is made clinically because laboratory and radiographic diagnosis is nonspecific and inconsistent. Thrombocytopenia is common due to platelet activation and consumption into the thrombi. Management is mainly supportive with intensive care management to ensure haemodynamic stability. Aspirin and corticosteroids have also been shown to be helpful, although the use of heparin is controversial.

Warfarin-induced skin necrosis

Microthromboembolic disease Cholesterol embolism Cholesterol embolism is a complication of widespread atherosclerotic disease. Rupture of an atherosclerotic plaque can occur spontaneously or, more commonly, at the time of vascular surgery or invasive procedures. Anticoagulant or fibrinolytic therapy can also weaken thrombi that usually prevent the release of cholesterol crystals. The characteristic presentation is with small limb-vessel occlusion with well-preserved peripheral pulses, which may occasionally lead to gangrene. Another common skin finding is livedo reticularis, where the cutaneous

Warfarin-induced skin necrosis is a rare condition that is due to microvascular thrombi provoked by a transient imbalance between procoagulant and anticoagulant factors. It most commonly affects the breasts, buttocks and thighs. It may occur on initiation of warfarin, especially in patients with heterozygous protein C or protein S deficiency because of the relatively shorter half-lives of the vitamin K-dependent proteins C and S compared with prothrombin. It is also seen in association with heparin-induced thrombocytopenia. It is prevented by bridging the initiation of warfarin with heparin and avoiding high loading doses. Treatment requires discontinuation of warfarin and starting therapeutic heparin. Intravenous infusions of protein C concentrate may also be used in the short term. Surgical débridement may be required in extensive cases. 855

Postgraduate Haematology

Haemostatic dysfunction associated with paraproteinaemia and amyloidosis Paraproteinaemia Bleeding and thrombotic complications both occur in association with paraproteinaemias, although abnormalities in laboratory tests are found much more frequently than clinical effects. Bleeding is more common in Waldenström macroglobulinaemia and amyloidosis than multiple myeloma. Among myeloma patients, bleeding is most common in those with IgA paraproteinaemia. The circulating paraprotein in these conditions can (i) cause hyperviscosity and lead to arterial and retinal bleeds due to abnormal wall shear stress; (ii) inhibit or increase clearance of FVIII and VWF leading to von Willebrand disease or, less commonly, acquired haemophilia; (iii) impair platelet aggregation; (iv) inhibit fibrin polymerization; and (v) have heparin-like anticoagulant function that can be reversed by protamine. Management of these conditions is directed towards the underlying cause. Plasma exchange is of help as a temporary measure.

Amyloidosis The pathophysiology of bleeding associated with systemic amyloidosis is multifactorial. The type of amyloidosis and the pattern of organ involvement are important determinants of the haemorrhagic tendency. AL amyloidosis, where liver and spleen involvement is frequent, is the commonest type associated with bleeding. Although FX deficiency is the most commonly found, decreased levels of all other factors, including VWF, have been reported. The coagulation factor deficiencies in these patients are thought to be due to adsorption onto amyloid fibrils. Abnormal fibrin polymerization and hyperfibrinolysis can also contribute to bleeding. Prolongation of the TT is the most common abnormality, seen in up to 90% of patients. A prolonged PT and APTT suggests FX deficiency. Platelet dysfunction may also be seen, due to the binding of the amyloid light chains to the platelet membrane. Deposition of amyloid fibrils in the blood vessel wall and perivascular tissue may lead to impaired vasoconstriction and vessel fragility. This is exemplified by cerebral amyloid angiopathy, which can lead to intracerebral haemorrhage especially in elderly nonhypertensive individuals. The microvascular involvement also explains the ‘raccoon eyes’ (bilateral periorbital purpura from coughing or prolonged inverted positioning for lower gastrointestinal procedures) and the ‘pinch purpura’ (skin pinching leading to purpura). Several treatments have been described, including factor replacement with FFP or PCCs, platelet transfusion, DDAVP and rFVIIa. Severe FX deficiency can be difficult to manage and first-line therapy is with PCCs or FFP. 856

Acquired inhibitors of coagulation factors Inhibitory autoantibodies to all coagulation factors have been described, although those against FVIII and VWF are most common.

Acquired haemophilia A Acquired haemophilia A has an annual incidence of about 1.5 per million and usually affects older patients with a median age of about 75 years. It affects males and females equally, except in younger patients where there is a female preponderance associated with pregnancy. Acquired haemophilia A presents with a typical bleeding pattern that is distinct from that seen in congenital haemophilia. Widespread subcutaneous bleeding is common, as are muscle bleeds and gastrointestinal and genitourinary bleeding. Neurovascular compression may be limbthreatening. Haemarthroses are relatively uncommon. Fatal bleeding, such as intracranial, pulmonary, gastrointestinal and retroperitoneal, occurs in 8–22% of cases (Table 43.8). Patients remain at risk of severe bleeding until the inhibitor has been eradicated even if they present with mild bleeding. Acquired haemophilia A is associated with an underlying autoimmune (systemic lupus erythematosus or rheumatoid arthritis), malignant, lymphoproliferative or dermatological (pemphigoid) disease or pregnancy in about half of cases (Table 43.9). If acquired haemophilia A presents in association with pregnancy,

Table 43.8 Bleeding symptoms in patients with acquired haemophilia A.* Site of bleeding

Subcutaneous/skin Muscle Subcutaneous only Gastrointestinal/ intra-abdominal Genitourinary Retroperitoneal/thoracic Other Postoperative Joint None Intracranial haemorrhage Fatal

All bleeds at presentation (%)

Bleeds requiring treatment (%)

N = 172 81 45 24 23

N = 65 23 32 Not applicable 14

9 9 9 0 7 4 3 9

18 5 23 11 2 Not applicable 0 No data

*Patients had often had multiple bleeds. Source: data from Collins et al. (2007) and Morrison et al. (1993).

Acquired coagulation disorders

Table 43.9 Diseases associated with acquired haemophilia A.

Number of patients Idiopathic (%) Collagen, vascular and other autoimmune diseases (%) Malignancy (%) Skin diseases (%) Possible drug reaction (%) Pregnancy (%)

Green & Lechner (1981)

Morrison et al. (1993)

Collins et al. (2007)

215 46 18

65 55 17

150 63 17

7 5 6

12 2 3

15 3 0

7

11

2

Source: data from Green & Lechner (1981), Morrison et al. (1993) and Collins et al. (2007).

it may recur in subsequent pregnancies and, if antenatal, the fetus may be affected. Early diagnosis and urgent treatment of bleeding are key to successful management. The diagnosis is suggested by the clinical presentation and an isolated prolonged APTT. FVIII inhibitors are time and temperature dependent and in mixing studies normal plasma must be incubated for 1–2 hours otherwise the diagnosis may be missed. The diagnosis is confirmed by a low FVIII and positive Bethesda titre (Figure 43.6). In some cases diagnosis is complicated because all the intrinsic factors may be low due to inhibition of FVIII in the factor-deficient plasma used to assay other intrinsic factors. Dilution of the antibody will result in increased levels of the non-specifically reduced factors while FVIII remains low. It is important to exclude a lupus anticoagulant because this can also be associated with an apparently low FVIII and an APTT that does not correct with normal plasma. Acquired FVIII inhibitors have complex kinetics, so that not all FVIII is inhibited in a Bethesda assay. It is therefore often not possible to measure the inhibitor titre accu-

Assess personal and family history of bleeding

Isolated prolonged aPTT Confirm prolonged aPTT Exclude heparin contamination

Suspect coagulation factor deficiency or lupus anticoagulant (LA)

Mixing tests with pooled normal plasma (immediate and incubated)

aPTT correction

Weak/no aPTT correction

Suspect clotting factor deficiency

Suspect acquired haemophilia or LA Time and temperature dependent

Measure FVIII, IX, XI, XII

Not time and temperature dependent

Measure FVIII, IX, XI, XII and inhibitor

Negative

Single factor deficiency

Acquired haemophilia

Tests for LA

Positive

Lupus anticoagulant

Figure 43.6 Diagnostic algorithm for an isolated prolonged APTT and possible acquired haemophilia A. 857

Postgraduate Haematology

rately. The FVIII level and inhibitor titre are not predictive of the severity of the bleeding or response to treatment. Treatment involves minimizing invasive procedures, protecting the patient from trauma, treatment of bleeds and eradication of the inhibitor. At present, the available therapies to treat bleeds are with bypassing agents, namely rFVIIa or the activated PCC FEIBA (factor eight inhibitor bypassing activity). Both agents appear to be equally efficacious. Multiple doses may be required to control bleeds and prevent recurrence. There is a risk of thrombosis associated with bypassing agents, particularly in elderly patients with risk factors for arterial thrombosis. FVIII is unlikely to be efficacious in acquired haemophilia A, although mild bleeds in patients with low-titre inhibitors and measurable FVIII may respond to desmopressin. Porcine FVIII has been used with good success in the past but is no longer available. The results of trials with a recombinant B domaindeleted porcine factor are awaited. If standard haemostatic treatment with bypassing agents fails, FVIII and immunoadsorption has been used with good effect, although this treatment modality is only available in a few specialized centres. Immunosuppression should be started as soon as the diagnosis is made and is usually with either steroids alone or steroids plus a cytotoxic agent such as cyclophosphamide or azathioprine. A response is usually seen after 2–3 weeks but full remission takes a median of about 5 weeks. If first-line therapy fails, rituximab or combinations of cytotoxic agents may be used. Occasionally responses have been reported with ciclosporin. Intravenous immunoglobulin does not increase the response rate to other immunosuppressive agents.

Acquired von Willebrand disease Acquired von Willebrand disease may be associated with an autoantibody, typically in the context of monoclonal gammopathy of undetermined significance, Waldenström macroglobulinaemia and other lymphoproliferative diseases, myeloproliferative disease and systemic lupus erythematosus. The antibody leads to either rapid clearance or functional inhibition of VWF. Low VWF:Ag, VWF:RCo and VWF:CB are found and measurement of the VWF propeptide may be useful in demonstrating rapid clearance. Mixing studies can be used to demonstrate the inhibitor. Low VWF levels are also seen in hypothyroidism probably due to decreased synthesis, whereas increased VWF is associated with hyperthyroidism. Increased proteolysis of VWF in highshear environments such as leaking cardiac values leads to a syndrome of acquired type 2A von Willebrand disease. VWF has also been reported in association with malignancy and it is suggested that it may be adsorbed onto malignant cells. Hydroxyethyl starches used as plasma expanders have been associated with bleeding secondary to VWF deficiency. Treatment options include DDAVP, VWF concentrates and, in patients with an inhibitory antibody, high-dose intravenous immunoglobulin. 858

Acquired factor V deficiency Acquired inhibitors to FV may arise spontaneously but are usually associated with exposure to topical thrombin preparations that have trace amount of bovine FV. Laboratory findings are of prolonged PT and APTT that do not correct with normal plasma. Patients may respond to FFP and, in resistant cases, platelets may be useful because surface-bound FV appears to bypass the inhibitor.

Acquired protein S deficiency Autoantibodies to protein S have been reported in association with infection, particularly chickenpox. Patients present with skin necrosis and DIC.

Prothrombin deficiency associated with lupus anticoagulant Although lupus anticoagulants are typically associated with thrombosis, occasionally cross-reactivity with prothrombin can lead to a bleeding disorder. Acquired deficiencies of other procoagulant and antifibrinolytic proteins have occasionally been described. Bleeding manifestations are variable and some patients with markedly abnormal laboratory tests do not bleed.

Selected bibliography Bevan DH (1999) Cardiac bypass haemostasis: putting blood through the mill. British Journal of Haematology 104: 208–19. Collins PW, Hirsch S, Baglin TP et al. (2007) Acquired hemophilia A in the United Kingdom: a 2-year national surveillance study by the United Kingdom Haemophilia Centre Doctors’ Organisation. Blood 109: 1870–7. De Paepe A, Malfait F (2004) Bleeding and bruising in patients with Ehlers–Danlos syndrome and other collagen vascular disorders. British Journal of Haematology 127: 491–500. Eby C (2009) Pathogenesis and management of bleeding and thrombosis in plasma cell dyscrasias. British Journal of Haematology 145: 151–63. Falanga A, Rickles FR (2003) Pathogenesis and management of the bleeding diathesis in acute promyelocytic leukaemia. Best Practice and Research. Clinical Haematology 16: 463–82. Green D, Lechner K (1981) A survey of 215 non-hemophilic patients with inhibitors to Factor VIII. Thrombosis and Haemostasis 45: 200–3. Hess JR, Brohi K, Dutton RP (2008) The coagulopathy of trauma: a review of mechanisms. Journal of Trauma 65: 748–54. Hess JR, Lindell AL, Stansbury LG et al. (2009) The prevalence of abnormal results of conventional coagulation tests on admission to a trauma centre. Transfusion 49: 34–9. Huth-Kuhne A, Baudo F, Collins P et al. (2009) International recommendations on the diagnosis and treatment of patients with acquired haemophilia A. Haematologica 94: 566–75.

Acquired coagulation disorders

Levi M, Toh CH, Thachil J, Watson HG (2009) Diagnosis and management of disseminated intravascular coagulation. British Journal of Haematology 145: 24–33. Levy JH, Dutton RP, Hemphill JC 3rd, Shander A et al. (2010) Multidisciplinary approach to the challenge of hemostasis. Anesthesia and Analgesia 110: 354–64. Lisman T, Leebeek FW, de Groot PG (2002) Haemostatic abnormalities in patients with liver disease. Journal of Hepatology 37: 280–7. Morrison AE, Ludlam CA, Kessler C (1993) Use of porcine factor VIII in the treatment of patients with acquired hemophilia. Blood 81: 1513–20.

Noris M, Remuzzi G (1999) Uremic bleeding: closing the circle after 30 years of controversies? Blood 94: 2569–74. Stainsby D, Maclennan S, Thomas D, Isaac J, Hamilton PJ (2006) Guidelines on the management of massive blood loss. British Journal of Haematology 135: 634–41. Zangari M, Elice F, Fink L, Tricot G (2007) Hemostatic dysfunction in paraproteinemias and amyloidosis. Seminars in Thrombosis and Hemostasis 33: 339–49.

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CHAPTER 44

Thrombotic thrombocytopenic purpura and haemolytic–uraemic syndrome (congenital and acquired)

44

Pier M Mannucci, Flora Peyvandi and Roberta Palla A. Bianchi Bonomi Hemophilia and Thrombosis Center, IRCCS Cà Granada, Ospedale Maggiore, Milan, Italy

Historical introduction, 860 Thrombotic thrombocytopenic purpura, 862 Pathology and pathogenesis, 862 Clinical and laboratory findings, 863 Differential diagnosis, 865

Natural history, 866 Treatment, 866 Haemolytic–uraemic syndrome, 868 Diarrhoea-related HUS, 868 Familial HUS, 869

Historical introduction A brief historical sketch is useful for delineating the progress made in understanding these complex syndromes, which are difficult to diagnose. Thrombotic thrombocytopenic purpura (TTP) was first described in 1924 by Moschowitz in a previously healthy 16-year-old girl who died after an acute illness presenting with a pentad of signs and symptoms (anaemia, thrombocytopenia, fever, hemiparesis and haematuria). Post-mortem examination showed widespread thrombi in the terminal circulation of several organs, composed mainly of platelets. Over the next three decades, other cases were described, mainly, but not exclusively, in young or adult women. Some of these cases occurred in isolation (idiopathic), others in association with diseases or conditions (Table 44.1). It was understood that anaemia was due to massive intravascular haemolysis, in turn due to fragmentation (schistocytosis) of red cells that were forced by blood flow to pass through partially occluded vessels in the terminal circulation, and that thrombocytopenia was caused by consumption of platelets due to their widespread deposition in microvascular thrombi. Thirty-one years after Moschowitz, the paediatric nephrologist Gasser described a syndrome that was called ‘haemolytic– uraemic syndrome’ (HUS) and which, in common with TTP, exhibited microangiopathic haemolytic anaemia, consumption thrombocytopenia and microvascular thrombosis, but which

Postgraduate Haematology: 6th edition. Edited by A. Victor Hoffbrand, Daniel Catovsky, Edward G.D. Tuddenham, Anthony R. Green © 2011 Blackwell Publishing Ltd.

860

Atypical (non-diarrhoea-related) HUS, 869 Concluding remarks, 869 Selected bibliography, 870

differed because it occurred in very young children, with absent or minimal neurological symptoms but severe signs of renal damage. Subsequently, when larger series of patients with HUS and TTP were examined, it became apparent that a clear distinction between the two syndromes was often difficult. Although thrombocytopenia, microangiopathic haemolytic anaemia and ischaemic symptoms due to widespread formation of thrombi in the terminal circulation of several organs were consistent features, age and the prevalence of neurological over renal symptoms could not clearly differentiate TTP from HUS and vice versa. There were, for instance, recurrent forms which, in the same individual, presented sometimes with prevalent neurological symptoms (TTP) and on other occasions with renal symptoms (HUS). The term ‘thrombotic microangiopathy’ (TMA) was then proposed, intended to emphasize the common pathology of the two syndromes, with no implication on the prevalence of neurological or renal symptoms. In the early 1980s, a major breakthrough in understanding the pathogenesis of TMAs strengthened the idea of the unitarian terminology. Even though it had been postulated for a long time that massive thrombus formation in the terminal circulation was due to the presence of ‘toxic’ substance(s) that aggregated platelets intravascularly, the putative aggregating agent had remained elusive. In 1982, Moake and others demonstrated, first in TTP and subsequently also in HUS, that the plasma of these patients contained highly thrombogenic forms of the multimeric glycoprotein von Willebrand factor (VWF), a major adhesive protein contained in endothelial cells, platelets and plasma. Abnormal VWF multimers of particularly high molecular weight (ultra-large) bind more avidly to platelet glycoprotein (GP)Ib and aggregate platelets in conditions of high shear

Thrombotic thrombocytopenic purpura and haemolytic–uraemic syndrome (congenital and acquired)

stress in the terminal circulation. Accordingly, it was postulated that ultra-large VWF was the ‘toxic’ aggregating agent involved in the formation of occlusive thrombi in both TTP and HUS. It remained to be explained why such ultra-large multimers, normally absent in the circulation, were present in patient plasma. The deficiency or dysfunction of one or more enzymes disposing of them physiologically was postulated but it was not until the late 1990s that Furlan and Tsai with their associates

independently showed that the link between ultra-large VWF multimers and TTP was a metal ion-dependent plasma metalloproteinase of 190 kDa, identified in 2001 by Zheng and colleagues as a new (the 13th) member of the ADAMTS (a disintegrin and metalloprotease with thrombospondin-1 repeats) family of metalloproteases and therefore called ADAMTS-13 (Figure 44.1). The only known physiological function of this protease, present in plasma and in platelets, is to regulate the size of VWF by cleaving ultra-large multimers as soon as they are secreted from endothelial cells into plasma (Figure 44.2), thereby preventing their circulation in plasma and role in platelet aggregation and thrombus formation. Most importantly, both the investigators made the intriguing observation that ADAMTS-13 was deficient in patients with TTP but measurable in normal amounts in those with HUS. This observation challenged the unitarian theory of TTP and HUS as different clinical manifestations of the same pathological process, and generated the paradigm that TTP is due to low levels of the VWF-cleaving protease, which however is not involved in the pathogenesis of HUS. As often in clinical medicine, the paradigm was not sustained by the progress of knowledge. Not all cases of TMA diagnosed

Table 44.1 Conditions and diseases associated with TTP. Pregnancy and post partum Infections (particularly HIV) Drugs (quinine and quinidine, ticlopidine, clopidogrel, ciclosporin, interferon alfa, statins) Chemotherapy (mitomycin, cisplatin, gemcitabine) Allogeneic bone marrow transplantation Connective tissue disorders (lupus erythematosus and scleroderma) Cardiac surgery

ADAMTS-13

Figure 44.1 Domain structure of ADAMTS-13. Pro, propeptide; TSP1, thrombospondin 1; CUB, complement components C1r/C1s, urinary epidermal growth factor, bone morphogenetic protein-1.

A disintegrin and metalloprotease with thrombospondin-1 repeats Disintegrin Zn+

TSP1

Cys-rich

TSP1

TSP1

Pro

1427 aa CUB

Figure 44.2 Interaction between VWF and ADAMTS-13 in plasma. VWF is secreted from endothelial cells as ultra-large multimers that anchor to endothelial cell surfaces and which are also released into the circulation. ADAMTS-13 cleaves a Tyr–Met bond in the A2 domain of VWF, severing the ultra-large multimers. Failure of cleavage leads to the persistence in plasma and on endothelial cells of ultra-large multimers, which tend to aggregate platelets, especially in conditions of high shear forces.

Endothelial cells

Prepro-vWF monomer

Pro-vWF dimer Multimers of regular size vWF polymer

Secretion into plasma

Cleavage

Shear Platelet

Partially unfolded vWF Protease

No cleavage

Platelet

Unfolded vWF of ultralarge size

Platelet Platelet aggregates

861

Postgraduate Haematology

as TTP owing to the prevalence of neurological symptoms have low or undetectable levels of ADAMTS-13. On the other hand, even if most cases diagnosed as HUS due to the prevalence of renal symptoms have normal levels of ADAMTS-13 (particularly cases typically occurring in association with bloody diarrhoea), there are unequivocal cases of HUS, particularly the forms called atypical or diarrhoea-negative, characterized by low or undetectable protease levels. With this as historical background, we have decided to describe TTP separately from HUS in this chapter.

Thrombotic thrombocytopenic purpura TTP is a rare disease, with an estimated incidence of 2–10 cases per million per year in all racial groups. Recently, greater awareness and perhaps improved diagnostic facilities give the impression that incidence is increasing. Even though both sexes may be affected, the syndrome is definitely more frequent in women (two-thirds of cases). There are two different forms of TTP. Congenital TTP, caused by mutations in the ADAMTS-13 gene, is inherited as an autosomal recessive condition and is often, but not exclusively, clinically manifested at birth or during childhood. Acquired TTP can be differentiated into two types: autoimmune forms, due to autoantibodies against ADAMTS-13; and those probably secondary to massive endothelial activation with release of ultra-large VWF multimers in amounts exceeding the system’s ability to degrade them, despite the presence of normal or only slightly reduced levels of ADAMTS-13 as measured with the currently available functional assay. Both these pathogenic situations are usually triggered by factors that cause massive endothelial activation. The most common physiological or pathological conditions present in the autoimmune forms are pregnancy, infections, autoimmune diseases and the use of drugs such as ticlopidine and clopidogrel. The most frequent conditions associated with the forms of TTP with normal or only slightly reduced levels of ADAMTS-13 (conventionally defined as levels above 10%) are metastatic tumours, organ transplantation (particularly allogeneic bone marrow transplantation and solid organ transplants) and the use of such drugs as ciclosporin, mitomycin and interferon alfa. Mortality was very high (80–90%) until plasma exchange therapy was introduced, and is still unacceptably high (10– 20%) despite the dramatic improvement due to the adoption of this therapeutic measure. Because of the variability of presenting symptoms and of the associated comorbid conditions (see Table 44.1), cases of TTP may be seen initially by a variety of physicians other than haematologists and pathologists, such as neurologists, nephrologists, oncologists and gynaecologists, and sometimes this hinders prompt recognition of the syndrome, an essential requisite for optimal management. 862

Pathology and pathogenesis As mentioned above, the pathological basis of TTP is the widespread formation in the microcirculation of platelet thrombi, associated with relatively little endothelial cell injury and fibrin formation but with abundant intra-thrombus VWF. Microthrombi are found in several organs (mainly brain, kidney, myocardium, lung and pancreas), whereas grossly detectable thrombi, arterial or venous, are lacking. The present pathogenetic model implies that endothelial cells, activated by varied and often unidentified triggering agents, secrete large amounts of ultra-large uncleaved VWF, which aggregates platelets directly in conditions of high fluid shear stress and leads to massive intravascular platelet aggregation, ischaemic organ damage, consumptive thrombocytopenia and schistocytic anaemia. As mentioned above, two main mechanisms cause ADAMTS-13 deficiency in TTP: mutations in the gene that encodes the protease and the occurrence of autoantibodies against ADAMTS-13. Congenital TTP Congenital TTP is very rare (1 in 1 million) and represents 5% of all TTP cases. It usually occurs immediately after birth or during childhood, although some exceptions have been reported where the disease became manifest in adulthood or was still absent in the third decade of life. Congenital TTP is caused by homozygous or double heterozygous mutations in the ADAMTS13 gene (located on chromosome 9q34) that affect protein secretion or function; it is inherited in autosomal recessive manner. To date, more than 80 mutations have been documented in patients with familial TTP. Analysis of the location of the mutations on the ADAMTS13 gene reveals no evident hotspots. However, 75% of the missense mutations are found in the N-terminal part of the molecule, up to the spacer domain. Most (60%) of the identified mutations are missense; the remaining 40% (nonsense, frameshift and splicing mutations) generates truncated forms of the protein. Of those mutations analysed by in vitro expression studies (only 20–30% of the reported gene mutations), the majority cause severe ADAMTS13 deficiency by decreasing its biosynthesis, intacellular trafficking and secretion and/or proteolytic activity. Also the spectrum of clinical presentation and course of congenital TTP is very wide. While some patients have neonatal disease onset and multiple recurrent episodes and develop progressive organ failure, others may only experience a single episode that develops in adulthood and which is very responsive to plasma infusion, leaving no residual organ damage. Moreover, there are cases from the same kindreds who present with very different clinical manifestations despite the same undetectable or barely detectable ADAMTS-13 activity and gene mutations: sibs with frequent and repeated episodes of TTP contrast with others who remain disease-free for prolonged periods or even lifelong.

Thrombotic thrombocytopenic purpura and haemolytic–uraemic syndrome (congenital and acquired)

Autoimmune TTP Autoimmune TTP accounts for the majority of clinical cases and occurs in adults in the absence of any obvious triggering event. However, it can also arise secondary to other conditions, such as autoimmune diseases, infections, drug intake, pregnancy, sepsis, tumours and bone marrow transplantation. Autoimmune TTP is due to anti-ADAMTS-13 antibodies that inhibit the proteolytic activity of ADAMTS-13 and/or bind the protease to accelerate its clearance from plasma through opsonization and/or other yet unclear mechanisms. Anti-ADAMTS-13 antibodies are usually of IgG type, although in few cases autoantibodies of IgA and/or IgM isotype were also found. Epitope mapping of anti-ADAMTS-13 antibodies showed that autoantibodies found in the majority of patients (88%) are polyclonal (IgG4), with a primary epitope in the Cys-rich spacer. Indeed these domains have been shown to be essential for efficient VWF-cleaving activity of ADAMTS-13 and crucial in the interaction between VWF and the protease. However, subsequent studies have shown that cases of TTP are observed in patients with IgG molecules that also react with the TSP1 2–8 and CUB domains of ADAMTS-13, suggesting that autoantibodies with specificity towards more distal C-terminal domains of ADAMTS-13 may impair its function in vivo, probably taking part in the modulation of VWF binding. The higher incidence of autoimmune idiopathic TTP in specific ethnic groups such as Afro-Caribbeans, as well as the report of idiopathic TTP in two monozygotic twins both developing anti-ADAMTS-13 antibodies, strongly argue for a genetic predisposition even in the acquired form of the disease. However, several observations fail to fit perfectly with this relatively straightforward mechanism of disease. Not all patients with bona fide TTP, as diagnosed by clinical criteria (thrombocytopenia and anaemia), have severe ADAMTS-13 deficiency and patients can present the characteristic features and clinical course of TTP without severe ADAMTS-13 deficiency or even with normal ADAMTS-13 activity levels. This may be explained by the inability of the functional assays available at present to reveal some types of ADAMTS-13 deficiency and/or by deficiency of VWF-cleaving proteases other than ADAMTS-13. Ultra-large VWF multimers, pivotal players in the presently accepted model of TTP, are not constantly detected in patient plasma. In some instances, there is an imbalance between their release into plasma from endothelial cells and excessive binding to platelets, so that even defective large multimers may be present in plasma. It is unclear why patients with sustained ADAMTS-13 deficiency, genetic or immunologically mediated, develop clinical symptoms and signs only sporadically. Still unknown factors that trigger excessive endothelial cell activation may be necessary for expression of the full clinical spectrum of the syndrome. In summary, the prevailing paradigm holds that TTP is often the consequence of the defective processing of highly thrombogenic ultra-large multimers of VWF, which are secreted

in excess by endothelial cells and which are not adequately disposed of because of congenital or immune-mediated dysfunction of ADAMTS-13 (see Figure 44.2). There are data suggesting that other possible mechanisms of disease, other than VWF and its cleaving processes, may be involved in the pathogenesis of TTP.

Clinical and laboratory findings TTP occurs frequently, unheralded, in previously healthy individuals (acute idiopathic), but also in association with physiological or pathological conditions (see Table 44.1). The presence of thrombocytopenia and haemolytic anaemia, common to all TMAs, is essential for the diagnosis of TTP. Like HUS, TTP is a clinical diagnosis and biopsies are not necessary. Platelet count is often very low in the acute phase, with values of 20 × 109/L or less, and petechiae are frequently seen. Signs of microangiopathic haemolytic anaemia (haematocrit usually 1.5 mg/dL), signs of renal involvement such as microscopic haematuria and proteinuria are frequent. High fever is not constant in TTP, despite the inclusion of this symptom in the original description. There is no or little alteration of coagulation and fibrinolysis, even though D-dimer levels are often moderately raised. ADAMTS-13 assays The possibility of using ADAMTS-13 data to manage TTP patients is based on the widespread availability of assays that are rapid, reliable and feasible for most clinical laboratories. Such tests includes assays of ADAMTS-13 activity, ADAMTS-13 antigen and neutralizing or non-neutralizing anti-ADAMTS-13 autoantibodies. Several assays of ADAMTS-13 activity have been developed. They are based on the degradation of purified, plasma-derived or recombinant VWF multimers or of synthetic VWF peptides by patient plasma and the direct or indirect detection of VWF cleavage products by ADAMTS-13 (Figure 44.3). A number of variables may interfere with assay results. 863

Postgraduate Haematology

1st step

Digestion of VWF substrate by ADAMTS13 L1591 R1668 78aa VWF multimers

G1481

R1668

D1596 R1668 73aa

A2

Dilutions of patient’s plasma (source of ADAMTS13) Addition or not of denaturing agents

Incubation (1–24 hours)

Direct or indirect measurement of ADAMTS13 activity by different techniques

1 All the assays measure ADAMTS-13 activity in static conditions and thus do not reflect the in vivo physiological blood flow conditions necessary for optimal ADAMTS-13 enzymatic activity. 2 Another important variable is that denaturating agents (i.e. guanidine HCl or 1.5 mol/L urea) are required to promote the susceptibility of VWF multimers to cleavage by ADAMTS-13. 3 The use of shorter peptides, instead of full-length VWF, in enzyme immunoassay-based methods has not solved the issue of intra- and inter-laboratory variability. Immunoenzymatic assays employing different monoclonal and polyclonal antibodies to monitor plasma antigen levels of ADAMTS-13 are available. With regard to the detection of anti-ADAMTS-13 autoantibodies, most of them are inhibitory and they can be titrated in vitro by classical mixing studies, using mixtures of heatinactivated plasma from patients and normal plasma at 1 : 1 dilution or several dilutions. Less frequently, autoantibodies are non-neutralizing and promote the clearance of ADAMTS13 from blood without inhibiting its activity. These nonneutralizing antibodies can be detected by Western blotting or ELISA assays. Clinical utility of ADAMTS-13 testing Analysis of a large cohort of patients with congenital TTP has revealed an age-dependent clustering of cases into two relatively distinct groups. As already mentioned before, about 50% of patients present within the first 5 years of life, but a second group remains without symptoms until 20–40 years of age (Figure 44.4). Examples of siblings with the same ADAMTS-13 mutations but markedly different clinical courses have also been reported. Whereas the first studies by Tsai and Lian and by Furlan and colleagues reported that all patients with acquired TTP had severe ADAMTS-13 deficiency, subsequent studies have shown that only a proportion of patients, ranging from 13 to 70%, develop TTP concomitant with severe ADAMTS-13 864

100 80

Per cent

2nd step

Figure 44.3 Principles of methods for the measurement of ADAMTS-13 activity. VWF multimers picture from Siedlecki CA, Lestini BJ, KottkeMarchant K et al. (1996) Sheardependent changes in the three-dimensional structure of human von Willebrand factor. Blood 88: 2939–50.

Substrate (source of VWF)

60 40 20 0

Figure 44.4 Age of disease onset in patients affected by congenital TTP showing percentages of patients (N = 78) with different age of TTP onset. Red indicates neonatal onset, orange intermediate onset (2 months to 18 years), green adult onset (>18 years) and light blue patients who reached adulthood without developing TTP episodes.

deficiency, thus challenging the previous observations that severely deficient activity of VWF-cleaving protease is a specific diagnostic marker for discriminating TTP from other microangiopathies. The current prevailing opinion is that while undetectable or very low plasma levels of enzymatic activity ( 40 GPLU are considered to be moderate-titre antibodies; >99th percentile has been employed also. The significance of low-titre antibodies is less clear. 892

laboratory features are variably present in APS. These include mild thrombocytopenia and an unusual dermatological feature, livedo reticularis. Cardiac valvular abnormalities occur in up to 30% of patients, but are usually subclinical. Most commonly the mitral or aortic valve is affected, often with valve thickening or incompetence. However, haemodynamic changes as a consequence of valvular damage are rare. Exceptionally, the syndrome may manifest as widespread microvascular occlusion, with multiorgan failure, so-called catastrophic APS. APS may occur with another chronic systemic autoimmune disease, usually systemic lupus erythematosus (SLE), when the term ‘secondary antiphospholipid syndrome’ is used. In primary APS there is no evidence for another relevant underlying condition.

Antiphospholipid antibodies Antiphospholipid antibodies are a family of antibodies reactive with proteins that have the property of binding to negatively charged phospholipids. The most important is β2-glycoprotein I (β2-GPI), as pathogenic antibodies (i.e. those most strongly associated with clinical events) are most frequently reactive with this protein. β2-GPI is a member of the complement control protein family. It has five domains. It is now known that antiphospholipid antibodies reactive with each of these five domains may occur but it is only those which recognize a specific epitope on domain I that are pathogenic. Antibodies to other proteins which have affinity for negatively charged phospholipid have been implicated in the syndrome, including protein C, protein S and annexin V. The lupus anticoagulant (LA) is an in vitro phenomenon in which the antiphospholipid antibody slows clot formation, thereby lengthening the clotting time. This is probably due to impairment of the assembly of the components of prothrombinase on phospholipid due to interference by the antibody (Figure 46.1). LA is due to antibodies reactive to β2-GPI/

Phospholipidbinding protein

Prothrombin

Platelet

Figure 46.1 Antiphospholipid antibody binding: explanation for the lupus anticoagulant phenomenon.

Acquired venous thrombosis

phospholipid or to prothrombin/phospholipid. The β2-GPIdependent antibodies also bind in anticardiolipin assays, as the glycoprotein is present in test serum and often in assay reagents. LA due to prothrombin-reactive antibodies may be negative in anticardiolipin assays. Therefore some subjects with APS have LA and anticardiolipin and some LA only. Others have anticardiolipin without LA, due to the presence of non-β2-GPI and non-prothrombin-dependent antibody or possibly to relative insensitivity of the coagulation assays for LA. Because antiphospholipid antibodies are so heterogeneous, a comprehensive laboratory approach is essential for their reliable detection. In most laboratories, ELISAs employing cardiolipin and coagulation-based assays for LA remain the principal diagnostic tools. ELISA for β2-GPI may offer improved specificity. The pathogenicity of IgA anticardiolipin antibodies is disputed and their detection is not utilized routinely in diagnosis.

Pathogenic mechanisms in APS The pathogenesis of pregnancy failure and thrombosis in APS is not fully understood. Animal experiments support a pathogenic role for antibodies to β2-GPI. Important candidate mechanisms for thrombosis are antibody-induced concentration of prothrombin on phospholipid surfaces in vivo resulting in enhanced thrombin generation, interference with the activated protein C anticoagulant pathway, increased von Willebrand factor activity, and increased monocyte and endothelial tissue factor expression. Increased platelet activation and inhibition of fibrinolysis by antiphospholipid antibodies have also been proposed. In relation to pregnancy failure, again numerous pathogenic mechanisms have been proposed. It is noteworthy that early miscarriage is a common feature and occurs before full placentation. This raises doubts whether a thrombotic mechanism is causal. Therefore, it is of considerable interest that a body of evidence is accumulating for a role of complement activation in pregnancy loss in APS. Of particular note in this context, heparin is effective in preventing embryo loss in a murine model of APS and it does so through inhibition of complement rather than via an anticoagulant mechanism. This raises the possibility that similar mechanisms may be relevant to early pregnancy loss in women with APS. Placental dysfunction is the hallmark of second- and third-trimester complications of intrauterine fetal death, intrauterine growth restriction, pre-eclampsia and placental abruption. The aetiology of the placental dysfunction has been related to placental infarction and an acute atherosis in the maternal spiral arteries, although these are not universal features. One interesting hypothesis for the cause of placental insufficiency is displacement of annexin V from trophoblast by antiphospholipid antibodies, with resultant acceleration of thrombin generation on the exposed negatively charged phospholipid.

Laboratory diagnosis of antiphospholipid antibodies In diagnosing APS it is essential to consider that antiphospholipid antibodies are not specific to the syndrome. In addition to transient antibodies, which may for example be triggered by intercurrent infection, some chronic infections are associated with antiphospholipid antibodies, such as syphilis and hepatitis C. Antiphospholipid antibodies may also be detected incidentally in healthy subjects and they occur in relation to use of some drugs, particularly chlorpromazine. These drug-induced and infection-related antibodies do not usually appear to be associated with the clinical thrombotic manifestations of APS. In some cases they appear to be neither β2-GPI nor prothrombin dependent. The diagnosis of APS relies on the demonstration of the persistent presence of either LA by coagulation tests or of antiphospholipid antibodies by solid-phase immunoassays for anticardiolipin and/or β2-GPI. Reliance on just one type of assay may lead to false-negative assessment of antiphospholipid antibodies. Overall, among the commonly applied assays, it appears that those for LA associate most strongly with clinical events. In relation to solid-phase assays, high-titre antibodies and IgG antibodies associate more strongly with clinical manifestations than do IgM and lower-titre antibodies. Coagulation assays (lupus anticoagulant tests) The LA assay is a double misnomer for it is neither a test for SLE nor for an in vivo anticoagulant. LA tests are indirect assays that rely on slowing of the clotting time of plasma through interference by antiphospholipid antibodies. The tests most frequently employed are the activated partial thromboplastin time (APTT), the dilute Russell’s viper venom time (DRVVT) and, less frequently now, the kaolin clotting time (KCT). Platelet activation causes exposure of negatively charged phospholipid at the cell surface and therefore contamination of test plasma with platelets must be minimized, as these will limit the sensitivity of tests particularly when plasma must be stored frozen prior to testing. Platelet depletion may be achieved in various ways, most commonly by careful double centrifugation. None of the above coagulation assays is specific for LA. Specificity and sensitivity are also reagent dependent. For example, some partial thromboplastin reagents are insensitive to LA. If the same reagent is employed for LA tests as in routine laboratory screening for coagulopathy, sensitivity to LA must be assured. Factors that lengthen or shorten clotting times (other than antiphospholipid antibodies) potentially intefere in LA tests. Examples are anticoagulant drugs and clotting factor deficiencies and inhibitors, which lengthen clotting times, and increased clotting factor levels, especially FVIII, which shortens the time to clotting in the APTT for example, potentially masking the presence of LA. 893

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In order to reduce the risks of false interpretation, in addition to prolongation of clotting time in a phospholipid-dependent coagulation test, the criteria for LA positivity also include (i) evidence of an inhibitor demonstrated by mixing studies and (ii) confirmation of the phospholipid-dependent nature of the inhibitor. In principle, laboratory tests should employ a detection or screening stage (prolongation of the clotting time) and a confirmation stage showing (i) failure of correction of the prolongation when normal plasma is added in order to exclude factor deficiency as the cause of the prolongation and (ii) that the prolongation is phospholipid dependent, for example by showing that addition of excess phospholipid corrects the clotting time. To achieve this, and because no LA test consistently shows 100% specificity and sensitivity, more than one test system should be used for detection of LA. The prothrombin time and thrombin time should also be performed as they are not usually affected by the presence of LA and the results assist in the interpretation of LA tests, for example when there is undisclosed anticoagulant therapy. The APTT is commonly employed as the initial screening test for LA. Its specificity for inhibitor detection is improved by inclusion of a mixing study with platelet-free normal pooled plasma. When prolongation of the APTT is due to coagulation factor deficiency, the clotting time corrects when the test is repeated on an equal mixture of patient and normal plasma, whereas the prolongation above normal may persist with LA, consistent with its inhibitory activity. However, correction in a mixing study does not exclude LA, as a weak antibody is obviously diluted out by addition of normal plasma and this may be sufficient to abolish its effect. Very occasionally LA causes enhancement of the prolongation of the APTT when normal plasma is added. This phenomenon has been called the lupus cofactor effect but is the exception rather than the rule. Inhibitors to clotting factors, usually FVIII, are associated with bleeding rather than thrombosis but also cause prolongation of the clotting times which may not be corrected by addition of normal plasma. Typically, FVIII inhibitors are time-dependent, unlike LA. Diagnostic confusion may arise due to LA that prolongs the APTT, causing erroneously low results in coagulation factor assays based on the APTT. A normal APTT is insufficient to exclude LA and additional tests must be performed. The DRVVT does not involve the clotting factors of the extrinsic system, unlike the APTT, nor FVII, unlike the prothrombin time. Any inhibition of coagulant-active phospholipid in the test by LA results in a prolonged DRVVT. However, as is the case with all LA tests, it is not specific. Deficiencies of clotting factors, for example FII and FX due to warfarin therapy, will also prolong the DRVVT. The specificity of the test is improved by repeating it in the presence of a high concentration of phospholipid, which should result in partial or complete correction of the prolonged clotting time if it is due to LA. This phospholipid is conveniently provided as platelet membranes in which negatively charged phospholipid is exposed by freezing 894

and thawing. In the presence of LA, the ratio of test to normal plasma clotting time is often in excess of 1.2, and corrects to less than 1.2, or at least partially, in the platelet neutralization procedure. As with all coagulation tests, because of variations in reagents and techniques it is essential that laboratories derive local normal ranges using a large number of plasma samples from healthy volunteers. In the KCT no additional phospholipid is employed. The test therefore resembles the APTT in that it involves the extrinsic and common pathways of coagulation but the sensitivity to LA is enhanced because the small amount of phospholipid present is only that derived from residual platelets in the test sample and plasma lipids. The test is affected by clotting factor deficiencies and anticoagulants, but specificity can be improved by use of normal plasma mixing at more than one ratio to test plasma. LA is identified when the KCT fails to correct even after relatively large proportions of normal plasma are added, whereas in factor deficiency the KCT is corrected with small amounts of normal plasma. Alternative tests for LA may be employed but are not in general use. They include the tissue thromboplastin inhibition test and clotting tests that use venoms other than Russell’s viper venom. Examples are Taipan and Textarin venoms. Many laboratories rely on commercial assay kits for LA testing. It is essential that steps are taken to ensure internal and external quality assurance. Solid-phase assays (for anticardiolipin and anti-β2-GPI antibodies) Solid-phase assays for antiphospholipid antibodies, such as the anticardiolipin ELISA, allow rapid processing of numerous serum samples and the results are not affected by factor deficiency or the use of anticoagulants. The introduction of international standards allows the calculation of anticardiolipin results in IgG or IgM antiphospholipid units (GPLU and MPLU, respectively) related to a given concentration of affinitypurified anticardiolipin immunoglobulin. Despite this, there remains a lack of precision, and comparability between laboratories using different assays is not ensured. Clinicians should be aware of the performance of the assay in use. The detection of anticardiolipin allows the diagnosis of APS in a subject with an appropriate clinical history, even when LA is absent. However, the anticardiolipin assay is not a substitute for the LA test, nor does it confirm that LA is present due to the antibody heterogeneity referred to above. Furthermore, the clinical significance of low-titre anticardiolipin is doubtful. Thus, in cases where the anticardiolipin titre is less than 30 GPLU and tests for LA are negative, a diagnosis of APS may not be conclusive. Under these circumstances it is particularly important to consider other causes of thrombosis or miscarriage. Specific assays for anti-β2-GPI antibodies have been developed, and several commercial kits are available. Anti-β2-GPI

Acquired venous thrombosis

antibody assays may show higher precision and better correlation with the thromboembolic complications in APS and SLE than assays for anticardiolipin, and are less likely to show transient positive results in association with infection. Antiprothrombin antibodies generally exhibit poor specificity for venous thrombosis and recurrent fetal loss, and may be found in patients with infection, and are not included in the consensus criteria for diagnosis of APS. The prevalence of antiphospholipid antibodies in subjects with thrombosis varies with selection criteria for testing. Because the risk of recurrent thrombosis appears to be great, antiphospholipid antibodies should be sought in subjects with arterial, venous or microvascular thrombosis where no other cause is apparent. Examples are younger subjects with ischaemic stroke in the absence of cardiovascular disease and subjects with unprovoked VTE. The prevalence of persistent antiphospholipid antibodies among women with recurrent first-trimester miscarriage is around 15%, although a proportion of these are low-titre antibodies. In women with recurrent miscarriage due to APS the prospective fetal loss rate may be as high as 90%. In contrast, the prevalence of positive tests for antiphospholipid antibodies in unselected women of childbearing age is around 3% and they are not sensitive predictors of poor pregnancy outcome in women with no history of pregnancy complications. Because miscarriage is a common phenomenon, screening for antiphospholipid antibodies is not indicated after a single event. Maternal antiphospholipid antibodies may be downregulated during pregnancy, so tests are best performed preconceptually when possible. A small proportion of women with antiphospholipid antibodies also have anti-Ro antibodies. Their detection is important as anti-Ro is associated with a 2% risk of complete heart block in the fetus and a 10% chance of neonatal lupus.

Management of APS Thrombosis There is wide variability in severity of prothrombotic states between individuals with APS. The management of patients with antiphospholipid antibodies and previous thrombosis remains contentious. Retrospective observational studies suggested that these patients should remain on indefinite oral anticoagulation, maintaining an International Normalized Ratio (INR) of 3–4. However, subsequent prospective randomized studies indicate that a lower target INR of 2–3 is effective in preventing recurrent thrombosis in the majority of patients with previous venous thrombosis. There is less certainty in arterial thrombosis; for example, clinical observation suggests that there is a significant subpopulation of APS with small-vessel thrombosis, some evident as lacunar infarcts on magnetic resonance imaging, who appear to require a target INR of 3–4 to prevent recurrent cerebral thrombosis.

Immunosuppressive therapy is not generally indicated in primary APS other than in the very rare case where thrombosis recurs despite intensive anticoagulant therapy. Corticosteroids and other immunomodulatory therapies have been administered. An exception may be catastrophic APS when combination treatment with antithrombotics, corticosteroids and other immunomodulatory therapies such as rituximab are administered as potentially life-saving emergency measures. Pregnancy The management of recurrent fetal loss is based on the use of anticoagulation with empirical doses of heparin, usually lowmolecular-weight heparin in the UK, often in combination with low-dose aspirin. Initial studies indicated that this approach increases the chance of a successful outcome of a healthy live birth from 30 to 70–80%, although the total number of cases in randomized studies is limited and trials have given conflicting results. Nevertheless, low-molecular-weight heparin prophylaxis has become standard, although some clinicians believe that combination antithrombotic therapy as a first line should be reserved for women with a previous history of fetal death while those with recurrent miscarriage should be given supporting care as first line, with aspirin or aspirin/heparin reserved for those with further pregnancy failure. Corticosteroids are ineffective and their use is associated with frequent maternal morbidity from hypertension, glucose intolerance and premature labour. Thrombophylaxis for those with APS and a previous history of thrombosis is based again on use of low-molecular-weight heparin, although there is neither consensus on dosing nor the need to monitor therapy.

Venous thromboembolism and cancer Cancer is a major risk factor for VTE. VTE is the major cause of morbidity and mortality after the cancer itself in patients with malignancy. It is estimated that almost 15% of cancer patients will have a thromboembolic event. Post-mortem studies have shown that thromboembolism is common in patients who die of cancer. Of all oncology patients, palliativecare inpatients have the highest risk of VTE with a DVT prevalence that may be as high as 50%, including bilateral deep vein thromboses. Chemotherapy may also increase that risk: thalidomide is a prominent example. The risk of thrombosis varies with type of cancer, ovarian, brain and pancreatic cancers having the highest rates. Lymphomas and leukaemias account for a significant proportion of thromboses as do colonic and lung cancers. In Trousseau syndrome, the cancer-related phlebitis is recurrent and migratory and affects both the superficial and deep veins, in contrast to the more common association of lower limb DVT in cancer. Unusual sites such as arms and neck, as 895

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well as superficial veins of the thorax and abdomen may be involved. Typically, such a patient has an occult tumour, usually adenocarcinoma, although it is also a feature of acute leukaemia. In Trousseau syndrome, conventional anticoagulation often fails to prevent recurrent thromboses.

Prothrombotic changes associated with malignancies In Table 46.3 the components of Virchow’s triad are used to illustrate the features that lead to oncology patients having such a high risk of thrombosis. There are specific abnormalities unique to cancer patients, the so-called tumour procoagulants. Tumour procoagulants are surface molecules on cancer cells that activate coagulation. The two principal tumour cell procoagulants are tissue factor and cancer procoagulant. The latter directly activates FX without involvement of FVII. The increasing number of tumour procoagulants that have been described are listed on the International Society for Thrombosis and Haemostasis registry. APS has also been reported in malignancy, particularly in association with paraproteins.

Difficulties in management Cancer patients with thrombosis are at particularly high risk of recurrent thrombotic events. Cohort and population-based studies have shown that the risk of recurrence after stopping anticoagulation in cancer patients is approximately double that in non-cancer patients. Treatment with anticoagulation in cancer patients is often unsuccessful, due to the high risk of recurrent VTE and major bleeding. Unfortunately, these risks are associated with oral anticoagulation intensities within the usual therapeutic range. The risks are greatest during the first

Table 46.3 Some pathogenic factors for thrombosis in cancer patients. Stasis Immobility Extrinsic pressure, e.g. oedematous limbs Vessel wall/endothelial perturbation Cytokine release from tumours Local tumour infiltration Central venous catheter Hypercoagulability Dehydration Cytokine-related prothrombotic changes Tissue factor/cancer procoagulant expression on tumour cells Disseminated intravascular coagulation Increased platelet activation

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few weeks of anticoagulation treatment and increase with cancer extent. Several studies have demonstrated significant increases in rates of bleeding, as high as 20% among cancer patients receiving oral anticoagulation. Within the palliative care setting, where patients have far more advanced disease, the bleeding incidence was higher, even with strict monitoring of anticoagulation. Oral anticoagulation in cancer patients is complicated further due to anorexia, vomiting, liver impairment and drug interactions. Moreover there may be frequent interruption to treatment due to thrombocytopenia and the need for invasive procedures, and venous access is often difficult in these patients especially if they have had previous chemotherapy through peripheral veins. The decision to use oral anticoagulation in this group of patients should not be taken lightly and safe treatment requires intensive monitoring of INR and the burden of repeated blood tests. Disillusionment with the use of oral anticoagulation in cancer patients has led to trials assessing low-molecular-weight heparins in cancer. The CLOT trial randomized over 650 cancer patients with symptomatic proximal DVT and/or pulmonary embolism to treatment with dalteparin for 5–7 days and then randomization to either oral anticoagulation maintaining a target INR of 2.5 or dalteparin. In the dalteparin arm a full treatment dose was given for 1 month and then reduced to 75–80% of this dose. After 6 months there was a significantly reduced rate of recurrent VTE in the dalteparin group of 8.8% compared with 17.4% in the group with oral anticoagulation. Bleeding was also significantly reduced. A similar reduction in VTE was found in patients with cancer who entered the LITE study where treatment dose tinzaparin was compared with oral anticoagulation after VTE. These data support the use of lowmolecular-weight heparin rather than warfarin in patients with cancer and VTE.

Thrombotic risk in polycythaemia rubra vera and essential (primary) thrombocythaemia If left untreated, patients with polycythaemia vera (PV) have a median survival of 18 months, with the majority dying of vascular occlusion. The occlusive lesions may involve the larger vessels or the microvasculature. In PV, large-vessel events involve arteries and veins equally whereas in essential thrombocythaemia (ET) arteries are more commonly involved. A high proportion of thromboses occur in the cerebral circulation, although thrombosis widely distributed in the arterial and venous systems, including the splanchnic vessels, has been well documented. Hepatic vein and portal vein thromboses are more commonly associated with PV than ET. Indeed it has been reported that some patients with apparently idiopathic hepatic or portal vein thrombosis have subclinical myeloproliferative disease based on the finding of erythropoietin-independent erythroid colony growth in marrow culture or presence of JAK2 mutation. In PV there is good evidence that the incidence of

Acquired venous thrombosis

vascular occlusion is positively related to the packed cell volume, with the lowest incidence in patients with good control of haematocrit. The role of thrombocytosis in risk of thrombosis in PV is disputed. The risk of thrombosis in PV also increases with advancing age and a past history of thrombosis. ET is often diagnosed in the subclinical phase but a number of studies suggest that overall survival is determined principally by occurrence of thrombosis. In one study the rate of thrombosis ranged from around 2% per patient-year in those aged less than 40 to 15% in those aged greater than 60. In around 50% of cases of ET. the JAK2 V617F mutation is detectable. Whether subjects with the mutation are more likely to develop thrombosis is a subject of current investigation. The pathogenesis of thrombosis in ET is poorly understood. Elevated platelet counts are implicated, with platelet counts greater than 600 × 109/L being associated with increased incidence of vascular occlusion. Although treatment to reduce abnormal counts will reduce the frequency of events, a proportion of patients will still experience thrombosis. In addition, some patients with ET will remain thrombosis-free with no treatment to reduce their platelet count. The microvascular events and vasomotor manifestations of both PV and ET almost certainly relate to quantitive and qualitative changes in platelets, for they are not seen in other forms of polycythaemia or thrombocytosis. It is possible that similar small-vessel occlusive and vasomotor changes seen in the feet and hands may also occur in other parts of the body. Erythromelalgia, a syndrome consisting of painful burning red extremities with normal peripheral pulses, is the characteristic vasomotor disturbance. Physical findings may be absent or there may be warmth, duskiness and mottled erythema of the involved areas. Livedo reticularis is occasionally found. In the digital vessels, usually of the toes but occasionally the fingers, the development of thrombosis can lead to digital ischaemia and gangrene. In relation to the cerebral circulation, a range of symptoms and signs including transient cerebral ischaemia, transient monocular blindness, migraine, headaches and seizures are seen. Both the cerebrovascular complications and erythromelalgia of myeloproliferative disease may respond promptly to low-dose aspirin and/or platelet cytoreduction. Conventional risk factors for cardiovascular disease are implicated in the pathogenesis of thrombosis in ET, including hypertension, smoking, hypercholesterolaemia and diabetes, and should be considered in deciding whether cytoreductive therapy is indicated.

Acute promyelocytic leukaemia In comparison with the severe haemorrhagic presentation in the majority of cases of acute promyelocytic leukaemia (APL), acute arterial thrombosis is a rare presenting feature. Thrombosis is associated with the microgranular variant of APL, which accounts for 25% of all cases, characterized by a paucity of

myeloid granules and a lobulated monocytoid nucleus. The cells stain positive with Sudan black and myeloperoxidase and there is a high incidence of CD34 positivity. Vascular occlusion is more common when a high white cell count (> 150 × 109/L) predisposes to leucostasis. It is thought that the leukaemic cells release procoagulants. It is important to recognize this variant of APL because the use of all-trans retinoic acid may increase the risk of thrombosis due to a further increase in peripheral white cell count exacerbating leucostasis.

Inflammation and thrombosis Systemic inflammation is a potent prothrombotic stimulus (Table 46.4). Inflammation upregulates procoagulant factors, downregulates physiological anticoagulants, inhibits fibrinolytic activity and increases the platelet count. Inflammatory mediators promote coagulation by causing endothelial cell activation and increasing the expression of tissue factor. Endotoxin, tumour necrosis factor (TNF) and interferon-1α induce tissue factor expression primarily on monocytes/macrophages and probably in atherosclerotic plaques as well. Thrombin, a key enzyme in coagulation, also has cytokine-like activities for it augments leucocyte adhesion and can activate endothelium, leucocytes and platelets. Thrombin-mediated signalling through protease-activated receptor (PAR)-1 on vascular cells has recently been shown to mediate production of chemokines such as MCP-1, leading to recruitment of inflammatory cells including monocytes. Activation of these cells by thrombin increases the expression of negatively charged phospholipids such as phosphatidylserine on the surface of the cells, promoting surface procoagulant activity. Interleukin (IL)-6 and its family of molecules mediate the acute-phase response. This increases liver synthesis of plasma proteins including fibrinogen and other coagulation proteins, Table 46.4 Effects of inflammation on haemostasis. Increased Tissue factor expression Surface procoagulant activity, negatively charged phospholipid Platelet reactivity Levels of fibrinogen and other coagulation proteins Decreased Thrombomodulin expression Endothelial cell protein C receptor Half-life of activated protein C Protein Z Fibrinolytic activity due to increased PAI-1 Endothelial gylcosaminoglycans PAI, plasminogen activator inhibitor. 897

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thus priming the blood coagulation system for action. IL-6 also increases platelet reactivity. Plasma levels of the recently discovered vitamin K-dependent anticoagulant protein Z, which acts by inhibiting FXa, fall during inflammation; thus protein Z appears to be a negative acute-phase protein. Of all the physiological anticoagulant pathways, the protein C pathway appears to be the most influenced by inflammation. Thrombomodulin and endothelial cell protein C receptor are both downregulated by inflammatory cytokines such as TNF-α. There is inhibition of the promoter of the thrombomodulin gene as well as active pinocytosis to remove existing surface molecules, while neutrophil elastase readily cleaves thrombomodulin from the endothelial cell surface. Moreover, thrombomodulin is very sensitive to oxidation of an exposed methionine by oxidants produced by leucocytes. These observations have led to studies demonstrating apparent therapeutic value of activated protein C in subjects with multiorgan failure due to severe sepsis. Endothelial cell activation occurs in inflammation. This causes downregulation of fibrinolytic activation by increased production of plasminogen activator inhibitor (PAI)-1. There may also be cleavage of glycosaminoglycans from the surface of the endothelium, so that there is loss of heparan sulphate and other molecules that activate antithrombin. As a consequence of these changes, conditions that provoke an inflammatory response are associated with increased risk of venous thrombosis. These include inflammatory bowel disease, Behçet disease, systemic tuberculosis, SLE and diabetes. Atherosclerosis can be considered a chronic inflammatory state and has recently been associated with an increased incidence of venous thrombosis and, more recently, the metabolic syndrome has been implicated. Furthermore, epidemiological studies indicate an excess of minor illness, including infections, in the weeks preceding episodes of VTE. The association between inflammation and thrombosis emphasizes the need for thrombophylaxis in patients hospitalized on medical wards. Implementation of pharmacological thrombophylaxis has been inadequate in such patients.

Haematological prothrombotic states due to non-malignant diseases of the blood and bone marrow

est site and affects 15–25% of patients with PNH. Presentation is with pain in the right upper quadrant, jaundice and abdominal distension due to hepatomegaly and ascites. Thrombosis in splanchnic veins results in persistent abdominal pain, and symptoms and signs of intestinal obstruction may occur if infarction of the bowel ensues. Thrombosis of the splenic vein leads to splenomegaly and occasionally splenic rupture can occur. Portal vein thrombosis produces ascites and the later development of oesophageal varices. Saggital sinus thrombosis is the most frequent neurological complication. Symptoms include severe headache. There may be evidence of raised intracranial pressure, impaired conscious level and focal neurological signs. Occasionally, painful discoloured skin lesions occur when the dermal veins are affected. These lesions rarely ulcerate. Occasionally, skin lesions can resemble purpura fulminans; these can affect large areas of skin with necrosis and demarcation. Pregnancy in PNH is associated with an increased risk of fetal loss (40%) as a result of thrombosis and haemorrhage. The pathogenesis of thrombosis in PNH remains uncertain. Platelets are capable of compensating for the decreased expression of decay accelerating factor (CD55) due to the presence of factor H, a similar protein, that is present within α-granules. In PNH there is also a deficiency of membrane inhibitor of reactive lysis (MIRL, CD59), a glycosylphosphatidylinositol (GPI) anchor-dependent protein. In the absence of CD59, platelet lysis is minimized by the release from the cell surface of excess membrane attack complex by exovesiculation. The externalized phosphatidyl serine on the microvesicles released into the circulation acts as the binding site for prothrombinase complex. It is likely that the release of procoagulant microvesicles contributes to the increased risk of venous thrombosis. Increased platelet activation and an increased sensitivity to aggregation by thrombin have also been described. Fibrinolysis is also affected in PNH. Urokinase-type plasminogen activator receptor is also a GPI-bound protein that is absent from PNH cells. It binds urokinase to the cell surface and converts plasminogen to plasmin.

Thrombotic thrombocytopenic purpura See Chapter 44.

Sickle cell disease Paroxysmal nocturnal haemoglobinuria (see Chapter 11) Venous thrombosis occurs in up to 40% of patients with paroxysmal nocturnal haemoglobinuria (PNH) and represents a significant cause of morbidity and mortality. Frequently it is the presenting feature. Thrombosis most commonly occurs in the hepatic veins, portal veins and saggital sinus. Hepatic vein thrombosis leading to Budd–Chiari syndrome is the common898

In sickle cell disease the pathophysiology involves microvascular and macrovascular occlusion with sickled cells. There is also considerable clinical and post-mortem evidence of cerebral, pulmonary and placental thrombosis, suggesting a prothrombotic tendency in these patients. Additional evidence for a prothrombotic state is enhanced thrombin generation, as shown by increased levels of prothrombin fragment 1+2 and thrombin– antithrombin, in patients with sickle cell disease in their steady

Acquired venous thrombosis

state when compared with age-matched controls. There is also evidence that sickled erythrocytes adhere more readily to vascular endothelium and strongly accelerate coagulation due to abnormal exteriorization of procoagulant anionic membrane phospholipids. Comparison of the coagulation and fibrinolytic pathways in sickle cell patients with ethnically matched controls has shown increased levels of von Willebrand factor during sickling, although it is not clear whether this is specific or part of the acute-phase reaction. Low levels of protein C, protein S and heparin cofactor II are well described. The aetiology of the reduction of these physiological anticoagulants is unclear. It may relate to impaired liver function from repeated hepatic sickling, whereas increased consumption has also been proposed as a cause. Low heparin cofactor II levels are also found in other chronic haemolytic anaemias such as thalassaemia intermedia, suggesting that chronic haemolysis may also lead to increased consumption of this protein. Nephrotic syndrome, sometimes associated with sickle nephropathy, leads to low antithrombin levels and a prothrombotic state.

Selected bibliography Agnelli G, Becattini C (2010) Acute pulmonary embolism. The New England Journal of Medicine 363: 266–74. Arbuthnot C, Wilde JT (2006) Haemostatic problems in acute promyelocytic leukaemia. Blood Reviews 20: 289–97. Cannegieter SC, Doggen CJ, van Houwelingen HC, Rosendaal FR (2006) Travel-related venous thrombosis: results from a large population-based case control study. PLoS Medicine 3: e307.

Cappellini MD (2007) Coagulation in the pathophysiology of haemolytic anaemias. Hematology. American Society of Hematology Education Program 74–8. Carrier M, Lee AY (2009) Prophylactic and therapeutic anticoagulation for thrombosis: major issue in oncology. Nature Clinical Practice. Oncology 6: 74–84. Esmon CT (2003) Inflammation and thrombosis. Journal of Thrombosis and Haemostasis 1: 1343–8. Goldhaber SZ (2010) Risk factors for venous thromboembolism. Journal of the American College of Cardiology 56: 1–7. Greaves M, Machin SJ, Cohen H et al. (2000) Guidelines on the investigation and management of the antiphospholipid syndrome. British Journal of Haematology 109: 704–15. Harrison CN (2005) Platelets and thrombosis in myeloproliferative diseases. Hematology. American Society of Hematology Education Program 409–15. Hillmen P (2008) The role of complement inhibition in PNH. Hematology. American Society of Hematology Education Program 116–23. Keeling D, Davidson S, Watson HG (2006) The management of heparin-induced thrombocytopenia. British Journal of Haematology 133: 259–69. Pierangeli SS, Chen PP, Raschi E et al. (2008) Antiphospholipid antibodies and the antiphospholipid syndrome: pathogenic mechanisms. Seminars in Thrombosis and Hemostasis 34: 236–50. Prandoni P, Kahn SR (2009) Post-thrombotic syndrome: prevalence, prognostication and need for progress. British Journal of Haematology 145: 286–95. Robertson B, Greaves M (2006) Antiphospholipid syndrome: an evolving story. Blood Reviews 20: 201–12.

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CHAPTER 47

47

Management of venous thromboembolism Beverley J Hunt Thrombosis and Haemostasis King’s College, London, UK Guy’s and St Thomas’ NHS Foundation Trust, London, UK

Introduction and epidemiology, 900 Mechanisms, 900 Diagnosis of venous thromboembolism, 901 Objective diagnosis with imaging techniques, 902 Investigation for concomitant cancer, 903 Therapeutic agents, 903 Thrombolytic therapy, 903 Anticoagulant therapy, 903 Novel anticoagulant drugs, 906 Initial treatment of venous thromboembolism, 906 Thrombolytic therapy, 906

Venous thrombectomy, 907 Inferior vena cava filters, 907 Anticoagulant therapy, 907 Primary prophylaxis (thrombophlyaxis), 908 Efficacy and safety of thrombophylaxis, 908 For how long should thrombophylaxis be given?, 909 Secondary prophylaxis, 909 Duration of secondary prophylaxis with oral vitamin K antagonists, 910

Introduction and epidemiology Venous thromboembolism (VTE), which comprises deep vein thrombosis (DVT) and pulmonary embolism (PE), is responsible for an estimated 60 000 deaths annually in the UK based on epidemiological modelling. The clinical incidence of VTE is about 1 per 1000 per annum in adults, with a slight preponderance in men. Two-thirds present as DVT and one-third as PE. The major outcomes are death, recurrent VTE, post-thrombotic syndrome, pulmonary hypertension and major bleeding due to anticoagulation. Within 1 month of diagnosis, about 6% of patients with DVT and 10% of those with PE die. The mortality rate for PE has been estimated to be as high as 30% in some patient groups due to the high failure rate of recognizing PE before death, with mortality rates being highest in those with cancer. Despite the incidence of VTE increasing with age (1 per 10 000 per annum under 30 years of age and 5–6 per 1000 per annum by the age of 80), deaths also occur in the younger age group due to failure to consider the diagnosis. The increased numbers of VTE with ageing may relate to the increasing presence of other illnesses and also an inherent increased coagulability with ageing.

Postgraduate Haematology: 6th edition. Edited by A. Victor Hoffbrand, Daniel Catovsky, Edward G.D. Tuddenham, Anthony R. Green © 2011 Blackwell Publishing Ltd.

900

Management in the case of bleeding or surgery, 911 Secondary prophylaxis with LMWH, 912 Treatment of patients with cancer, 912 Venous thromboembolism in pregnancy, 912 The post-thrombotic syndrome, 913 Chronic thromboembolic pulmonary hypertension, 913 Selected bibliography, 913

Approximately 60% of cases of fatal PE are hospital acquired, i.e. 32 000 per annum in the UK with 25 000 being preventable with appropriate thromboprophylaxis. Interestingly, more VTE is diagnosed in the 3 months following hospitalization than during admission. As a rule of thumb, in the natural history of post-orthopaedic surgery, the median time for DVT to present is 7 days after the event, with PE presenting 21 days postoperatively. Thus both cases and fatalities usually occur after hospital discharge, which is why ‘hospital-acquired VTE’ rather than ‘hospital VTE’ is considered appropriate. However, within hospital PE accounts for 10% of deaths.

Mechanisms Virchow’s triad described in 1859 remains the best model of venous thrombogenesis. Changes in blood flow, vessel wall or blood coagulability comprise the triad, and usually a combination of these are present. The multi-hit hypothesis of thrombosis recognizes that more than one factor is usually operating to cause VTE, although the number of factors required to cause thrombosis decreases with ageing. The major risk factors are given in Table 47.1. Immobility is the key factor in those who do not have major thrombophilias or vessel wall abnormalities, and increases the risk of VTE 10-fold. Just sitting still for 90 min reduces blood flow in the popliteal vein by 40%. The term ‘seated immobility syndrome’ (also known as SIT syndrome) has been coined to encompass VTE precipitated by prolonged

Management of venous thromboembolism

Table 47.1 Common risk factors for venous thromboembolism according to Virchow’s triad.

Table 47.2 Clinical scores used for predicting the probability of DVT prior to further testing.

Reduced or altered flow Immobility Seated immobility thrombosis (SIT) syndrome: travellers thrombosis, e-thrombosis Previous venous thromboembolism Surgery Trauma

Risk factors, symptoms or signs

Prothrombotic changes Increasing age Pregnancy and the puerperium COC, HRT and estrogen receptor-modulating drugs Cancer Genetic thrombophilia Antiphospholipid syndrome Myeloproliferative disease Obesity Surgery Trauma Damage to vein walls Surgery Trauma COC, combined oral contraceptive; HRT, hormone-replacement therapy.

sitting, the term thus encompassing traveller’s thrombosis and ‘e-thrombosis’ (caused by sitting at a computer workstation).

Diagnosis of venous thromboembolism Diagnosis relies on the use of objective diagnostic methods. Over 80% of DVT episodes are clinically silent and often confined to the calf veins. The concern is that such asymptomatic DVT can become symptomatic and/or embolize. There is no way of predicting which patients will develop symptomatic VTE, and it is well recognized that significant numbers of massive fatal PE occur without warning and as a result of asymptomatic DVT. Thus the clinical approach to VTE is to maintain a high index of suspicion and to treat until the diagnosis is refuted or confirmed by objective testing. Clinical presentation of PE depends on the size, location and number of emboli and the patient’s underlying cardiorespiratory reserve. The classic triad of chest pain, haemoptysis and dyspnoea is present in less than 20% of patients, while 97% of patients with PE have at least one of the following: pleuritic chest pain, dyspnoea, respiratory rate above 20/min. Tachycardia and tachypnoea are the most frequent signs. A right-sided S3

Active cancer (treatment ongoing or terminated within 6 months or palliative care) Paralysis, paresis, recent plaster cast on the leg Recent confinement to bed of more than 3 days or major surgery within 12 weeks Localized tenderness along the deep venous system Swelling of the entire leg Calf circumference > 3 cm larger than the asymptomatic leg (measured 10 cm below the tibial tubercle) Pitting oedema (more on the symptomatic side) Collateral flow in superficial veins (not varicose veins) Previous DVT Alternative diagnosis as or more likely than DVT

Points 1 1 1 1 1 1

1 1 1 –2

In the case of symptoms from both legs, the leg with the most pronounced symptoms is used. Score > 2 points, high probability; score 1–2 points, medium probability; score < 1 point, low probability. Source: Anderson et al. (2003) with permission.

sound, widely split second sound, the murmur of tricuspid regurgitation and an accentuated S2 closure sound may be found. Fever is present in 14%. Massive PE has a mortality of 18–33% and may present with shock, dyspnoea and confusion. Chest radiography may show either nothing specific or the Westermark sign (dilatation of the pulmonary artery proximal to the emboli with sharp cut-off), while ECG may not show the classic signs of right ventricular strain such as P pulmonale and S1Q3T3. Arterial blood gases and transthoracic echocardiography have too low sensitivity The main role of these tests is to exclude other conditions, such as acute myocardial infarction. However, risk factors for VTE and clinical symptoms and signs with the highest predictive value can be assigned points that are added to obtain a clinical ‘score’ (Tables 47.2 and 47.3) that can be used as the first step in a diagnostic strategy to reduce costs and the requirement for invasive diagnostic procedures. The next step is the measurement of fibrin D-dimers, which are split products of fibrin degraded by plasmin, where the D-fragments of two fibrin molecules still are covalently bound and often also in complex with the E-fragment. Several methods can be used, such as enzyme-linked immunosorbent assay (ELISA), whole-blood agglutination of erythrocytes, latex agglutination or immunofiltration. ELISA methods have the best sensitivity but are not practical for near-patient testing, whereas the others are semi-quantitative or qualitative methods. There are also quantitative automated latex methods. The 901

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Table 47.3 Clinical scores used for predicting the probability of PE prior to further testing. Risk factors, symptoms or signs

Points

Signs of DVT (swelling, tenderness) Heart rate > 100 Immobilization > 2 days or recent surgery (< 4 weeks) Previous objectively verified DVT Haemoptysis Cancer Pulmonary embolism as or more likely than other diagnoses

3 1.5 1.5 1.5 1 1 3

Score > 6 points, high probability; score 2–6 points, medium probability; score < 2 points, low probability. Source: Wells et al. (2001) with permission.

manual latex methods have lower sensitivity and negative predictive value for exclusion of DVT or PE. Each hospital should provide information on the sensitivity and specificity of the test chosen for D-dimers. In general, a D-dimer assay is used for its negative predictive value (i.e. to exclude VTE) when the clinical score indicates low clinical probability. A positive result cannot be used for confirmation of the diagnosis owing to low specificity. For this reason, D-dimer is not a useful test in pregnancy or after surgery or in acute illness or the elderly. Whenever the clinical probability is high and/or the D-dimer test is positive, confirmation using diagnostic imaging techniques is necessary, aiming to use the most non-invasive method. In patients with massive PE and hemodynamic instability, rapid risk assessment is necessary and so bedside echocardiography has become the most popular tool. Spiral multislice chest computed tomography (CT) is also useful for identifying patients who may benefit from thrombolysis or embolectomy. Cardiac biomarkers, including troponin and the natriuretic peptides, are sensitive markers of right ventricular function. Low levels of troponin, B-type natriuretic peptide (BNP) and NT-terminal proBNP are highly sensitive for identifying patients with an uneventful clinical course. Multislice chest CT is useful not only for diagnosing or excluding PE but also for risk assessment. A right-to-left ventricular dimension ratio above 0.9 on the reconstructed CT four-chamber view identifies patients at increased risk of early death.

Objective diagnosis with imaging techniques Venography was the gold standard for diagnosing DVT, but the diagnostic accuracy is dependent on a high-quality examination. The veins and possible thrombi have to be well visualized and interpreted by an experienced radiologist, as artefacts may be difficult to differentiate from thrombi. A non-ionic contrast medium with low osmolarity should be used to minimize side902

effects and the risk of a post-venographic DVT. Caution is still needed with regard to renal failure. Ultrasonography of the leg veins is non-invasive and has no contraindications and thus has largely replaced venography as the method of choice. Diagnostically, it depends on the skill of the operator and has lower sensitivity than venography, especially for the veins of the calf. The simplest variant is compression ultrasonography, where a cross-section of the vein is compressed with the transducer. A non-compressible vein is diagnostic for DVT. The pelvic veins are not accessible with this method and recurrent thrombosis is difficult to discern. The most sophisticated variant of the technique is colour Doppler, which can determinate flow in several venous segments simultaneously, although generally the sensitivity is higher for proximal than for distal DVT. Low clinical probability in combination with a negative ultrasonographic examination, including the common and superficial femoral veins, the popliteal vein and the trifurcation in the proximal part of the calf, is considered sufficient to exclude DVT. However, with medium clinical probability, a negative ultrasonographic examination should be repeated within 1 week and, if still negative at that point, DVT is satisfactorily excluded. Presentation with high clinical probability and negative ultrasonography requires further investigation with venography. Suspected recurrent DVT is better served by venography as a first-line investigation, for ultrasound has low sensitivity in this setting. Objective verification of suspected PE has typically been obtained with ventilation–perfusion (V/Q) lung scanning, following a normal chest radiograph. Perfusion scanning with technetium-99m (99mTc) albumin macroaggregates is a sensitive but not very specific method. The specificity is improved by adding ventilation scanning, usually with xenon-133 (133Xe) aerosol or 99mTc-labelled carbon particles (Technegas), considering a V/Q mismatch (negative ventilation and positive perfusion scan) indicative of PE. However, an embolus may cause bronchospasm and thereby a ventilation defect and, more importantly, a V/Q mismatch may occur with several other conditions, including tumours, pneumonia, atelectasis, chronic obstructive pulmonary disease and others, and it does not differ between old and new emboli. The interpretation of lung scans is difficult, especially with patients with low or intermediate probability for PE, and thus other methods should be preferred for concurrent cardiopulmonary disease. Treatment can safely be withheld if the perfusion scan is negative or with a lowprobability scan in combination with low clinical probability. A high-probability lung scan, which should require perfusion defects corresponding to at least two segments, together with high clinical probability has a positive predictive value of more than 90%. Spiral CT of the lungs has good interobserver agreement and the number of inconclusive examinations is definitely lower than with V/Q scanning. With multislice techniques and a slice thickness of 2 mm, adequate assessment of 93% of the

Management of venous thromboembolism

segmental arteries and of 62% of the subsegmental arteries is achieved. Although the diagnostic accuracy for subsegmental PE is low with spiral CT, follow-up studies have shown that it was safe to withhold treatment on presentation of a negative examination. If there is a high clinical probability for PE and a negative perfusion scan or negative spiral CT, one possibility is to investigate for thrombi in the legs with bilateral ultrasonography or otherwise to proceed with pulmonary angiography. With contrast medium of low osmolarity, pulmonary angiography was not associated with mortality and only 0.4% of serious complications in four studies with more than 3000 patients. Although pulmonary angiography is considered the gold standard for diagnosis of PE, there are both false-positive and false-negative results, and the interobserver variability is poor when the PE is located in subsegmental arteries. Nevertheless, the method appears to exclude with great certainty those PE that require therapy. A novel possibility with high specificity and sensitivity for DVT and potential for PE is magnetic resonance imaging (MRI) direct thrombus imaging, which requires no contrast but relies on the formation of methaemoglobin in hypoxic trapped red cells within venous thrombi. This acts as an endogenous contrast agent when imaged using a T1-weighted magnetic resonance sequence – appearing as high signal.

Investigation for concomitant cancer The prevalence of cancer is approximately 20% in many cohort studies of patients with VTE. In a minority the malignancy is occult at the time of VTE. However, there is no evidence that screening for malignancies in those with VTE decreases the mortality and morbidity in those who are detected to have cancer. Instead, directed examinations should be performed when suspicion is raised by clinical signs or symptoms. Underlying cancer should be considered in patients with bilateral DVT, concomitant deep and superficial vein thrombosis at separate locations or recurrent VTE despite anticoagulation.

Therapeutic agents All agents discussed below have in common the obvious sideeffect of bleeding.

Thrombolytic therapy Streptokinase and recombinant tissue plasminogen activator are widely available fibrinolytic agents. Streptokinase, a glycoprotein purified from the supernatant of β-haemolytic streptococci, in complex with plasminogen, cleaves other plasminogen molecules to plasmin. This indirect action results in complex pharmacokinetic characteristics. Furthermore, streptokinase is immunogenic, which may cause allergic reactions as well as a

reduced or absent response on repeated use or after a recent infection with streptococci. Recombinant tissue plasminogen activator is an autologous glycoprotein, produced commercially with recombinant DNA technology, and it has a direct enzymatic effect on plasminogen that is strongly enhanced by the presence of fibrin.

Anticoagulant therapy For over 50 years, therapeutic anticoagulation was limited to the use of unfractionated heparin (UFH) and oral vitamin K antagonists. Although both are highly effective, both have major drawbacks, most notably their unpredictable pharmacokinetics, so that their use has become synonymous with monitoring. The arrival of new oral anticoagulants with predictable pharmacokinetics has led to great excitement. Heparin Heparin was first isolated between 1916 and 1922 by the medical students Jay McLean and Emmett Holt at Johns Hopkins University. As the source of the preparation was canine liver, it was named after the Greek word for liver, ηπαρ or hepar. In the 1930s it was used as an anticoagulant in human trials and its use became widespread after the Second World War. UFH is a naturally occurring glycosaminoglycan produced by mast cells and basophils. The pharmaceutical product is derived from tissues rich in mast cells such as porcine intestine or bovine lung. UFH is a polymer of repeating disaccharide units, primarily comprising sulphated glucosamine and uronic acid with a heterogeneous mixture of differing chain lengths; most preparations have a mean molecular mass of 13–15 kDa. A specific pentasaccharide sequence of the heparin molecule binds to antithrombin, inducing a conformational change and a 1000-fold increase in antithrombin activity. In turn, antithrombin inhibits thrombin and factor (F)Xa. Only the pentasaccharide sequence is required to generate anti-FXa activity, whereas a longer sequence of 18 saccharides, including the pentasaccharide, is necessary to bind thrombin to cause thrombin inhibition. Heparins are only active when administered parenterally. UFH can be given intravenously or subcutaneously; intramuscular administration should be avoided as it can result in large haematomas. At typical therapeutic doses, the half-life of intravenous UFH is 45–60 min, demanding continuous intravenous infusion. Given subcutaneously UFH has a lower bioavailability than intravenous heparin; activity starts at 2 hours and lasts approximately 10 hours, necessitating twice-daily dosing. A slight fall in platelet count (< 30% compared with baseline) occurs in up to one-third of patients within the first 4 days of starting heparin. This reversible dose-dependent phenomenon is not associated with bleeding or thrombotic complications and does not require cessation of heparin. More marked falls in platelet count (> 50% compared with baseline) should raise the possibility of heparin-induced thrombocytopenia (HIT). HIT 903

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is a life-threatening complication of heparin treatment and occurs in 0.3–6.5% of those receiving UFH. The onset is typically between 5 and 10 days after starting UFH unless heparin has previously been administered, in which case it may begin earlier. The cause is development of an IgG antibody to a complex of heparin and platelet factor 4, which is capable of activating and depleting platelets. Because the thrombocytopenia is due to platelet activation, patients with HIT are paradoxically at high risk of both venous and arterial thrombosis. Other features may include necrotizing skin changes at heparin injection sites or a history of anaphylactoid reactions to heparin injection. A clinical scoring system for suspected HIT is useful in determining the need for further investigation. The diagnosis of HIT may be confirmed by specific ELISA or by functional platelet studies. The treatment is immediate cessation of all types of heparins (including line flushes) and initiation of an alternative anticoagulant such as danaparoid, hirudin or fondaparinux at therapeutic doses. Platelet transfusion is contraindicated, and warfarin should not be started until the platelet count has recovered, as it may cause microvascular necrosis. Prolonged use of UFH can lead to osteoporosis by increasing osteoclast activity and reducing osteoblast numbers; 2% of patients receiving treatment-dose UFH for more than 9 months developed vertebral fractures. The risk of osteoporosis is considerably lower for low-molecular-weight heparin than for UFH. Low-molecular-weight heparin (LMWH) is produced by enzymatic or chemical depolymerization of UFH. Since its introduction in the late 1970s, LMWH has largely superseded UFH by virtue of its more predictable pharmacokinetic profiles, less frequent dosing and lower incidence of adverse effects. LMWH consists of a mixture of glycosaminoglycan chains of molecular mass 3000–5000 Da. LMWH has the same principal mode of action as UFH, but the inhibition of thrombin is reduced compared with the inhibition of FXa, as the former effect requires longer polysaccharide chains for binding to thrombin. The bioavailability after subcutaneous injection is better than for UFH and the effect is more predictable, due to less binding to plasma proteins. The half-life is also longer (3–5 hours), so that a once-daily subcutaneous injection is sufficient. Intravenous administration of LMWH is rarely used. Treatment with UFH requires monitoring with the activated partial thromboplastin time (APTT), whereas with LMWH monitoring is rarely needed, with the main exception being renal failure when inhibition of activated FX (anti-FXa) is measured. Side-effects such as HIT and osteoporosis are less common with LMWH. Although there are differences in molecular range, sulphation, ratio of FXa to thrombin inhibition, and pharmacokinetic characteristics between different LMWH peparations, there is no evidence for clinically important differences between them. Fondaparinux is a synthetic drug consisting of the antithrombin-binding pentasaccharide sequence found in the 904

heparin molecule. The pentasaccharide binds the antithrombin molecule, causing a conformational change that enhances its activity against FXa. Because there are no additional saccharides to provide the necessary bridging function for thrombin binding, fondaparinux has no activity against thrombin. Fondaparinux is given as a subcutaneous injection and has a half-life of 17 hours, making it suitable for once-daily dosing. Clearance is exclusively renal, so dose adjustment in renal failure is required and it is not recommended in those with a creatinine clearance below 30 mL/min. Fondaparinux does not bind to platelet factor 4, so has no capacity to cause HIT. Danaparoid is a heparinoid that consists of the glycosaminoglycans heparan sulphate, dermatan sulphate and chondroitin sulphate but without any heparin. Accordingly, it has low cross-reactivity with antibodies induced by heparin and it is therefore mainly used in patients with HIT. It is administered intravenously or subcutaneously and has an effect that is similar to UFH in the prevention and treatment of VTE. Vitamin K antagonists The vitamin K anatagonists or coumarins were first isolated by Karl Paul Link at the University of Wisconsin in the 1930s. The observation that cows bled to death after eating mouldy clover had led Link’s team to isolate the anticoagulant factor from the contaminated clover. Link later developed a synthetic coumarin derivative. This was patented by the Wisconsin Alumni Research Foundation who named it warfarin as a contraction of the organization’s acronym, WARF, and the word coumarin. The use of coumarins became widespread in the 1940s. There are two groups of vitamin K antagonists, the more commonly used coumarin derivatives and the indanediones, and their availability varies between countries. They exert their effect via inhibition of the enzymes vitamin K epoxide reductase and vitamin K reductase, which are required for the regeneration of KH2 from vitamin K epoxide. The latter is generated when the vitamin K-dependent coagulation factors (FII, FVII, FIX and FX, as well as the physiological inhibitors protein C and protein S) undergo post-translational modification with γ-carboxylation of approximately 10 glutamic acid residues in the N-terminal Gla domain. During treatment with vitamin K antagonists, typically three of these residues remain in the non-carboxylated state, which reduces the ability of these coagulation factors to bind calcium and to localize the coagulation process to phospholipid surfaces. The vitamin K antagonists are bound to plasma proteins, mainly albumin (98–99%), and are metabolized by the hepatic cytochrome P450 enzymatic system. Interactions with other drugs or food may occur at many levels (Table 47.4) and constitute a major disadvantage. There are a number of common genetic polymorphisms of the CYP2C9 enzyme primarily responsible for warfarin metabolism. The most common polymorphisms, CYP2C9*2 and CYP2C9*3, are each seen in around

Management of venous thromboembolism

Table 47.4 Mechanisms for drug interactions with vitamin K antagonists with typical examples of such drugs. Enhancement of vitamin K pathway: vitamin K, lipid emulsions Reduced endogenous synthesis of vitamin K: induced by antibiotics Accelerated catabolism of coagulation factors: thyroid hormone, androgens Decreased synthesis of coagulation factors: clofibrate Decreased warfarin absorption due to binding: colestyramine Increased absorption of warfarin: acarbose Inhibition of cyclic interconversion of vitamin K: second-/ third-generation cephalosporins Inhibition of cytochrome P450 (CYP3A4): clarithromycin Inhibition of cytochrome P4502C9, S-enantiomer: sulfinpyrazone Stereoselective inhibition of hydroxylation, R-enantiomer: cimetidine Non-stereoselective clearance: amiodarone Induction of hepatic enzymes: barbiturates, rifampicin Displacement of protein binding: etoposide Potentiation of the warfarin receptor effect: clofibrate Antiplatelet effect: acetylsalicylic acid

10% of whites, and are associated with a reduced warfarin requirement. A small proportion of individuals (< 1%) have a hereditary resistance to all coumarins mediated by reduced affinity of the coumarin receptor and require very large doses of coumarins (> 30 mg warfarin daily) to achieve an anticoagulant effect. Because of coumarin’s unpredictable pharmacokinetics, treatment requires regular monitoring. This was initially with the prothrombin time (PT). With the widely adopted use of a standardized calibration system, the International Normalized Ratio (INR), the reliability of comparisons of treatment intensities at different laboratories has been improved. The INR from an individual sample is calculated using the formula INR = (PTpatient/PTcontrol)ISI. Rare side-effects of vitamin K antagonists are skin necrosis, purple toe syndrome, rash and toxic hepatitis, whereas the adverse effect of warfarin on bone mineral density is debated. Skin necrosis can occur in approximately 1 in 5000 patients in early initiation of treatment, and is due to an imbalance between mildly depressed vitamin K-dependent coagulation factors and a more pronounced initial reduction of protein C and protein S, resulting in a hypercoagulable state with thrombus formation in small veins and venules in the dermis and subcutaneous fat. The purple toe syndrome is a very painful, burning, dark-blue discoloration of the toes and sides of the feet, probably due to cholesterol embolization from atherosclerotic plaques that have become friable owing to reduced fibrin deposition or haemorrhage into the plaques several weeks after initiation of anticoagulation. Skin rashes may be papular, vesicular or urticarial

and often very itchy. Vitamin K antagonists also have a teratogenic effect, with skeletal malformations, optic atrophy and mental impairment, occurring in a small percentage of babies of exposed mothers. Direct thrombin inhibitors All are only active parenterally and are administered mainly by intravenous infusion. Hirudin is a 65-amino-acid polypeptide, originally discovered in the saliva of the medicinal leech Hirudo medicinalis. Two recombinant derivatives are available, lepirudin and desirudin. Hirudin is an irreversible direct thrombin inhibitor, binding to both the active and substrate-recognition sites of the thrombin molecule. As the clearance of hirudin is exclusively renal, the half-life is highly dependent on renal function. With normal renal function, hirudin has a half-life of 60 min after intravenous administration, but in those with endstage renal failure the half-life can be as long as 300 hours. Hirudin is licensed for the treatment of thrombosis associated with HIT. Unfortunately, 40–70% of patients develop hirudin antibodies after a week of treatment; although they are rarely inhibitory, they may bind to hirudin and reduce renal clearance, resulting in a prolonged half-life. Bivalirudin is a recombinant 20-amino-acid polypeptide analogue of hirudin. It binds to the substrate-binding site of thrombin, but unlike hirudin can be cleaved by thrombin itself, making bivalirudin a reversible thrombin inhibitor. Bivalirudin has a half-life of 25 min following intravenous injection. Because only a proportion of excretion is renal, clearance is less dependent on renal function than for hirudin. Argatroban is a smallmolecule reversible thrombin inhibitor derived from the amino acid arginine with a half life of around 40 min; clearance is primarily hepatic. Because of their short half-lives, argatroban and bivalirudin have been used principally in the management of HIT and during percutaneous coronary procedures in patients at risk of HIT. All these parenteral direct thrombin inhibitors require therapeutic monitoring, usually by APTT, with a typical target ratio of 2.0–2.5. The ecarin clotting time is a better alternative, but is less widely available. There is no specific reversal agent for these direct thrombin inhibitors. Options in bleeding patients include blood product support, activated prothrombin complex concentrates or recombinant activated FVII. Hirudin is not cleared by haemodialysis. Oral direct thrombin inhibitors

Dabigatran Dabigatran is a novel synthetic small-molecule direct thrombin inhibitor that binds reversibly to both fibrin-bound and free thrombin and has predictable pharmacokinetics and so does not require monitoring. It has a plasma half-life of 14–17 hours, permitting once-daily dosing. Clearance is predominantly renal, and dose adjustment in renal failure may be necessary. 905

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Dabigatran causes prolongation of APTT, but not in a linear dose-dependent fashion; more accurate monitoring can be achieved using the ecarin clotting time. Dabigatran is licensed for thrombophylaxis after orthopaedic hip and knee replacement, and in clinical trials it has been shown to be non-inferior to warfarin in the management of VTE; patients randomized to dabigatran had fewer minor bleeds but more dyspepsia and more drug discontinuation. A Phase III study, RE-LY, evaluated the efficacy and safety of two different doses of dabigatran relative to warfarin in over 18 000 patients with atrial fibrilation. Dabigatran 110 mg was non-inferior to warfarin for the primary efficacy end point of stroke or systemic embolization, whereas dabigatran 150 mg was significantly more effective than warfarin or dabigatran 110 mg. Major bleeding occurred significantly less often with dabigatran 110 mg than warfarin; dabigatran 150 mg had similar bleeding to warfarin. Oral direct factor Xa antagonists Several oral direct FXa antagonists (i.e. they do not require antithrombin to exert their activity) are undergoing clinical trials; indeed rivaroxaban is now licensed for thrombophylaxis after orthopaedic joint replacement.

Rivaroxaban Rivaroxaban has an oral bioavailability of 60–80% and has a rapid onset of action, reaching peak concentration at 3 hours and a half-life of 5–9 hours. Excretion is predominantly renal. In clinical trials where it was used as thrombophylaxis after orthopaedic hip and knee surgery, it was found to have superior efficacy to enoxoparin. Results from trials in other areas are awaited.

Novel anticoagulant drugs There are a number of other novel oral anticoagulants in the pipeline, thus ensuring that the days of dependence on warfarin and heparin are numbered. Apixaban is an orally bioavailable small-molecule direct FXa inhibitor undergoing Phase III trials whose excretion is predominantly biliary and so it may be useful in renal failure. Others of interest include tecarfarin, a warfarin variant that is not metabolized by cytochrome P450 and thus has the potential to give more time in range.

Initial treatment of venous thromboembolism Before treatment is started, blood samples for haemoglobin, platelet count, APTT and PT should be obtained for baseline assessment of the risk of bleeding. In addition, with anticipated treatment with LMWH, serum creatinine, and with thrombolytic therapy plasma fibrinogen and blood group and save, should also be performed. Thrombophilia screening is of no utility at this stage for the results do not usually affect immediate patient management. Moreover, false-positive results may 906

be obtained because levels of physiological anticoagulants are usually reduced at the time of an acute thrombus due to consumption.

Thrombolytic therapy The most serious consequence of VTE is fatal PE. Many patients die before a diagnosis of PE has been made. Massive PE with unstable haemodynamics has a high mortality, but the prognosis improves dramatically with rapid lysis of the emboli. The benefit-to-risk ratio of thrombolysis in proximal DVT is as yet uncertain but it is recommended for unstable patients with PE, although these patients represent less than 5% of all patients hospitalized for PE. The streptokinase/urokinase PE thrombolysis trials showed that thrombolytic therapy successfully decreases pulmonary artery pressures acutely, with improvements in the lung scan and arteriogram at 12 and 24 hours, although there was no overall decrease in mortality in those receiving thrombolysis compared with those receiving heparin therapy. The use of thrombolytic treatment in patients with sub-massive PE is controversial. Contraindications to thrombolysis include active internal bleeding, a stroke within 2 months, and an intracranial process such as neoplasm or abscess (Table 47.5). Relative contraindications include surgery within 10 days, uncontrolled hypertension Table 47.5 Contraindications to thrombolytic therapy. Absolute contraindications Active internal bleeding Known haemostatic disorder Cerebrovascular accident, craniocerebral trauma or neurosurgery within 6 months Active intracranial process Relative contraindications Major surgery, biopsy, puncture of non-compressible vessels, vaginal delivery, external heart massage or major trauma within 7 days Vascular or eye surgery within 3 weeks Gastrointestinal bleeding, acute pancreatitis or pericarditis within 3 months Uncontrolled hypertension (diastolic ≥ 110 mmHg) Age > 80 years Diabetic retinopathy In addition, caution should be observed during the first trimester of pregnancy, the first 20 hours of menstruation, bacterial endocarditis and where there are abnormal screening tests for haemostasis. Patients with previous treatment with streptokinase within 3–4 months are unlikely to respond to repeated treatment due to antibody formation. Adherence to these precautions and contraindications should be more strict for the treatment of DVT than of massive PE.

Management of venous thromboembolism

and pregnancy. Haemorrhagic complications are higher in patients with a recent invasive procedure such as pulmonary angiogram or placement of an inferior vena cava (IVC) filter. There is a reported incidence of intracranial haemorrhage of approximately 2%, with higher rates in the elderly and those with poorly controlled hypertension. The major haemorrhage rate ranges from 11 to 20%. Heparin is discontinued 30 min before thrombolytic therapy and restarted 2–4 hours after its discontinuation. Regional or local infusion of fibrinolytic agents has not proved to be more effective, for bleeding complications are still prevalent. Antipyretics such as paracetamol may be given for febrile reactions and steroids for more pronounced allergic reactions, but prophylaxis with the latter does not provide any additional benefit. With major haemorrhage, the infusion should be stopped and blood losses replaced. For patients undergoing streptokinase therapy, the situation is more complex. Because of the indirect pharmacokinetics, a paradoxical effect may occur if the infusion of streptokinase is slowed or stopped. More plasminogen will then become available for transformation to plasmin by already circulating streptokinase–plasminogen complexes. It is therefore vital to reduce the proteolytic potential, which can be done with aprotinin, an antiplasmin agent. Conventional inhibitors of fibrinolysis, such as tranexamic acid, are not effective as they only counteract plasminogen conversion to plasmin but do not inhibit already formed plasmin. With very low levels of fibrinogen and major haemorrhage, infusion of fibrinogen (concentrate or cryoprecipitate) is also indicated. Plasma fibrinogen level should be monitored every 12–24 hours. If the level is not reduced whatsoever, it is unlikely that a thrombolytic effect is achieved. The target is approximately 0.2–1.0 g/L, but levels higher than 0.2 g/L do not exclude the risk of haemorrhage, and therapeutic levels do not provide a guarantee for removal of the thrombus.

Venous thrombectomy There are insufficient data to evaluate the long-term benefit of surgical removal of the thrombus. The procedure is often combined with formation of an arteriovenous fistula to improve the flow, but there is not enough evidence to show that this increases the patency rate of the vein. Bleeding is common, often requiring transfusion, and a fatal outcome has been reported in up to 14%. The benefit of the treatment is that relief of symptoms is immediate, and it may be considered in the rare cases when loss of the limb is imminent.

Inferior vena cava filters A temporary or permanent filter provides a tool to prevent further PE by reducing the risk during the first 12 days from 4.8% to 1.1%. However, PE can still occur in patients with IVC filters, and there is also a small risk of thrombosis at the site of

filter insertion. The primary indication for an IVC filter is the prevention of PE in patients with established VTE who have a contraindication to anticoagulation. Once the filter is in place, there is a tendency for clinicians to delay anticoagulation. However, anticoagulation should be considered in all patients with an IVC filter once a temporary contraindication to anticoagulation has passed. A large randomized trial showed no reduction in mortality or long-term risk of PE after 2 years of permanent indwelling catheters; indeed there was an increased risk of recurrent thrombosis.

Anticoagulant therapy The objective of anticoagulant therapy in the acute phase is to prevent further progression of the thrombus as well as (further) PE. It is not sufficient to start treatment with vitamin K antagonists alone, because of the delay of about 5 days until they provide full anticoagulation. A once-daily treatment dose of subcutaneous LMWH can be given and will have immediate effect and so patients with DVT are usually managed as outpatients, unless they have renal failure where intravenous UFH is required. With UFH an initial intravenous bolus should be given and treatment is continued with intravenous infusion until anticoagulation with a coumarin, usually started simultaneously, has become effective. Treatment with UFH is targeted at an APTT of 2–2.5 times the upper limit of normal, which is measured 4 hours after starting the infusion and then at least once daily. With APTT below the target range, another bolus dose is given and the infusion rate is increased. Conversely, with APTT above the treatment range, the infusion may be halted for 30 min and should be restarted at a reduced rate. Failure to reach the therapeutic APTT range is associated with a poor antithrombotic effect, whereas a possible association between long APTT results and bleeding complications is less evident. Occasionally, there may be difficulties in reaching a therapeutic APTT with inherited and acquired deficiencies of antithrombin. If the APTT is not prolonged despite increased doses of UFH, and especially if there is clinical deterioration, the antithrombin level should be measured and, if there is a deficiency, the addition of antithrombin, either by using fresh frozen plasma or antithrombin concentrates, should be considered. More often, a therapeutic APTT is not achieved despite normal antithrombin levels, but there is no evidence of clinical progression. This condition is believed to be caused by high levels of acute-phase proteins, such as FVIII or histidine-rich glycoprotein, which counteract the prolongation of APTT but apparently not the antithrombotic effect of heparin. Measurement of anti-FXa in these patients often demonstrates therapeutic levels and should be taken into account so that undue increment of the dose of heparin is avoided. If the baseline APTT is prolonged, the reason may be presence of a lupus anticoagulant (mildly to moderately prolonged APTT) or congenital deficiency of FXII (severely prolonged 907

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APTT for homozygous patients), and monitoring of treatment with UFH is often difficult. The alternatives are monitoring with anti-FXa or treatment with LMWH, which does not require monitoring. Patients with suspected PE can be managed in the same way with LMWH subcutaneously and vitamin K antagonists orally. It is thus possible to give this treatment on an outpatient basis, but patient safety has so far not been demonstrated for outpatient management of PE in a randomized trial. Treatment for DVT with LMWH can be undertaken safely without hospitalization. Randomized trials have demonstrated equal effect and safety with LMWH injected once or twice daily. For hospitalized patients, LMWH is associated with a lower incidence of haemorrhage than treatment with UFH. In the case of very ill patients or with a high risk of bleeding, it still may be preferable to treat with UFH, infused intravenously, in view of the shorter half-life if the treatment has to be discontinued abruptly and protamine sulphate used to neutralize residual UFH. Interestingly, initial treatment with LMWH instead of UFH is associated with a reduced mortality during 3 months of follow-up due to an increased survival in the subgroup of patients with concomitant cancer. Although the dose of LMWH is adjusted according to body weight, there are conflicting data regarding whether the dose should be increased linearly or ‘capped’ in obese patients. Patients with mild to moderate renal failure (serum creatinine 200–400 µmol/L) should have anti-FXa levels after 2–3 days at the time of peak concentration and, if above 1 unit/mL, the dose should be reduced. For patients with severe renal failure UFH is preferred. Patients with VTE should be mobilized early, preferably within 1 day, unless thrombolytic therapy is given. Mobilization does not increase the risk of new PE, and pain and swelling of the leg diminish more rapidly than if the patients are immobilized for 1 week. Patients should wear compression stockings as soon as they are out of bed.

Primary prophylaxis (thrombophlyaxis) A systematic review by the US Agency for Healthcare Quality and Research ranked PE as the most common preventable cause of hospital death, and thrombophylaxis as the number one strategy to improve patient safety in hospitals. Despite good clinical evidence of the efficacy and cost-effectiveness of thrombophylaxis, there has been failure of its implementation internationally. Most inpatients have several risk factors for VTE. The baseline risk is estimated to be 5–15% for those adults who are acutely unwell in medical beds, with risks of about 50–60% in hip replacement surgery and severe stroke and nearly 100% in spinal cord injury/polytrauma cases. The arguments for using thrombophylaxis in all those at risk include the following. 908

• Despite the recognition of specific risk factors, it is impossible to predict exactly which individuals within a group at risk will have VTE. • Screening with ultrasound has low sensitivity for DVT and is not cost-effective. • Over 80% of DVT are clinically silent and often confined to the calf vein. The concern is that such asymptomatic DVT can become symptomatic and/or embolize. There is no way of predicting which patients will develop symptomatic VTE without warning and as a result of asymptomatic DVT. Therefore in view of our inability to predict the exact individuals in a high-risk group who will develop VTE, the standard approach is to administer thrombophylaxis to all those at risk.

Efficacy and safety of thrombophylaxis Many randomized clinical trials over the past 35 years have demonstrated the efficacy and safety of thrombophylaxis, summarized elegantly and regularly by the American College of Chest Physicians. The National Institute for Health and Clinical Excellence (NICE) have produced guidelines for the UK. Thrombophylaxis can be separated into mechanical and pharmacological methods. Mechanical methods Mechanical approaches include antiembolic stockings, intermittent pneumatic compression devices, mechanical foot pumps and IVC filters. All except IVC filters reduce the risk of DVT. IVC filters reduce the risk of embolism from an established DVT and can therefore be considered secondary prophylaxis. Antiembolic stockings apply graded circumferential pressure on the legs, greatest distally, which increase the velocity and volume of blood flow in the deep veins. They have a lower grade of compression compared with graduated compression stockings used in patients with post-thrombotic syndrome, and need to be fitted correctly to work effectively. Intermittent pneumatic compression causes sequential compression of the legs in a wave-like milking effect. Foot pumps squeeze the plexus of veins known as the venae comitantes of the lateral plantar artery and thus reduce venous stasis. The poor quality, low number and poor end points of the studies of mechanical thrombophylaxis have been widely criticized. A recent well-conducted study showed that thigh-length stockings do not alter the risk of DVT after stroke but did have a 5% risk of skin breaks, ulcers, blisters and skin necrosis. Mechanical methods are contraindicated in patients with critical limb ischaemia, limb fractures and severe neuropathy, while ill-fitted stockings may cause oedema, arterial ischaemia and superficial thrombosis. The key end point of the efficacy of thrombophylaxis is reduction in death rate due to PE, and to ascertain this requires thousands of patients in one or several trials, which would need

Management of venous thromboembolism

to include post-mortem studies. Such studies have not been performed for mechanical thrombophylaxis. However, the main advantage of mechanical methods is that they do not produce an increased bleeding risk. Stockings and intermittent pneumatic compression act in a synergistic manner when combined with pharmacological agents in surgical patients. However, there is no evidence to suggest that there is any benefit when IVC filters are added to appropriate thrombophylaxis. Insertion is common in trauma patients who have a very high risk of VTE, but this has never been shown to be protective in a trial. Pharmacological UFH and LMWHs have been clearly shown to reduce the risk of death due to PE. The modern trials of pharmacological thrombophylaxis use a composite primary outcome measure of symptomatic and asymptomatic VTE. A meta-analysis of 46 trials of UFH thrombophylaxis in surgery showed that UFH, when compared with placebo, reduced the all-cause mortality from 4.2% to 3.2%. Fatal PE were reduced from 0.8% to 0.3%, symptomatic PE from 2.0% to 1.3% and DVT from 22% to 9%. However, bleeding events were significantly increased from 3.8 to 5.9%. A problem with UFH is the need for frequent dosing. Thricedaily dosing of UFH 5000 units was more effective in reducing the rate of VTE and especially PE, but the risk for major bleeding is significantly increased compared with twice-daily dosing. UFH has been largely superseded by LMWHs whose longer half-lives allow once-daily administration, although UFH remains the first choice in renal dysfunction, in which LMWH accumulates, causing increased bleeding, due to its long half-life and renal excretion. LMWH has been shown to reduce the risk of VTE by a similar amount as UFH (60–70%), with a similar bleeding risk compared with placebo. Hospital-acquired VTE is more common in medical than surgical patients because medical patients are the commonest admissions. Indeed 70–80% of hospital-acquired fatal PE occurs in medical patients. Apart from being an older cohort, 40% of medical patients have more than one risk factor for VTE, including previous VTE, cancer, stroke, heart failure, chronic obstructive airways disease, sepsis and bed rest. Approximately 5000 acutely ill medical patients have been entered into randomized controlled trials comparing LMWH with placebo and studies comparing LMWH with UFH. For example, the MEDENOX study showed that enoxaparin 40 mg was superior to both enoxaparin 20 mg and placebo given for a median of 6 days to acutely ill medical patients. There was a significant reduction in DVT between 6 and 14 days in those patients who received enoxaparin 40 mg to 5.5%, compared with 15% who received placebo (P < 0.001). There were no significant differences in bleeding between the groups. Fondaparinux is also licensed for thrombophylaxis. Postoperative adjusted-dose oral vitamin K antagonists are unfashionable in Europe due to the

unpredictable pharmacokinetics of warfarin leading to a significant risk of both over- and under-anticoagulation. The evidence for using antiplatelet drugs in thrombophylaxis is based on methodologically flawed studies and has been shown to be inferior to LMWH. For example, in a trial of joint replacement randomized to aspirin or LMWH, the relative reduction in VTE risk of LMWH was 63% over aspirin. Moreover, aspirin is associated with an increased risk of bleeding, especially when combined with other methods. Its use in thrombophylaxis is not recommended in all the major guidelines.

For how long should thrombophylaxis be given? The acute-phase response that produces a prothrombotic state persists for weeks after surgery. Although the risk of postoperative DVT is highest within the first few weeks after surgery, VTE complications may occur later. Extended prophylaxis for up to 35 days has been shown to reduce VTE in those with ongoing significant risk factors for VTE, and so LMWHs are licensed for extended use for 28–35 days in major hip, abdominal or cancer surgery, reducing the risk of VTE by a further 60% from 2.7% to 1.1%.

Secondary prophylaxis After a VTE, if initial treatment with UFH or LMWH is not followed by a period of secondary prophylaxis, the risk of recurrence over 90 days is 29%. Secondary prophylaxis is usually given with vitamin K antagonists, commonly with warfarin. In some European countries, phenprocoumon or acenocoumarol is prescribed predominantly. Phenprocoumon has a half-life of 160 hours compared with 20–55 hours for warfarin, which may cause difficulties when the drug has to be eliminated quickly in case of overdose or haemorrhage. Acenocoumarol has a half-life of only 11 hours, and this does not result in a requirement for twice-daily dosing. Comparative studies with warfarin and acenocoumarol have demonstrated a similar effect and safety profile. Treatment with a vitamin K antagonist should be started concomitantly with the initial anticoagulant therapy, or once the diagnosis of PE or DVT is established. The optimal intensity is an INR of 2.0–3.0, at least during the first 6 months after an episode of VTE. It may take 3–10 days to reach this target range and the maintenance dose varies from 1 to 20 mg daily. The time to reach the therapeutic range and to find the individual maintenance dose is shortened with higher initial dosing (warfarin 15 mg daily versus lower doses, or warfarin 10 mg vs. warfarin 5 mg) and by using a nomogram or computer software for the dose adjustments. The higher induction dose of 15 mg should be avoided in patients with a high risk of bleeding and is contraindicated in patients with deficiency of protein C or 909

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S because of the increased risk of warfarin-induced skin necrosis. The initial treatment with UFH or LMWH may be discontinued when the vitamin K antagonist has been given for at least 5 days and an INR of at least 2.0 has been achieved for two consecutive days. The secondary prophylaxis with vitamin K antagonists is then continued for a period that sometimes can be decided very shortly after the diagnosis of VTE, but which often requires reassessment due to the results of evaluation for thrombophilia or other investigations that may favour a longer treatment period. Conversely, bleeding complications or poor compliance may prompt a shortening of the planned treatment period. In order to improve the quality of anticoagulant therapy, it is crucial that the patient is followed properly as an outpatient. Treatment is monitored by measuring the INR. Dose adjustments become more precise, with less INR results outside the therapeutic range, if decision support aids, such as nomograms, algorithms or computer software, are used. Staff in charge of dose adjustments should be trained and follow a large number of patients regularly. Well-motivated patients should be encouraged to perform self-testing with capillary blood sampling using a ‘point-of-care’ instrument and to be in charge of dose adjustments, with advantages in terms of less clinical complications. This requires some training and visits to an anticoagulant clinic a few times per year for comparison of the results with those obtained at the laboratory. All patients who receive vitamin K antagonists should be informed about the following (printed information should also be provided). • The risk of bleeding. • Whom to contact whenever such symptoms occur. • The risk of using acetylsalicylic acid and non-steroidal antiinflammatory drugs, which impair platelet function, and about the alternatives that can be used. Although paracetamol interacts with warfarin, this is not clinically significant. • Vast numbers of drugs interact with vitamin K antagonists so that the possibility of an interaction should be questioned for each new drug prescribed. Several herbal medicines, including St John’s wort, ginseng and garlic, reduce the concentration of warfarin in blood. • The effect of large amounts of dark green vegetables, which contain considerable amounts of vitamin K and may therefore neutralize the anticoagulant effect, but stressing that a regular intake of moderate amounts of vegetables is recommended.

Duration of secondary prophylaxis with oral vitamin K antagonists The optimal duration of secondary prophylaxis is dependent on a risk–benefit analysis between avoidance of haemorrhagic complications and recurrent VTE. Meta-analyses have shown that a longer duration reduces the risk of recurrence, but treat910

Table 47.6 Factors associated with a higher risk of recurrence of venous thromboembolism. Higher risk Triggering risk factor not identified Proximal DVT and/or PE Presence of cancer Thrombophilic defects Deficiency of antithrombin, protein C or protein S Homozygosity for FV Leiden or prothrombin G20210A Antiphospholipid antibodies Follow-up of the thrombotic disease Persistently increased D-dimers Remaining thrombus on ultrasound examination Lower risk Triggering risk factor removable or permanent Distal DVT Thrombophilic defects Heterozygosity for FV Leiden Heterozygosity for prothrombin G20210A Follow-up of the thrombotic disease Normalized level of D-dimers Normalized ultrasound

ment for more than 6–12 months is associated with an increased risk of bleeding. Factors associated with a higher risk of recurrence are shown in Table 47.6. For the majority of patients, treatment of at least 6 months is recommended. Shorter treatment is indicated for a distal DVT and temporary risk factor, such as trauma, surgery or oral contraceptives (6 weeks), or for patients with a high risk of bleeding (3 months). In randomized trials on the duration of secondary prophylaxis, there has typically been an annual incidence of 2–4% of major haemorrhage. One option to reduce this risk is to reduce the intensity of anticoagulant therapy, and studies on coumarins targeted at an INR of less than 2.0 have shown a favourable risk–benefit profile. Large randomized trials on low-intensity warfarin therapy in the extension of secondary prophylaxis after at least 3–6 months of treatment, targeted at the usual INR of 2.0–3.0, have shown that a prophylactic antithrombotic effect is obtained but perhaps less than with full intensity, although the annual incidence of major haemorrhage was only 0.9–1.0% and may be an option in some low-risk patients. However, secondary prophylaxis targeted at an INR of 1.5–2.0 was not tested in patients with active cancer, for whom even standard vitamin K antagonist therapy is often insufficient (see below). A decision in favour of prolonged anticoagulation should be weighed against the cost and burden for the patient of monitor-

Management of venous thromboembolism

Table 47.7 Suggested duration of secondary prophylaxis in relation to presence of risk factors. Condition

Duration

Thrombophilic defects not identified or unknown First event, distal DVT, provoked by 6 weeks temporary risk factor 6 months or longer First event, distal DVT with idiopathic or permanent risk factor, or any proximal DVT or PE As above with increased risk of 3 months bleeding Single life-threatening event 12 months or longer First event, active cancer Until cancer resolved Second event, contralateral DVT As after the first event Second event, ipsilateral or PE 12 months or longer More than two events Indefinitely Thrombophilic defects identified* Deficiency of antithrombin Deficiency of protein C or protein S Homozygous form of thrombophilic defect Double heterozygous for thrombophilic defects Antiphospholipid syndrome Hyperhomocysteinaemia Elevated FVIII activity FV Leiden mutation, heterozygous Prothrombin polymorphism, heterozygous Life-threatening event and any defect

Indefinitely 12 months or longer Indefinitely† Indefinitely Indefinitely As without‡ 6 months or longer As without the defect As without the defect Indefinitely

*Independent of the event being provoked by a temporary or permanent risk factor, whenever the thrombophilic defect is permanent. † Possibly excluding the homozygous form of prothrombin G20210A. ‡ Consider the use of B vitamins thereafter. Source: modified from Schulman (2003) with permission.

ing the treatment, effects on the quality of life in view of restrictions regarding diet and other concomitant medications and, finally, the fact that about 1% of patients annually will have a major haemorrhage. Suggestions for the duration of secondary prophylaxis are given in Table 47.7. However, a significant minority of patients may wish to stop coumarins and instead take thrombophylaxis at times of haemostatic stress, for example prophylactic doses of LMWH at times of surgery or during long-haul air travel. Although it is not clear that the risk of recurrence is higher after the second episode of VTE than after the first, recurrent

DVT in the ipsilateral leg causes additional damage to the venous valves and increases the risk of post-thrombotic syndrome significantly. Patients with PE have a higher risk of recurrent symptomatic PE than do patients with initial DVT, and extension of secondary prophylaxis may thus be justified after recurrent PE. Patients with antiphospholipid syndrome have a high risk of recurrence without anticoagulation and therefore anticoagulation should be extended. Cessation of vitamin K antagonists is often associated with an increment in prothrombin fragment 1+2 and D-dimers, independent of whether they were stopped abruptly or gradually, and so there seems to be no clinical benefit in stopping gradually.

Management in the case of bleeding or surgery Excessive anticoagulation may be identified as an isolated prolonged INR above 4 or in combination with bleeding. Although elimination of one dose of the vitamin K antagonist, possibly followed by a reduction of the maintenance dose, may suffice in most cases, the addition of oral vitamin K1 1 mg reduces the risk of bleeding. The reason for excessive anticoagulation should be investigated and may reveal an interaction with other drugs or irregular dosing. In the case of major or life-threatening bleeding, reversal with vitamin K1 is too slow, as only a weak effect is noticed after 2 hours and the full effect occurs after 6 hours. Replacement of the deficient vitamin K-dependent coagulation factors is necessary, but the volumes of fresh plasma required are so large (typically 2–3 L to reduce the INR from 4.0 to 1.5 in a patient with body weight of 80 kg) that there is a substantial risk of volume overload. Prothrombin complex concentrate is the agent of choice: it carries no risk of volume overload and has undergone viral inactivation to eliminate the risk of transmission of HIV or hepatitis B or C. A few cases with thromboembolic complications in association with the use of prothrombin complex concentrate have been reported, but the risk appears small as no complications were observed in prospective cohorts. In patients with bleeding at INR levels within or below the therapeutic range, cancer should be suspected and investigations of the source are indicated. For minor surgery, such as tooth extractions or dermatological surgery, anticoagulant therapy can be maintained within the therapeutic range. Randomized trials have shown an excellent prophylactic effect against bleeding complications after dental extractions if the patients were prescribed mouth rinses with the fibrinolytic inhibitor tranexamic acid every 6 hours. For major surgery, the INR has to be reduced to below 1.5, either by complete interruption or by reduction in the vitamin K antagonist. Substitution with LMWH or UFH is given perioperatively until therapeutic INR has been regained. 911

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Secondary prophylaxis with LMWH Secondary prophylaxis with UFH twice daily subcutaneously, adjusted to prolong the APTT 1.5 times the normal value at 6 hours after injection, has been shown to be as effective as vitamin K antagonists. However, this never gained any popularity due to the requirement for two doses and adjustments. A meta-analysis of studies on secondary prophylaxis, comparing LMWHs with vitamin K antagonists, has shown that they are equally effective and safe. LMWH injected once daily subcutaneously may be the drug of choice for patients with a short treatment duration (6 weeks or less), with pronounced difficulties in achieving stable INR values, or when monitoring of the vitamin K antagonist is problematic. The dose of LMWH should be approximately 50% of the initial treatment dose and monitoring with anti-FXa is not required unless there is impairment of renal function or the patient is obese. The risk of osteoporosis with long-term LMWH is minimal and HIT is very rare.

Treatment of patients with cancer In cohorts of unselected patients with VTE, the prevalence of cancer is about 20%. Even occult cancer increases the risk of thrombosis for several years until it becomes clinically manifest. The diagnosis and treatment of patients with VTE and known cancer differs somewhat from what has been described above. The diagnosis of DVT with ultrasonography or phlebography may become false positive due to obstruction of the iliac vein by a pelvic tumour mass or metastases, and CT may therefore be the diagnostic method of choice. Compression of a pulmonary artery by a tumour mass or tumour emboli may simulate perfusion defects by PE, both on lung scanning and on pulmonary angiography. Measurement of D-dimers may not be of any value in these patients, who typically have elevated levels even in the absence of VTE. Initial treatment should preferably be given with LMWH in view of the survival benefit in patients with cancer over the following 3 months in comparison with those treated with UFH. Although patients with cancer have massive VTE more frequently than those without cancer, thrombolytic therapy is very hazardous, as the risk of bleeding is aggravated by necrosis in highly vascularized tumour tissue, erosion of normal tissues and thrombocytopenia due to bone marrow invasion by tumour or aplasia after chemotherapy. In some of these patients, the risk of bleeding is so pronounced that in the case of PE or massive DVT there is an indication for an IVC filter. Long-term anticoagulation with coumarins in patients with cancer is unsatisfactory, because there is an increase in the annual risk of recurrence of 14–18% compared with patients without cancer and, paradoxically, an increase in major bleeding of 7–11%. For secondary prophylaxis, LMWH at a dose of 912

75% of the initial treatment dose may be a better alternative than vitamin K antagonists. Long-term LMWH may prolong survival in patients with cancer and VTE, and perhaps also in those without VTE. This does probably not apply to terminally ill patients but rather to those with an expected survival of at least 1 year. However, many questions remain to be answered: the mechanism for this effect, whether the effect is confined to certain types of cancer, the minimum treatment period required and the optimal dose of LMWH. Secondary prophylaxis should continue as long as active cancer is present, unless the severity of bleeding complications precludes treatment.

Venous thromboembolism in pregnancy The incidence of VTE in association with pregnancy is 0.5–1.0 per 1000 with one to two fatal cases of PE in 100 000 pregnancies. DVT occurs commonly in the left iliofemoral vein. This may generate a clinical picture that is different from the one generally seen in DVT. The only symptoms may be abdominal cramps, sciatic back pain or fever. Objective diagnosis is even more important in the pregnant patient, as failure to treat an undiagnosed VTE as well as treatment of a false-positive diagnosis may cause harm. Pressure from the uterus on the pelvic veins may simulate the effect of thrombosis on ultrasonography, particularly in the third trimester. These examinations are more accurate if the mother is resting on her side. The radiation dose from phlebography, ventilation–perfusion lung scanning, spiral CT or pulmonary angiography is so small that the possible risk for the child of future malignancy caused by radiation or hypothyroidism due to free iodine is substantially less than the risk of missing the diagnosis. These examinations should therefore be performed if they are considered to be important for the diagnosis, but obviously with all available precautions. MRI or direct thrombosis imaging is not known to cause any harm to the fetus and, if available, may be a useful diagnostic tool. D-dimers are not helpful in these patients, who often have increased levels, especially during twin pregnancies or pre-eclampsia. Thrombolytic therapy may be considered for massive DVT or PE, but the risk of bleeding is particularly high if this is used shortly before or during the delivery or the puerperium. Thrombectomy for iliac or iliofemoral DVT can be performed safely, but there are no studies to show that this gives any better results than anticoagulant therapy. Despite the lack of clinical trials of LMWHs during pregnancy, they are widely used because of their ease of administration and good safety profile. There is debate as to whether twice-daily administration is better than once daily, for the halflife of LMWH is shorter in pregnancy due to improved renal clearance. It is not clear whether anti-FXa levels need to be monitored in pregnancy; there have been no clinical trials to

Management of venous thromboembolism

assess the level of anti-FXa activity required in pregnancy, so target levels are extrapolated from non-pregnant studies. After VTE some physicians continue with the treatment dose until the end of pregnancy, but usually secondary prophylaxis is carried on with a reduced dose of LMWH. Vitamin K antagonists should be avoided if possible because of an increased risk of fetal haemorrhage and because of teratogenic effects. Unlike coumarins, UFH and LMWH do not cross the placenta. At the time of delivery, blood samples are taken on admission for platelet count, APTT and PT to evaluate the risk of bleeding, and LMWH administration ceased. According to current guidelines, based on expert opinion, regional anaesthesia cannot be commenced or terminated within 24 hours of a treatment dose of LMWH or 12 hours after the previous prophylactic dose of LMWH. After acute VTE in pregnancy, after birth the prophylaxis is continued for at least 6 weeks and with a total duration that is at least as long as would have been chosen for a nonpregnant patient with the same risk factors. Alternatively, coumarins can be given after delivery, and LMWH is then stopped when the INR is within 2.0–3.0 for at least 2 days and after a minimum of 5 days. Neither LMWH nor vitamin K antagonists are secreted in breast milk in any amounts that can have an effect on the baby, although it seems sensible to ensure that the neonate receives intramuscular vitamin K to prevent haemorrhagic disease of the newborn. Women with inherited antithrombin deficiency require higher doses of heparin and regular anti-FXa activity monitoring. Administration of antithrombin concentrate should be considered in association with delivery to allow for safe cessation of LMWH.

The post-thrombotic syndrome Thrombi that do not undergo thrombolysis or which are not removed rapidly will destroy venous valves in the healing process, where they are transformed into fibrous tissue. Venous return of blood becomes impaired, accompanied by increased venous pressure. Permeation of fluid to the extravascular space causes oedema and creates a perivascular barrier to plasma proteins, which reduces the transport of nutrients to the tissues and results in skin atrophy and, in the most severe cases, venous ulcers. This process takes between 1 and 5 years, sometimes even longer, and is seen in 30% of patients 2 years after an event. The tools available to reduce the risk of developing the postthrombotic syndrome include the following. • Thrombolytic therapy, which has limited use due to the risk of haemorrhage. • Ensure that more than 50% of the time on coumarins is in the target range. • Graduated compression stockings for 2 years after an episode of DVT reduce the incidence of post-thrombotic syndrome by half. It is not known if shorter or longer treatment would have

similar or more pronounced effects. Knee-high stockings are sufficient for the vast majority of patients; they should be of compression class II (20–30 mmHg), fitted individually and should be worn whenever the patient is not recumbent.

Chronic thromboembolic pulmonary hypertension A prospective long-term follow-up study of patients after PE showed that 3.8% of patients at 2 years had evidence of chronic thromboembolic pulmonary hypertension, suggesting that it is a relatively common complication of PE. The diagnosis should be considered in those with dyspnoea after PE. Pulmonary endarterectomy is the first treatment of choice in patients with severe pulmonary hypertension, and it reduces pulmonary vascular resistance dramatically, although the perioperative mortality is 5–9%. Five-year survival of over 80% has been described.

Selected bibliography Anderson DR, Kovacs MJ, Kovacs G et al. (2003) Combined use of clinical assessment and D-dimer to improve the management of patients presenting to the emergency department with suspected deep vein thrombosis (the EDITED Study). Journal of Thrombosis and Hemostasis 1: 645–51. Baglin T (2010) Defining the population in need of thromboprophylaxis – making hospitals safer. British Journal of Haematology 149: 805–12. British Committee for Standards in Haematology (2005) Guidelines on oral anticoagulation (warfarin): third edition, 2005 update. British Journal of Haematology 132: 277–85. British Committee for Standards in Haematology (2006) Guidelines on the use of vena cava filters. British Journal of Haematology 134: 590–5. British Committee for Standards in Haematology (2006) The management of heparin-induced thrombocytopenia. British Journal of Haematology 133: 259–69. British Thoracic Society Standards of Care Committee Pulmonary Embolism Guideline Development Group (2003) British Thoracic Society guidelines for the management of suspected acute pulmonary embolism. Thorax 58: 470–84. Caprini JA (2010) Risk assessment as a guide for the prevention of the many faces of venous thromboembolism. American Journal of Surgery 199 (1 Suppl.): S3–10. Review. Cohen AT, Tapson VF, Bergmann JF et al. (2008) Venous thromboembolism risk and prophylaxis in the acute hospital care setting (ENDORSE study): a multinational cross-sectional study. Lancet 371: 387–94. Connolly SJ, Ezekowitz MD, Yusuf S et al. (2009) Dabigatran versus warfarin in patients with atrial fibrillation. New English Journal of Medicine 361: 1139–51. 913

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Geerts WH, Bergqvist D, Pineo GF et al. (2008) Prevention of venous thromboembolism: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th edition). Chest 133 (6 Suppl.): 381S–453S. Hunt BJ (2009) The prevention of hospital-acquired venous thromboembolism in the United Kingdom. British Journal of Haematology 144: 642–52. Pengo V, Lensing AW, Prins MH et al. (2004) Incidence of chronic thromboembolic pulmonary hypertension after pulmonary embolism. New England Journal of Medicine 350: 2257–64. Prandoni P, Kahn SR (2009) Post-thrombotic syndrome: prevalence, prognostication and need for progress. British Journal of Haematology 145: 286–95. Schulman S (2003) Care of patients receiving long-term anticoagulant therapy. New England Journal of Medicine 349: 675–83. Thromboembolic Disease in Pregnancy and the Puerperium: Acute Management (Green-top 28). Available at http://

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www.rcog.org.uk/files/rcog - corp/uploaded - files/GT28 ThromboembolicDisease2007.pdf Thromboprophylaxis during Pregnancy, Labour and after Vaginal Delivery (Green-top 37). Available at http://www.rcog.org.uk/ files/rcog - corp/uploaded - files/GT37Thromboprophylaxis 2004.pdf van der Heijden JF, Hutten BA, Büller HR, Prins MH (2002) Vitamin K antagonists or low-molecular-weight heparin for the long term treatment of symptomatic venous thromboembolism. Cochrane Database of Systematic Reviews (1): CD002001. Wadelius M, Chen LY, Lindh JD et al. (2009) The largest prospective warfarin-treate dcohort supports genetic forecasting. Blood 113: 784–92. Wells PS, Anderson DR, Rodger M et al. (2001) Excluding pulmonary embolism at the bedside without diagnostic imaging: management of patients with suspected pulmonary embolism presenting to the emergency department by using a simple clinical model and D-dimer. Annals of Internal Medicine 135: 98–107.

CHAPTER 48

Congenital platelet disorders Maurizio Margaglione1 and Paul RJ Ames2

48

1

University of Foggia, Foggia, Italy Royal Preston Hospital, Preston UK

2

Introduction, 915 Thrombocytopenias, 916 Congenital non-inherited thrombocytopenia, 916 Inherited thrombocytopenias, 917 Thrombocytopathies, 919

Disorders of platelet adhesion, 919 Disorders of platelet signalling transduction, 921 Disorders of platelet aggregation, 921 Therapy, 924 General measures, 924

Introduction In haemostasis, a sequence of local events culminates in spontaneous arrest of bleeding from a traumatized blood vessel. The normal haemostatic mechanisms are sufficient to seal any vascular discontinuity. Three closely linked biological systems are involved: platelets, blood vessels and coagulation proteins. Platelets, anucleate cells that are derived from the cytoplasm of bone marrow megakaryocytes, circulate in the bloodstream for about 7–10 days. In the resting state platelets do not normally interact with endothelial cells or other blood cells but do so when the vessel wall is disrupted. Adhesion to exposed subendothelial components allows platelet activation and release of active substances (e.g. ADP) from intracellular organelles that amplify and propagate activation and, finally, platelet aggregation that plugs the ruptured blood vessel. At this primary stage of haemostasis platelets stop bleeding from damaged blood vessels transiently and provide the surface which promotes blood coagulation that strengthens the platelet plug (the secondary phase of haemostasis). Clinically, abnormalities of platelet adhesion and aggregation exhibit a history of spontaneous and/or easy bruising and a prolonged skin bleeding time. The characteristic clinical features of abnormal bleeding due to platelet deficiencies are distinct from those seen in disorders of plasma coagulation factors (Table 48.1). The occurrence of superficial bleeding in patients with platelet disorders as opposed to the deeper bleeding in patients with clotting factor disorders is a useful clinical pointer, but it must be remembered

Postgraduate Haematology: 6th edition. Edited by A. Victor Hoffbrand, Daniel Catovsky, Edward G.D. Tuddenham, Anthony R. Green © 2011 Blackwell Publishing Ltd.

Drugs, 924 Platelet transfusions, 924 Other measures, 924 Conclusions, 925 Selected bibliography, 926

that the activities of platelets and coagulation factors are closely related. Bleeding associated with platelet abnormalities manifests as haemorrhages from small vessels. Petechiae usually develop on the skin and the visible mucous membranes, but they may be distribuited throughout the body including internal organs. Characteristically, bleeding resulting from platelet diseases is immediate and transient, tends to stop promptly with local pressure and does not recur when the pressure is removed. When this pattern of bleeding occurs in the neonatal period, infancy and childhood, a congenital platelet disorder should be suspected. However, the disease may be clinically silent and the patient may enter adult life before bleeding occurs. The family history may be of great importance, providing a characteristic pattern of inheritance, but it should be remembered that a negative family history does not exclude an inherited platelet abnormality, i.e. the family history is usually negative in autosomal recessive traits. A comprehensive medical history and a careful clinical examination of the patient presenting with a haemorrhagic disorder is crucial for the correct diagnosis. Subsequent laboratory tests chosen on the basis of the clinical information obtained will lead to a precise definition, where possible, of the platelet abnormality. Congenital defects of platelets may give rise to bleeding syndromes of varying severity and are difficult to classify because of the rarity of many forms, the extreme heterogeneity, and also because of incomplete knowledge about a variety of diseases. Table 48.2 classifies congenital platelet disorders into two main groups: thrombocytopenias and thrombocytopathies. Each group is further divided according to specific criteria based on functional and biochemical defects. Such a classification, as others, should be viewed as tentative, because some disorders may be characterized by multiple pathogenetic factors whereas others are grouped together for convenience of classification rather than on their known pathophysiology. 915

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Table 48.1 Main specific clinical differences between diseases of coagulation factors and platelet disorders. Findings

Disorders of coagulation

Platelets/vessels

Onset of bleeding

Delayed after trauma

Mucosal bleeding Petechiae Deep haematomas Ecchymoses Haemarthrosis Bleeding from superficial cuts and scratches Sex of patient

Rare Rare Characteristic Large and solitary Characteristic Minimal

Spontaneous or immediately after trauma Common Characteristic Rare Small and multiple Rare Persistent; often profuse

80–90% male

Equal

Table 48.2 Classification of congenital platelet disorders. Thrombocytopenia Non-inherited thrombocytopenia Drugs and chemical agents Isoimmune thrombocytopenia Infiltration of bone marrow Infections Other causes Inherited thrombocytopenias Thrombocytopenias with reduced platelet size Thrombocytopenias with normal platelet size Thrombocytopenias with increased platelet size Thrombocytopathies Disorders of platelet adhesion Disorders of platelet signalling transduction Disorders of platelet aggregation

Thrombocytopenias Thrombocytopenia defines a subnormal number of platelets in the circulating blood, usually below 100 × 109/L. Acute thrombocytopenia is the most frequent cause of severe bleeding and the risk of haemorrhage is inversely proportional to the platelet count, with spontaneous bleeding occurring frequently at a platelet count below 20 × 109/L. When automated methods are used, ‘artefactual thrombocytopenia’ or pseudothrombocytopenia can be observed. Different mechanisms can cause a falsely low platelet reading. This may happen in patients with a wide variety of clinical disorders. Non-technical factors inducing

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pseudothrombocytopenia include paraproteinaemias, cold agglutinins, giant platelets, previous contact of platelets with foreign surfaces (i.e. dialysis membrane), lipaemia and EDTAinduced platelet clumping. The possibility of a pseudothrombocytopenia must be ruled out through manual counting and/ or examination of an adequately stained blood film before concluding that a patient has true thrombocytopenia.

Congenital non-inherited thrombocytopenia Congenital thrombocytopenia not ascribed to inherited causes may be the result of a series of pathogenetic mechanisms, most likely attributable to deficient marrow platelet production or enhanced platelet destruction. Drugs and chemical agents The maternal use of drugs and chemical agents may cause thrombocytopenia by suppressing platelet production, by damaging platelets directly or by inducing the formation of platelet antibodies. Maternal ingestion of ethanol, thiazides, chlorpropamide, tolbutamide, estrogens, steroids and other drugs may selectively suppress thrombopoiesis. Alkylating agents, antimetabolites or chemotherapeutic drugs capable of crossing the placenta may lead to severe thrombocytopenia in newborns as the result of a predictable suppression of the bone marrow. The maternal ingestion of quinine, quinidine, hydralazine or selected antibiotics known to induce immune-mediated thrombocytopenia may induce congenital immune-mediated thrombocytopenia. In these newborns, thrombocytopenia usually improves rapidly and very rarely gives rise to severe or fatal haemorrhages. Alloimmune thrombocytopenia Immune thrombocytopenia may also result from the placental transfer of platelet antibodies formed as the result of active immunization of the mother by fetal platelet isoantigens, if the fetus has inherited a paternal platelet-specific antigen that induces antibody formation. Transplacental passage of the maternal antibody, usually of the IgG isotype, may induce severe thrombocytopenia in the fetus. This usually occurs when the mother has PlA1-negative platelets and the fetus carries PlA1-positive platelets. Newborns may show petechiae and purpura, or more severe bleeding at the time of birth, or may appear normal at delivery and then manifest severe bleeding within the first week after birth. In this case, thrombocytopenia usually improves within 1 month but severe and fatal intracranial haemorrhages may occur. Bone marrow infiltration Congenital thrombocytopenia caused by bone marrow infiltration is extremely rare, being limited to cases of disseminated reticuloendotheliosis and congenital leukaemia. Thrombocytopenia with or without associated myeloid and

Congenital platelet disorders

erythroid depression occurs in children with numerous infiltrative disorders, including solid tumours, myelofibrosis, Gaucher disease, Niemann–Pick disease and the mucopolysaccharidoses. Infections Thrombocytopenia, usually mild but sometimes very severe, is commonly seen in infected newborns and several mechanisms are probably responsible. In fact, impaired platelet production as a result of invasion of megakaryocytes, the destruction of circulating platelets and the formation of antigen–antibody complexes may explain many instances of thrombocytopenia associated with viral infections. Maternal infection with toxoplasma, cytomegalovirus, rubella, herpesviruses or hepatitis varicella, as well as recent maternal vaccinations (rubella), may induce congenital thrombocytopenia. Other causes Occasionally, children born to women with chronic idiopathic thrombocytopenic purpura manifest congenital thrombocytopenia. It is conceivable that autoantibodies causing the disease in the mother cross the placenta and bind fetal platelets. Other causes of congenital thrombocytopenia due to increased platelet consumption or destruction include common maternal disorders such as pre-eclampsia, systemic lupus erythematosus or other autoimmune diseases, especially if the woman has antiphospholipid antibodies. A moderate to severe thrombocytopenia has been observed in association with giant cavernous haemangioma, first described by Kasabath and Merritt, in which the consumption of platelets occurs primarily within the tumour. Thrombocytopenia is often found associated with coagulation abnormalities typical of disseminated intravascular coagulation and the severity tends to parallel the size of the vascular tumour.

Inherited thrombocytopenias Several inherited diseases may present with thrombocytopenia as an accompanying feature of generalized bone marrow failure (e.g. Fanconi anaemia) or metabolic diseases causing marrow infiltration (e.g. Gaucher disease). These and other complex clinical syndromes (e.g. Noonan syndrome) are not discussed in this chapter. Inherited thrombocytopenias are very rare. In some of these diseases thrombocytopathy may coexist with thrombocytopenia (e.g. Bernard–Soulier syndrome, Wiskott– Aldrich syndrome). These diseases are discussed in the appropriate section according to the most prominent defect. As proposed by the Italian Working Group, both platelet size and the presence (syndromic) or absence (non-syndromic) of other clinical features, different from those deriving from platelet abnormalities, are helpful criteria for classifying inherited thrombocytopenias.

Inherited thrombocytopenias with reduced platelet size Wiskott–Aldrich syndrome (WAS) is a rare (1 in 250 000) X-linked recessive disorder characterized by eczema, susceptibility to infections associated with defects in cellular and humoral immunity, and thrombocytopenia with reduced platelet size. The gene responsible for WAS maps to Xp11.4 and codes for a protein, WASp, expressed only in haemopoieticderived cells and which is involved in the transduction of signals from the receptor to the actin cytoskeleton. Intermittent bleeding, recurrent bacterial and viral infections, and progressive eczema occur during the first months of life. Death at an early age commonly results from intracranial haemorrhage, infection or lymphoreticular malignancy. A number of kindreds have been reported in whom X-linked thrombocytopenia (XLT) occurred alone or in association with partial manifestations of WAS. XLT is caused by a mutation within the WAS gene. Patients with XLT mainly suffer from an isolated bleeding tendency.

Inherited thrombocytopenias with normal platelet size Congenital deficiency of megakaryocytes is a rare form of thrombocytopenic purpura in the newborn and may occur with skeletal, renal or cardiac malformations. Isolated congenital amegakaryocytic thrombocytopenia is an autosomal recessive syndrome leading to bone marrow aplasia later in childhood. This disorder is associated with abnormalities in the expression or function of the thrombopoietin receptor c-Mpl, and a series of mutations in the c-Mpl gene (THPO) have been identified. Most commonly, associated skeletal anomalies are present. Bilateral agenesis of the radius is the most commonly associated abnormality, so-called thrombocytopenia with absent radius (TAR) syndrome. The TAR syndrome is an autosomal recessive disease characterized by severe, even fatal, haemorrhagic manifestations. Although abnormalities in the thrombopoietin/ c-Mpl signalling pathway have been suggested, no mutations have been found in genes coding for these proteins. In some infants, the ulna and humerus may also be absent and other skeletal abnormalities may occur. In addition, proximal radio-ulnar synostosis, syndactyly and other skeletal abnormalities are reported in association with an autosomal dominant congenital amegakaryocytic thrombocytopenia in patients carrying heterozygous mutations of the HOXA11 gene. Less commonly, patients manifest cardiac and other minor defects. Schulman–Upshaw syndrome, which is caused by mutations in the ADAMTS13 gene, is characterized by thrombotic thrombocytopenic purpura with neonatal onset (congenital microangiopathic haemolytic anaemia), thrombocytopenia and frequent relapses, and response to fresh plasma infusion.

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Inherited thrombocytopenias with increased platelet size Thrombocytopenias with increased platelet size are characterized by the presence of an increased platelet volume with a reduced number of platelets (megathrombocytopenia) and are the commonest inherited forms of thrombocytopenias. Among them, a series of diseases have been characterized at the molecular level, whereas other clinical entities still await clear identification of the molecular defect. Giant platelets and moderate thrombocytopenia are most frequently found in certain populations of Mediterranean extraction and may also be associated with other inherited or congenital syndromes, such as May– Hegglin anomaly, Bernard–Soulier syndrome and stomatocytosis. May–Hegglin anomaly is a rare autosomal dominant disease characterized by giant platelets and basophilic inclusions (Döhle bodies) within granulocytes. Large Döhle bodies are seen in peripheral blood and in bone marrow granulocytes, and most patients show mild neutropenia but no significant susceptibility to infection. About 50% of patients have significant thrombocytopenia deriving from ineffective thrombopoiesis, but the occurrence of life-threatening haemorrhage is rare. In patients suffering from May–Hegglin anomaly the platelets show not only a greatly increased (twice normal) volume but most of them also display bizarre morphology and hypergranularity. Closely related to the May–Hegglin anomaly is a group of other autosomal dominant diseases known as Sebastian, Fetchner and Epstein syndromes. Their main characteristic is the presence in almost all patients of giant platelets with thrombocytopenia. In addition, patients may develop sensorineural hearing loss, cataract and glomerulonephritis, and most of them display Döhle bodies. The exact classification of patients according to the presence or absence of the above-mentioned clinical signs is shown in Table 48.3. The molecular basis of these syndromes has been elucidated. All these clinical entities are caused by mutations that occur within a gene (MYH9) located on the long arm of chromosome 22 (22q13.1). This gene codes for the heavy chain of non-muscle myosin IIA (NMMHC-IIA), a protein involved in the contractile activity of the cytoskeleton. Grey platelet syndrome (GPS) is a rare disorder inherited as an autosomal dominant trait, although recessive transmission has been shown. It is characterized by large platelets with a

selective deficiency in the number and content of α-granules. As a result of this, platelets are either markedly hypogranular or agranular and display deficiency of α-granule proteins, such as fibrinogen, von Willebrand factor, thrombospondin, β-thromboglobulin and platelet factor 4. Recently, a missense mutation has been identified at position 759 in the GATA1 gene that induces an amino acid change (Arg216Gln) which segregates in an X-linked fashion, suggesting that the GATA1 gene is required for regulation of platelet α-granules. Thrombocytopenia is usually pronounced and severe bleeding may occur. Mediterranean macrothrombocytopenia is an asymptomatic disorder with moderate isolated thrombocytopenia and large platelets inherited as an autosomal dominant trait. The condition is characterized by mild or no clinical manifestations and normal bone marrow megakaryocytosis, platelet survival and in vitro platelet functions. Platelets from some patients suffering from Mediterranean macrothrombocytopenia show reduced expression of the GPIb/IX platelet receptor and heterozygous mutations within the GPIbα (GP1BA) or GPIbβ (GP1BB) genes have been described. Therefore, in these patients Mediterranean macrothrombocytopenia may be classified as a heterozygous form of Bernard–Soulier syndrome (see below). The remaining patients with Mediterranean macrothrombocytopenia do not show a reduction in the content of the GPIb/IX platelet receptor and the pathogenesis of this form remains to be clarified. A thrombocytopenia resulting from enhanced platelet destruction may be present in type 2B von Willebrand disease. Patients suffering from this syndrome show a qualitative abnormality of plasma von Willebrand factor (VWF) such that VWF binds inappropriately to circulating platelets. The molecular bases are mutations within the VWF gene, usually in exon 28, that give rise to increased reactivity of VWF with the platelet receptor GPIb/IX. Clearance of the resulting VWF–platelet complexes leads to thrombocytopenia and the selective loss of the largest VWF multimers from plasma. In general, the degree of thrombocytopenia is moderate and bleeding is of variable severity. A mutation in the VWF gene has been identified in a kindred previously labelled Montreal platelet syndrome, a very rare platelet disorder characterized by giant platelets, reduced platelet count and spontaneous in vitro platelet aggregation; this

Table 48.3 Main clinical differences among syndromes due to mutations within the MYH9 gene. Disease

Macrothrombocytopenia

Renal desease

Hearing loss

Cataract

Leucocyte inclusion*

MHA SBS EPS FTS

Present Present Present Present

Absent Absent Present Present

Absent Absent Present Present

Absent Absent Absent Present

Present Present Absent Present

*Ultrastructural differences in leucocyte inclusions exist (see Balduini et al. 2003). EPS, Epstein syndrome; FTS, Fetchner syndrome; MHA, May–Hegglin anomaly; SBS, Sebastian syndrome. 918

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kindred has now been reclassified as having type 2B von Willebrand disease. Type 2B von Willebrand disease should be distinguished from the rare pseudo von Willebrand disease (or platelet-type von Willebrand disease). The defect in this autosomal dominant condition results from mutations in the gene encoding the GPIbα subunit of the platelet receptor GPIb/IX. The few mutations identified give rise to a gain of function of the platelet receptor and increase the affinity for VWF, resulting in spontaneous binding of platelets to VWF and leading to shortened platelet survival and thrombocytopenia. The reasons why most patients suffering from platelet-type von Willebrand disease show variable enlarged platelets remain unclear. Other very uncommon causes of macrothrombocytopenia are Jacobsen syndrome and Paris–Troussau syndrome. Both are syndromic diseases inherited as autosomal dominant traits and characterized by chromosomal deletion encompassing the same region of chromosome 11 (11q23.3–q24.2). A heterozygous deletion of the FLI1 gene has been identified in unrelated children with deletions of 11q23 and Paris–Trousseau thrombocytopenia. Interestingly, in patients suffering from Jacobsen syndrome without thrombocytopenia, the chromosomal deletion did not include the FLI1 gene, suggesting that deletion of this gene is responsible for the thrombocytopenia that occurs in most patients with Jacobsen and Paris–Trousseau syndromes.

surface, become activated, release the contents of their storage organelles, and aggregate to each other. Abnormalities in any of these stages – adhesion, activation, secretion and aggregation – may give rise to congenital disorders of platelets. Patients suffering from any of these diseases usually show a bleeding diathesis with a prolonged bleeding time and a normal platelet count.

Disorders of platelet adhesion The interaction of platelet receptors with elements of the subendothelium, collagen, fibronectin and blood components allows platelet adhesion to the subendothelium, but it also occurs through the bridging effect of VWF (Figure 48.2). A series of receptors have been identified on the platelet surface and they interact with one or more of these elements. The most important receptors are GPIb/IX and α2β1 (previously known as GPIa/IIa). The α2β1 receptor is one of the receptors on the platelet surface that binds collagen and is a member of the integrin β1 subfamily. Other receptors for collagen are GPIV and GPVI. Binding of molecules to these receptors leads to the subsequent binding to other receptors, which serves to reinforce adhesion and to generate intracellular signals, such as calcium mobilization and protein phosphorylation. Bernard–Soulier syndrome Bernard–Soulier syndrome (BSS) is a bleeding disorder characterized by giant platelets on the blood smear, mild or moderate thrombocytopenia, and prolongation of the skin bleeding time disproportionate to the thrombocytopenia. BSS is a recessively inherited autosomal disorder and consanguinity is common in reported kindreds. Based on reported data, the frequency of BSS has been estimated to be approximately 1 in 1 million population. Bleeding may be severe and fatal haemorrhages may occur.

Thrombocytopathies Platelets have a complex ultrastructure comprising a multitude of molecules and the malfunctioning of any of these may give rise to a specific disease (Figure 48.1). Platelets participate in haemostasis by adhering to exposed elements of the subendothelial matrix. They then spread onto the subendothelial

Glanzmann thrombasthenia Defects of thrombopoietin signalling: CAMT

Dense granule disorders

Ibα Ibβ

β3

IX

Bernard–Soulier platelet-type vWD

V

αIIb

Ibα Ibβ IX

c-mpl

Defects of cytoskeleton function: MHA, WAP, etc.

α-Granule disorders

Figure 48.1 Schematic representation of the complex structure of platelets. Abnormalities of any platelet complex can lead to an alteration in a specific function.

α2

Receptor defects: ADP, TXA2, etc. GPIV Signalling defects and enzyme deficiencies

GPVI

β1

Defective binding to or signalling with collagen

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(a)

(b)

Endothelium (c)

(d)

Endothelium

Cutaneous haemorrhages and muscle and visceral bleeding are common. Epistaxis and menorrhagia may be difficult to control. Haemarthrosis has also been reported. Platelet counts range from as low as 20 × 109/L to near normal and, on the peripheral blood film, over 80% of platelets are usually larger than 2.5 µm, often up to 8.0 µm in diameter. The number of bone marrow megakaryocytes is usually normal. Patients presenting with BSS show absent platelet agglutination in response to ristocetin (in the presence of human VWF) and normal aggregation, ATP secretion and thromboxane (Tx) B2 formation in response to a variety of aggregating agents, and delayed response to thrombin. Biochemical and cellular factors contributing to these abnormalities have been clarified. Because of the defective binding with VWF, platelets in BSS do not agglutinate in response to ristocetin and have substantial reduction in their ability to adhere to sites of vascular injury where subendothelial VWF becomes exposed. As a consequence, plug formation, the primary haemostatic response, is impaired in BSS and increased and prolonged bleeding occurs. Variable levels of the GPIb–IX–V complex have been detected on the platelet surface of patients with BSS, some patients with BSS exhibiting nearly normal amounts (variant type of BSS). Despite the difference in glycoprotein content, the clinical bleeding problems and platelet functional and morphological abnormalities of these patients were indistinguishable from the classical BBS phenotype. The GPIb–IX–V receptor complex provides the principal site mediating the interaction of platelets with the adhesive VWF. The entire cDNA sequences encoding the protein chains comprising this receptor have been deduced, allowing studies on the molecular basis of the syndrome. This complex consists of four proteins: the disulphide-linked α-chain (135 kDa) and β-chain 920

Figure 48.2 Platelets circulate in the bloodstream and do not interact with endothelial cells (a) unless the vessel wall is damaged (b). Adhesion of platelets to injured blood vessel wall (c) and spreading on it (d) are possible through the interaction of platelet receptors with their ligands and VWF–collagen.

(25 kDa) of GPIb and the non-covalentely associated subunits GPIX (22 kDa) and GPV (82 kDa). They all share structural and functional features suggesting a common evolutionary origin. Different transcripts encode the four polypeptide chains and, with the exception of that of GPIbβ, genes show continuous (intron-depleted) open reading frames. In addition, each element contains one or more homologous 24-amino-acid leucine-rich glycoprotein repeats. The genetic heterogeneity in the glycoprotein content of BSS patients shows that multiple molecular abnormalities may lead to a similar clinical disorder, and implies that BSS may be the result of defects within the subunits that hamper the coordinate expression of the complex on the platelet membrane. In this respect, BSS would resemble abnormalities of other multi-subunit complexes, in which a defect in a single subunit prevents the assembly and surface expression of the complex. In addition to quantitative and qualitative abnormalities of the GPIbα gene, the recognized BSS phenotype has also been documented in patients with detrimental mutations within platelet GPIX and GPIbβ genes. Very few patients with a defect in one of the platelet receptors for collagen have been described. These patients show mild bleeding disorders and a selective impairment, at a variable extent, in collagen response, adhesion to subendothelial surfaces and collagen-induced platelet aggregation. Platelets have two major receptors for collagen, the α2β1 integrin, with a major role in adhesion of platelets to subendothelial surfaces, and GPVI, mainly involved in platelet activation. Thus, it is conceivable that collagen binding to platelets occurs through a multistep mechanism involving first the attachment of platelets to exposed collagen of the subendothelium in flowing blood by means of the α2β1 receptor and then platelet activation through a second receptor, GPVI.

Congenital platelet disorders

Disorders of platelet signalling transduction Platelets that adhere to the subendothelial surface become activated and begin the production or release of several intracellular messengers, which modulate a series of platelet responses such as calcium mobilization, protein phosphorylation and production of arachidonic acid. Activated platelets also release substances stored in their granules, some of which act in the recruitment of additional platelets and lead to the formation of the primary haemostatic plug (Figure 48.3). Several signalling mechanisms are involved in events that govern platelet responses, starting with platelet adhesion to injured blood vessel and leading to secretion and aggregation. Available evidence suggests that specific abnormalities in platelet signalling mechanisms may be the basis of platelet dysfunction, and the term ‘platelet signalling disorders’ defines a group of heterogeneous abnormalities in platelet secretion and signal transduction. Congenital defects of platelet signalling mechanisms are grouped together for convenience of classification rather than by the pathophysiology of specific diseases. Patients suffering from these defects represent the vast majority of subjects presenting with inherited thrombocytopathies. Many platelet receptors are coupled to G proteins: P2Y1 and TxA2 receptors and thrombin act via Gαq; prostaglandin recep-

(a)

tors activate whereas P2Y12 receptors inhibit adenylate cyclase through Gs and Gi2 respectively. Platelet activation by means of binding to receptors gives rise to hydrolysis of phosphoinositide by phospholipase (PL)C, leading to the formation of inositol trisphosphate, which in turn functions as a messenger to release calcium from intracellular stores. In addition, hydrolysis of phosphoinositide leads to the formation of diacylglycerol that activates protein kinase C. The activation of protein kinase C is thought to play a major role in platelet secretion and in the activation of the αIIbβ3 complex. Although defects in PLC activation, calcium mobilization and protein phosphorylation have been suggested in several patients, unique patients have been reported with deficiency in platelet Gαq, Gs hyperfunction and reduced expression of PLCβ2. Following stimulation, platelet PLA2 mobilizes arachidonic acid from the phospholipid pool. The arachidonic acid is then metabolized by cyclooxygenase and thromboxane synthase to form TxA2, a strong platelet-aggregating agent that is necessary for the secretion response. A defect in arachidonic acid mobilization and TxA2 production has been identified in some patients. Few patients with impaired liberation of arachidonic acid have been described. Platelets in individuals with this defect show abnormal aggregation and secretion in response to a series of stimulating factors but normal production of TxA2 in response to arachidonic acid. Several patients have been reported with deficiency of cyclooxygenase and show a slightly prolonged bleeding time and impaired platelet aggregation. In addition, a few patients show a defect in TxA2 formation and present with a variable bleeding diathesis.

Disorders of platelet aggregation (b)

ADP

ADP

ADP

(c)

Figure 48.3 Platelets, after adhering to the subendothelial surface (a), become activated, release intracellular messengers (b) and aggregate each other leading to the formation of the primary haemostatic plug (c).

Platelet aggregation may be defined as the interaction of activated platelets with one another and occurs after adhesion of platelets to the wall of the injured blood vessel. A series of factors are capable of inducing platelet aggregation and may be classified as primary and secondary platelet-aggregating agents. Primary aggregating agents are those factors, such as ADP, adrenaline and thrombin, able to directly induce platelet aggregation independently of their ability to release intraplatelet ADP or to induce the production of prostaglandins. Secondary aggregating agents are factors that induce aggregation of platelets through their ability to provoke the release reaction of ADP or the synthesis of prostaglandins. Accordingly, disorders due to impairment of platelet aggregation may be classified into defects of primary aggregation (Glanzmann thrombasthenia), selective impairment of platelet receptors (ADP, adrenaline) and defects of secondary aggregation (storage pool disease, selective impairment of platelet receptors such as thromboxane and collagen). In vitro, a series of substances are employed to challenge platelets and the manner in which they respond to these stimuli may be helpful for identifying specific thrombocytopathies (Table 48.4). 921

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Table 48.4 Platelet response to aggregating agents in different thrombocytopathies. Disorder

ADP

Adrenaline

Collagen

Arachidonic acid

Ristocetin

Bernard–Soulier Pseudo von Willebrand disease

Normal Normal

Normal Normal

Normal Normal

Normal Normal

ADP receptor defect Adrenaline receptor defect Collagen receptor defect Defect of signal transduction

Impaired Normal Normal Variable impairment Absent Impaired Impaired

Impaired Impaired Normal Variable impairment Absent Impaired Impaired

Impaired Normal Impaired Variable impairment Absent Impaired Impaired

Impaired Normal Normal Variable impairment Absent Variable Impaired

Absent Increased at low doses Present Present Present Present

Glanzmann thrombasthenia δ-Storage pool disorder Thromboxane receptor defect

Defects of primary aggregation: Glanzmann thrombasthenia Glanzmann thrombasthenia (GT) is a bleeding diathesis marked by prolonged bleeding time, normal platelet count, and absence of platelet aggregation in response to platelet agonists such as ADP, collagen, arachidonic acid and thrombin. Platelet agglutination induced by ristocetin and VWF is normal. This congenital bleeding disorder is associated with impaired or absent clot retraction. GT is one of the less common of the congenital bleeding disorders (prevalence 1 in 1 million) and is transmitted as an autosomal recessive trait; consanguinity has been reported in affected kindreds. The clinical features are those expected with platelet dysfunction: easy and spontaneous bruising, mucosal membrane bleeding, subcutaneous haematomas, and petechiae. Rarely, patients suffer from intra-articular bleeding with resultant haemarthroses. Fatal haemorrhages have been reported. Quantitative or qualitative (variant GT forms) abnormalities of the platelet αIIbβ3 integrin (also known as GPIIb/IIIa complex) have been shown to be responsible for this disorder. Mutations within the genes that code for αIIbβ3 subunits have been described in GT patients. As with other integrins, αIIb and β3 subunits are prominent integral components of the platelet membrane that form heterodimers containing specific sites for platelet–platelet cohesion. The αIIbβ3 integrin serves as a platelet receptor for fibrinogen, fibronectin, vitronectin and VWF. In addition, the αIIbβ3 integrin modulates, to some extent, calcium influx, cytoplasmic alkalinization, tyrosine kinase phosphorylation and clot retraction. The clinical heterogeneity of GT has been stressed on the basis of platelet function testing or using crossed immunoelectrophoresis, Western blot, flow cytometry and fibrinogen binding. Type I GT is characterized by the lack of surfacedetectable αIIbβ3 complex and a profound defect in platelet aggregation and clot retraction. At variance with type I, platelets of patients suffering from type II GT have detectable, but mark922

Present Present Present

edly reduced, amounts of the αIIbβ3 receptor on their surface, usually 10–20% of normal values. Platelets manifest sufficient amounts of receptors to allow microaggregate formation, although there is still a profound defect in the ability to form large aggregates. Clot retraction is only moderately impaired. In addition, a series of patients with a variant form have been described who present near-normal levels of the αIIbβ3 complex, which is dysfunctional in that platelets, when activated, can neither aggregate nor bind fibrinogen. Extreme variability in the clinical symptoms is present, even among patients with similar degrees of platelet abnormality and prolongation of the bleeding time. In general, no aggregation abnormalities are detected in heterozygotes, but a decreased amount of the αIIbβ3 integrin has been reported, platelet content being approximately half of normal. Selective impairment of platelet receptors Defects of platelet ADP receptors have been characterized in a few patients and all suffered from a bleeding diathesis. On the platelet surface, different types of ADP receptors have been identified, including two G protein-coupled receptors, P2Y1 and P2Y12. The P2Y1 receptor is responsible for the shape change of platelets and transient platelet aggregation, giving rise to centralization of platelet granules and formation of filopodia, while the P2Y12 receptor is involved in amplification of the response and in the stabilization of platelet aggregates through full activation of the αIIbβ3 integrin. The P2Y12 receptor defect is inherited as an autosomal recessive trait and most patients so far identified were born from consanguineous parents. In these patients, blunted platelet aggregation in response to ADP, with a retained shape change, has been reported. Only one patient has been briefly reported with a defect of the P2Y1 receptor. This patient showed impaired platelet aggregation in response to ADP and other agonists. The selective impairment of adrenaline (α2-adrenergic) receptors has been associated with bleeding. A number of indi-

Congenital platelet disorders

viduals with an impaired aggregation response to adrenaline and a congenital defect of platelet α2-adrenergic receptors have been described and some of them presented a history of easy bruising. Defects of secondary aggregation Secondary aggregation disorders are more frequent than primary aggregation disorders and the most common in this category are the storage pool deficiency (SPD) syndromes. SPD syndromes may be classified in a system that takes into account the content of both dense and α granules (Figure 48.4). Thus δ-SPD identifies patients who show low platelet content of dense granules only. Patients with deficiency of both types of granules are designated αδ-SPD. Finally, patients presenting with reduced or absent platelet content of α-granules but normal levels of dense granules (α-SPD) are categorized as patients with GPS. The disorder is heterogeneous and the term SPD includes a group of disorders having as their common feature a diminution in secretable substances stored in platelet granules. A storage pool disease is found as an associated defect in most of the patients carrying other rare syndromes, such as WAS and the TAR syndrome. However, the majority of patients presenting with an SPD have no associated diseases and are otherwise normal. The clinical features of this type of secondary aggregation disorder are those expected with a platelet function defect and consist of easy and spontaneous bruising, mucocutaneous haemorrhages, haematuria and epistaxis. Patients with δ-SPD or αδ-SPD usually have absent ADP- and adrenaline-induced secondary aggregation waves, although the primary waves are present. Patients with δ-SPD may present a severe or partial ADP Serotonin β-Thromboglobulin Dense granule

Platelet factor 4 Fibronectin

α-Granule

Platelet-derived growth factor von Willebrand factor Fibrinogen Factor V

Figure 48.4 In platelets, granules are the storage site for substances that are important in the haemostatic process. The α-granules contain proteins involved in adhesion (fibronectin, VWF), cell–cell interaction (P-selectin) and in promoting coagulation (FV, platelet factor 4), whereas the content of dense granules is important in recruiting additional platelets (ADP, serotonin).

granule deficiency. Collagen-induced aggregation is absent or markedly reduced, whereas ristocetin-induced agglutination is normal. In several kindreds, the disorder appeared to be inherited as an autosomal dominant trait. However, in other families the type of inheritance could be not determined. In the absence of other congenital abnormalities or an associated α-granule deficiency, SPD is inherited as an autosomal dominant trait. The other forms of δ-SPD coincident with other congenital abnormalities, such as TAR, are usually inherited as autosomal recessive traits, or as X-linked traits as in the case of WAS. An SPD is usually present in Hermansky–Pudlak syndrome and in Chédiak–Higashi syndrome. Hermansky–Pudlak syndrome (HPS) is a rare autosomal recessive inherited disorder characterized by the presence of oculocutaneous albinism, absence of platelet dense granules, and infiltration of ceroid-pigmented reticuloendothelial cells in the lung and the colon, leading to pulmonary fibrosis and inflammatory bowel disease. HPS results from the abnormal formation of intracellular vesicles. The impaired function of specific organelles indicates that the causative genes encode proteins operative in the formation of lysosomes and vesicles. HPS displays genetic heterogeneity and, so far, mutations in eight orthologous genes have been found in patients with this disease: HPS1, HPS2 (AP3B1), HPS3, HPS4, HPS5, HPS6, HPS7 (DTNBP1) and HPS8 (BLOC1S3). In Chédiak–Higashi syndrome (CHS) a variable degree of oculocutaneous albinism and a poor resistance to respiratory and cutaneous infections are usually found. The infections are generally fatal during infancy or in early childhood but patients may also die of a chronic lymphohistiocytic infiltration known as the accelerated phase during the second or third decades of life. CHS is extremely rare and is inherited as an autosomal recessive trait. Unlike HPS, CHS does not seem to display locus heterogeneity and the only gene proved to cause CHS, LYST, is located on chromosome 1q42.1–42.2. HPS and CHS both present findings typical of an SPD: bleeding of mucosae, epistaxis and spontaneous soft-tissue bruising. On the other hand, in Quebec platelet disorder, a rare autosomal dominant bleeding disease associated with abnormal proteolysis of αgranule proteins, platelets show increased stores of urokinase plasminogen activator and genetic studies implicate a major role for a mutation in an uncharacterized cis element near the urokinase plasminogen activator gene. Intraplatelet generation of plasmin is thought to trigger degradation of stored α-granule proteins, increasing risks for a number of bleeding symptoms, including delayed-onset bleeding after haemostatic challenges that responds only to fibrinolytic inhibitor therapy. Recently, Paterson et al. clarified the pathogenisis of the Quebec platelet disorder: a duplication of the PLAU gene. Several patients have been described with selective impairment of the TxA2 receptor, a mild lifelong disorder, exhibiting mucosal bleeding and easy bruising and defective platelet aggregation in respone to several agents but not thrombin. Autosomal dominant inheritance has been suggested in some cases. 923

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Therapy As a rule, there is no specific treatment for the vast majority of congenital platelet disorders. In congenital thrombocytopenias due to maternal use of drugs or chemical agents, thrombocytopenia recovers after a few days to a few weeks, as in the case of thiazide drugs. In surviving thrombocytopenic newborns infected with rubella or cytomegalovirus, platelets levels may return to normal after several months. In newborns with alloimmune congenital thrombocytopenia, platelets return to normal values in 14–21 days. Only severe cases need to be treated with washed platelets, corticosteroids or exchange transfusions. In general, the main treatments in patients with congenital platelet diseases are general measures aimed at avoiding bleeding and the use of supportive therapeutic approaches for controlling haemorrhage. However, since types and severity of bleeding vary in different patients, therapeutic approaches have to be individualized.

General measures Education of patients is of great importance. Patients and their parents have to be instructed to avoid trauma. Regular dental care may be helpful in preventing gingival bleeding. Drugs that impair platelet functions, such as acetylsalicylic acid-containing medications, should be avoided. In women, oral contraceptives can be used to prevent menorrhagia. Local measures, such as the application of firm pressure in the case of epistaxis, will usually suffice in the event of mild bleeding.

Drugs Antifibrinolytic agents are useful as an adjunctive measure to prevent and control mild or moderate bleeding and may stop menorrhagia and other mild bleeding manifestations from mucous membranes, such as epistaxis. In the case of more serious bleeding, antifibrinolytic drugs such as tranexamic acid may be used, either 15–25 mg/kg orally three times daily or 10 mg/kg i.v. three times daily. Desmopressin (1-desamino-8-d-arginine vasopressin or DDAVP) is the mainstay of therapy of patients with congenital platelet defects. Depending on the platelet defect, administration of DDAVP may shorten the bleeding time. Intravenous, subcutaneous or intranasal administration of DDAVP increases factor VIII and VWF transiently by releasing them from storage sites and its use has been suggested to be of value in some patients presenting with congenital platelet defects, such as those with BSS, May–Hegglin anomaly, GPS, SPD and GT. The drug is usually administered at a dose of 0.3 µg/kg in 50 mL of saline by slow intravenous infusion (over 30 min) in order to avoid possible hypotensive effects. In general, since the response to DDAVP varies among patients but is constant in each patient, a test dose may be of value in identifying those patients who 924

will benefit from this treatment in order to prevent or control future bleedings. The use of recombinant activated factor VII (rFVIIa) has been proved to be helpful in the treatment of bleeding in haemophilic patients with inhibitors. Recently, administration of rFVIIa has been employed to stop bleeding in patients with congenital platelet disorders, such as BSS, SPD and GT. In addition, in thrombasthenics this drug has been employed to avoid bleeding during surgical procedures. However, adverse reactions have been described, such as venous thromboembolism and arterial ischaemia. Based on the evidence from the literature, rFVIIa is currently approved in Europe for prophylaxis and treatment of bleeding in patients with GT with antibodies to GPIIb/IIIa and/or HLA, and past or present refractoriness to platelet transfusion. The recommended dose is a bolus injection of 90 µg/kg at 2-hour intervals for a minimum of three doses. A second generation of synthetic thrombopoietin agonists, including AMG 531 and eltrombopag, have shown great potential in the treatment of forms of thrombocytopenia, including immune thrombocytopenic purpura and chemotherapyinduced thrombocytopenia. It is conceivable that thrombopoietin agonists would be helpful in congenital thrombocytopenia with reduced plasma levels of endogenous thrombopoietin.

Platelet transfusions Platelet transfusions, using platelets from HLA-matched donors when available, are employed to control severe haemorrhage in thrombocytopenic patients or in individuals with thrombocytopathies. Since the risk of post-traumatic as well as of spontaneous bleeding increases as the platelet count falls, spontaneous haemorrhage becomes common as platelet count drops below 20 × 109/L and is extremely likely at counts below 5 × 109/L. Extensive clinical experience has shown that control of bleeding is possible on achievement of an adequate elevation in the circulating platelet count. However, the coexistence of platelet dysfunction has to be taken into account in order to correctly calculate the dosage of platelets required. Platelet transfusions are effective in controlling bleeding but may be responsible for transmission of infectious diseases, febrile reactions or development of alloimmunization. In patients carrying thrombocytopathies, platelet transfusions should be employed only for the treatment of severe bleeding because of the risk of alloimmunization. The occurrence of platelet alloimmunization is more frequent in patients lacking a membrane glycoprotein, such as in BSS and GT. Alloantibodies develop because the deficient proteins are recognized as foreign and, in turn, induce refractoriness to platelet transfusion.

Other measures Splenectomy has generally no effect in congenital thrombocytopathies and in thrombocytopenias but has proven effective in

Congenital platelet disorders

patients with WAS. Allogeneic bone marrow transplantation may provide, in theory, an effective cure for inherited disorders involving platelet count or function, restoring normal megakaryocytopoiesis. Transplants have been successfully performed with complete correction in patients with WAS and severe GT. However, the risks of such a drastic procedure still outweigh those related to bleeding tendency and it is therefore rarely required in patients suffering from congenital platelet disorders.

Conclusions Over the last few years, a series of improvements to better understand the pathogenesis of congenital platelet disorders have been achieved. In several congenital platelet diseases, the

Table 48.5 Genes involved in congenital platelet disorders.

gene responsible has been identified and several patients have been characterized at molecular level (Table 48.5). This information has allowed more accurate comprehension of congenital thrombocytopenias and thrombocytopathies. Careful collection of personal and family clinical data, physical examination and appropriate laboratory testing are of value for the evaluation of a patient presenting with bleeding due to congenital platelet disorders (Figure 48.5). Using this approach, in many instances it is possible to correctly identify the platelet defect. However, despite recent advances in knowledge, in most patients with a congenital bleeding disorder and impairment of platelet function, the underlying molecular mechanisms are still unknown. In the near future, one of the challenges will be to ameliorate our understanding of congenital platelet disorders in order to obtain powerful tools for prevention, diagnosis and therapy of bleeding. On the other hand, gene therapy may offer

Disorder

Gene

Locus

Wiskott–Aldrich syndrome X-linked thrombocytopenia Congenital amegakaryocytic thrombocytopenia Congenital amegakaryocytic thrombocytopenia and radioulnar synostosis Schulman–Upshaw syndrome May–Hegglin anomaly and Sebastian, Epstein and Fetchner syndromes Mediterranean macrothrombocytopenia Jacobsen and Paris–Troussau syndromes Bernard–Soulier syndrome

WAS WAS MPL HOXA11

Xp11.4–p11.21 Xp11.4–p11.21 1p34 7p15.2

ADAMTS13 MYH9

9q34 22q13.1

GP1BA FLI1 GP1BA GP1BB GP9 GP1BA P2RY12 P2RY1 P2RX1 ADRA2A ITGA2 ITGA2B ITGB3 HPS1 HPS2 (AP3B1) HPS3 HPS4 HPS5 HPS6 HPS7 (DTNBP1) HPS8 (BLOC1S3) LYST near PLAU locus TBXA2R

17pter–p12 11q24.1–q24.3 17pter–p12 22q11.21–q11.23 3q21.3 17pter–p12 3q24–q25 3q25.2 17p13.3 10q24–q26 5q11.2 17q21.32 17q21.32 10q23.1–q23.3 5q14.1 3q24 22cen–q12.3 11p14 10q24.32 6p22.3 19q13.32 1q42.1–q42.2 10q24 19p13.3

Pseudo von Willebrand disease ADP receptor defect

Adrenaline receptor defect Collagen receptor defect Glanzmann thrombasthenia Hermansky–Pudlak syndrome

Chédiak–Higashi syndrome Quebec platelet disorder Thromboxane receptor defect

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Postgraduate Haematology

Thrombocytopenia Prolonged bleeding time

Drugs, toxics, infections, antibodies No

Yes Stop Resolution No

Yes

Other defects of primary haemostasis No Laboratory platelet investigation

No further evaluation

Yes Kidney, liver disease, vWD, leukaemia, etc.

Abnormal Platelet disease

Figure 48.5 Schematic algorithm for the initial screening of patients with congenital platelet disorders.

a cure for congenital platelet disorders. Hopefully, improvements in such approaches will allow treatment of the majority of patients suffering from severe congenital platelet disorders.

Selected bibliography Alamelu J, Liesner R (2010) Modern management of severe platelet function disorders. British Journal of Haematology 149: 813–23. Almeida A, Khair K, Hann I et al. (2003) The use of recombinant factor VIIa in children with inherited platelet function disorders. British Journal of Haematology 121: 477–81. American Society of Haematology ITP Practice Panel (1997) Diagnosis and treatment of idiopathic thrombocytopenic purpura: recommendations of the American Society of Haematology. Annals of Internal Medicine 126: 319–26. Arai M, Yamamoto N, Moroi M et al. (1995) Platelets with 10% of the normal amount of glycoprotein VI have an impaired response to collagen that results in a mild bleeding tendency. British Journal of Haematology 89: 124–30. Balduini CL, Noris P, Belletti S et al. (1999) In vitro and in vivo effects of desmopressin on platelet function. Haematologica 84: 891–6. Balduini CL, Iolascon A, Savoia A (2002) Inherited thrombocytopenias: from genes to therapy. Haematologica 87: 860–80. Balduini CL, Cattaneo M, Fabris F et al. (2003) Inherited thrombocytopenias: a proposed diagnostic algorithm from the Italian Gruppo di Studio delle Piastrine. Haematologica 88: 582–92. 926

Bellucci S, Damaj G, Boval B et al. (2000) Bone marrow transplantation in severe Glanzmann’s thrombasthenia with antiplatelet alloimmunization. Bone Marrow Transplantation 25: 327–30. Bernaciak J, Szczaluba K, Derwinska K et al. (2008) Clinical and molecular–cytogenetic evluation of a family with partial Jacobsen syndrome without thrombocytopenia caused by an ∼5 Mb deletion del(11)(q24.3). American Journal of Medical Genetics 146A: 2449–54. Bizzaro N (2007) Pseudothrombocytopenia. In: Platelets (A Michelson, ed.), 2nd edn, pp. 999–1008. Elsevier Science, Boston. Bolton-Maggs PHB, Chalmers EA, Collins PW et al. (2006) A review of inherited platelet disorders with guidelines for their management on behalf of the UKHCDO. British Journal of Haematology 135: 603–33. Cattaneo M (2002) Desmopressin in the treatment of patients with defects of platelet function. Haematologica 87: 1122–4. Cattaneo M (2005) The P2 receptors and congenital platelet function defects. Seminars in Thrombosis and Hemostasis 31: 168–73. Clark RH, Taylor LL, Wells RJ (1984) Congenital juvenile chronic myelogenous leukaemia: case report and review. Pediatrics 73: 324–6. D’Andrea G, Coalizzo D, Vecchione G et al. (2002) Glanzmann’s thrombasthenia: identification of 19 new mutations in 30 patients. Thombosis and Haemostasis 87: 1034–42. Diamandis M, Paterson AD, Rommens JM et al. (2009) Quebec platelet disorder is linked to the urokinase plasminogen activator gene (PLAU) and increases expression of the linked allele in megakaryocytes. Blood 113: 1543–6. Favier R, Jondeau K, Boutard P et al. (2003) Paris–Trousseau syndrome: clinical, hematological, molecular data of ten new cases. Thombosis and Haemostasis 90: 893–7. Freson K, Jaeken J, Van Helvoirt M et al. (2003) Functional polymorphisms in the paternal expressed XLalphas and its cofactor ALEX decrease their mutual interaction and enhance receptor-mediated camp formation. Human Molecular Genetics 12: 1121–30. Gabbetta J, Vaidyula VR, Dhanasekaran DN et al. (2002) Human platelet Galphaq deficiency is associated with decreased Galphaq gene expression in platelets but not in neutrophils. Thombosis and Haemostasis 87: 129–33. Galbusera M, Noris M, Remuzzi G (2009) Inherited thrombotic thrombocytopenic purpura. Haematologica 94: 166–70. Geddis AE (2009) Congenital amegakaryocytic thrombocytopenia and thrombocytopenia with absent radii. Hematology/Oncology Clinics of North America 23: 321–31. George JN, Caen JP, Nurden AT (1990) Glanzmann’s thromboasthenia: the spectrum of clinical disease. Blood 75: 1383–95. Hirata T, Kakizuka A, Ushikubi F et al. (1994) Arg60 to Leu mutation of the human thromboxane A2 receptor in a dominantly inherited bleeding disorder. Journal of Clinical Investigation 94: 1662–7. Introne W, Boissy RE, Gahl WA (1999) Clinical, molecular, and cell biological aspects of Chediak–Higashi syndrome. Molecular Genetics and Metabolism 68: 283–303. Jackson SC, Sinclair GD, Cloutier S et al. (2009) The Montreal platelet syndrome kindred has type 2B von Willebrand disease with the VWF V1316M mutation. Blood 113: 3348–51.

Congenital platelet disorders

Lillicrap D (2007) Von Willebrand disease – phenotype versus genotype: deficiency versus disease. Thrombosis Research 120 (Suppl. 1): S11–S16. Lopez JA, Andrews RK, Afshar-Kharghan V et al. (1998) Bernard– Soulier syndrome. Blood 91: 4397–418. Mao GF, Vaiduyala VR, Kunapoli SP et al. (2002) Lineage-specific defect in gene expression in human platelet phospholipase C-beta2 deficiency. Blood 99: 905–11. Miller JL (1996) Platelet-type von Willebrand disease. Thrombosis and Haemostasis 75: 865–9. Molineux G, Newland A (2010) Development of romiplostim for the treatment of patients with chronic immune thrombocytopenia: from bench to bedside. British Journal of Haematology 150: 9–20. Nair S, Ghosh K, Kulkarni B et al. (2002) Glanzmann’s thrombasthenia: updated. Platelets 13: 387–93. Nurden AT, Nurden P (2007) The grey platelet syndrome: clinical spectrum of the disease. Blood Reviews 21: 21–36. Ochs HD, Filipovich AH, Veys P et al. (2008) Wiskott–Aldrich syndrome: diagnosis, clinical and laboratory manifestations, and treatment. Biology of Blood and Marrow Transplantation 15 (1 Suppl.): 84–90. Oury C, Lanaerts T, Peerlinck K (1999) Congenital deficiency of the phospholipase C coupled platelet P2Y1 receptor leads to mild bleeding disorder. Thrombosis and Haemostasis 82 (Suppl.): 20–1. Paterson AD, Rommens JM, Bharaj B et al. (2010) Persons with Quebec platelet disorder have a tandem duplication of PLAU, the urokinase plasminogen activator gene. Blood 115: 1264–6. Pham A, Wang J (2007) Bernard–Soulier syndrome: an inherited platelet disorder. Archives of Pathology and Laboratory Medicine 131: 1834–6. Poon MC, d’Oiron R, Hann I et al. (2001) Use of recombinant factor VIIa (NovoSeven) in patients with Glanzmann thrombasthenia. Seminars in Hematology 38 (Suppl. 12): 21–5. Rao AK, Willis J, Kowalska MA et al. (1988) Differential requirements for adrenaline induced platelet aggregation and inhibition

of adenylate cyclase. Studies in familial α2-adrenergic receptor defect. Blood 71: 494–501. Rao AK, Ghosh S, Sun L et al. (1995) Effect of mechanism of platelet dysfunction in response to DDAVP in patients with congenital platelet function defects. A double blind placebo-controlled trial. Thombosis and Haemostasasis 74: 1071–8. Rao AK, Jalagadugula G, Sun L (2004) Inherited defects in platelet signalling mechanisms. Seminars in Thrombosis and Hemostasis 30: 525–35. Rivera J, Lozano ML, Navarro-Núñez L, Vicente V (2009) Platelet receptors and signalling in the dynamics of thrombus formation. Haematologica 94: 700–11. Roberts I, Stanworth S, Murray NA (2008) Thrombocytopenia in the neonate. Blood Reviews 22: 173–86. Serrarens-Janssen V, Semmekrot B, Novotny V et al. (2008) Fetal/ neonatal allo-immune thrombocytopenia (FNAIT): past, present, and future. Obstetrical and Gynecological Survey 63: 239–52. Shotelersuk V, Gahl WA (1998) Hermansky–Pudlak syndrome. Models for intracellular vesicle formation. Molecular Genetics and Metabolism 65: 85–96. Sola-Visner M, Sallmon H, Brown R (2009) New insights into the mechanisms of nonimmune thrombocytopenia in neonates. Seminars in Perinatology 33: 43–51. Stroncek DF, Rebulla P (2007) Platelet transfusions. Lancet 370: 427–38. Tubman VN, Levine JE, Campagna DR et al. (2007) X-linked grey platelet syndrome due to a GATA1 Arg216Gln mutation. Blood 109: 3297–9. van den Bemt PM, Meyboom RH, Egberts AC (2004) Druginduced immune thrombocytopenia. Drug Safety 27: 1243–52. Wei ML (2006) Hermansky–Pudlak syndrome: a disease of protein trafficking and organelle function. Pigment Cell Research 19: 19–42. Weiss HJ, Lages B, Vicic W et al. (1993) Heterogeneous abnormalities of platelet dense granule ultrastructure in 20 patients with congenital storage pool deficiency. British Journal of Haematology 83: 282–95.

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CHAPTER 49

Primary immune thrombocytopenia Drew Provan and Adrian C Newland

49

Queen Mary University of London, London, UK

Introduction, 928 Clinical features, 928 Reaching a consensus on terminology, 929 Pathophysiology, 929 ITP is multifactorial, 929 Antiplatelet antibodies and their targets, 930 Which platelet epitopes are involved?, 930 The role of Helicobacter pylori in the development of ITP, 930 T cells may also be involved, 931 Thrombopoietin levels in ITP, 931 Natural history of ITP, 932

Diagnosis, 932 Bone marrow examination, 933 Management, 933 Short-term treatment, 934 Long-term treatment, 934 Role of splenectomy for long-term control of the platelet count, 934 Patients with refractory ITP, 935 Is drug treatment needed?, 935 Rituximab, 935 Combination chemotherapy, 936 New treatments: TPO receptor agonists, 936

Introduction Primary immune thrombocytopenia (ITP), previously called idiopathic thrombocytopenic purpura, is an autoimmune bleeding disorder that affects both children and adults. Until fairly recently, it was considered an autoantibody disorder in which platelets, opsonized with antiplatelet antibody, were removed prematurely by the reticuloendothelial system. More recently, studies have shown that in many patients there is a relative platelet underproduction that contributes to the thrombocytopenia seen in this disease. Many patients have no clinical problems but bleeding may occur. ITP is unpredictable in its clinical course. To date, treatment has aimed at reducing the platelet destruction, mainly through immunosuppression. However, new thrombopoietin receptor agonists have been developed that enhance bone marrow platelet production. These newer targeted treatments are likely to be associated with less toxicity due to immunosuppression than traditional therapies.

Clinical features The two principal forms of ITP, paediatric and adult, are quite distinct in their underlying cause and presentation. In

Postgraduate Haematology: 6th edition. Edited by A. Victor Hoffbrand, Daniel Catovsky, Edward G.D. Tuddenham, Anthony R. Green © 2011 Blackwell Publishing Ltd.

928

ITP in children, 936 ‘Watch and wait’ policy, 936 General measures for persistent and chronic ITP in children, 936 Treatment options in childhood ITP, 937 ITP in pregnancy, 937 Laboratory investigation of ITP in pregnancy, 937 Management of ITP in pregnancy, 937 Delivery, 937 Treatment options in pregnancy, 938 Selected bibliography, 938

children, ITP may follow a viral illness or immunization. The profound thrombocytopenia may be associated with extensive petechiae, purpura and bruises. There may also be bleeding from mucous membranes such as the nose or mouth. Despite the severity of the clinical features, most children need little treatment, and undergo spontaneous remission in the majority of cases. Around 15% will develop a more chronic form of the disease. ITP in adults is much more insidious. There is generally no prodromal illness and the patient may be aware of petechiae or excessive bruising, and seek medical attention. ITP is often diagnosed by chance, for example during hospital admission for surgery or full blood count (FBC) is checked for other reasons. This is particularly true if the platelet count is not very low and there are no skin manifestations of thrombocytopenia. ITP, particularly in adults, is heterogeneous. Many patients suffer few clinical problems related to their thrombocytopenia while others have major bleeding from the outset. The platelet count alone appears to be an unreliable predictor of outcome, and the clinical symptoms and signs of ITP are influenced by patient age, general health, comorbidities, medication and many other factors that have not been identified. In addition, there may be an acquired platelet dysfunction caused by antibody binding to an important region of the glycoprotein molecules on the platelet surface. Autoantibodies reacting with glycoprotein (GP)IIb/IIIa may affect platelet aggregation, and anti-GPIb/IX autoantibodies can impair platelet adhesion to the subendothelial matrix, causing unexpectedly severe bleeding for the level of platelet count. Other autoantibodies such as antiphospholipid antibodies occur in up to 30% of patients with

Primary immune thrombocytopenia

Table 49.1 New Consensus Terminology definitions. Immune thrombocytopenia Diagnosis ‘Acute’ and ‘chronic’ replaced by Severe ITP

Refractory

Primary (old term ‘idiopathic’) Secondary (SLE, etc.) Requires platelet count < 100 × 109/L ‘Newly diagnosed’, ‘persistent’ and ‘chronic’ (see Figure 49.1) Where there is clinically relevant bleeding irrespective of platelet count Failed splenectomy or relapse and severe ITP or risk of bleeding

Newly diagnosed

0

Persistent

3

Chronic

12 months

Figure 49.1 Stages of ITP using the new Consensus Terminology. After diagnosis until 3 months the patient is described as having newly diagnosed ITP. If the ITP persists beyond 3 months, it is termed persistent ITP and once the 12-month milestone has been reached the disease is described as chronic ITP.

Pathophysiology ITP, and these may affect platelet and vascular function. However, in general, unlike the thrombocytopenia that accompanies bone marrow failure, serious bleeding is not common in patients with ITP. The reason for this is not completely understood, but is believed to be partly due to the large proportion of young (metabolically active) platelets present in ITP compared with other causes of thrombocytopenia. The other factor which is often overlooked is that the large platelets in ITP are not counted accurately by automated counters. The true platelet count in patients with ITP is therefore higher than the reading given when using an automated analyser.

Reaching a consensus on terminology Until recently, comparing data from clinical studies in ITP has been made extremely difficult due to the inconsistent way in which terminology has been used. For example, the term ‘refractory’ has been interpreted in different ways. For some it implies the post-splenectomy patient who has failed to respond or who has relapsed. Others interpret the term to mean those who do not respond well to treatment irrespective of splenectomy status. Similarly, deciding what constitutes a ‘complete response’ to therapy differs from study to study, making comparisons of drug treatments fraught with problems. In order to address this, a group of experts held a consensus meeting and agreed on the terminology that should be used in ITP. These have been published and will be used in all drug trials, and in the clinic, from now on. The key terminologies are listed in Table 49.1. Chronic ITP implies disease that has been present for 12 months or more from diagnosis. Previously, the term ‘chronic’ applied to individuals who had ITP for 6 months or longer. However, because patients may remit between 6 and 12 months, the term ‘chronic’ has now been redefined to refer to those individuals whose ITP has been present for 12 months or greater (Figure 49.1).

ITP in adults is a typical organ-specific autoimmune disease and, in common with other autoimmune diseases, there is a skewing of the Th1/Th2 response towards Th1 (proinflammatory). These are disorders in which there are antibodies or cells (B cells, T cells, antigen-presenting cells, or others) that react against self antigens. These may cause disease if the target tissue is damaged. The binding of antibodies to target cells results in clearance of the antigen from the body. The normal adaptive response, such as that seen in microbial infection, results in complete removal of the non-self antigen. In autoimmune disease, however, there is continual production and incomplete clearance of the antigen, leading to perpetuation of the immune attack. In ITP the antigen is platelet glycoprotein, found on megakaryocytes and platelets. Autoimmune disorders occur in around 5% of the population, although not all are symptomatic. In all, there are more than 80 distinct disorders, most of which are uncommon, apart from rheumatoid disease and autoimmune thyroiditis. Autoimmune diseases are clinical syndromes in which there is loss of tolerance to self, mediated by activation of T or B lymphocytes or other cells.

ITP is multifactorial From studies of other autoimmune diseases, it is quite clear that ITP has a multifactorial basis, and that loss of tolerance to a self antigen alone is not sufficient to generate the autoimmune disorder. Patients probably require (i) a specific set of genetic determinants, such as polymorphisms within major histocompatibility complex (MHC), CTLA4, or other genes; (ii) dysregulation of the immune response (involving dendritic cells, T or B cells, or all three); and (iii) an environmental ‘trigger’. The trigger may be infectious, for example a viral infection. Autoimmune disease arises only when all these determinants are present in an individual at the same time. This is further reinforced by the observation that self-reactive lymphocytes are 929

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commonly found in normal individuals. For example, siblings of patients who have autoimmune disorders are more likely to have autoantibodies themselves, with no overt evidence of autoimmune disease. This may be because they have not been exposed to the environmental trigger required to tip the balance towards autoimmune disease.

Antiplatelet antibodies and their targets The autoantibodies involved in ITP are generally IgG, but IgA and IgM autoantibodies have been reported. Opsonized platelets are removed prematurely by the reticuloendothelial system via an Fc-dependent mechanism. In addition, the autoantibodies may impair megakaryocyte growth and development, platelet release and may also induce apoptosis of megakaryocytes. The overall result of this is failure of platelet production. There are various assays for measuring antiplatelet antibodies. Looking for the presence of platelet-associated IgG is of no value since this is found in non-immune as well as immune thrombocytopenia. More sophisticated assays, such as the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay, has greater specificity (90%) albeit with low sensitivity (50–65%). Using MAIPA, platelet-associated IgG and antigen capture assays, several platelet antigens have been characterized. These include GPIIb/IIIa (αIIbβ3, the fibrinogen receptor) and GPIb/IX (the von Willebrand receptor), which appear to be the most frequently involved. Less commonly, GPIa/IIa, GPIV and GPV are involved. Recent reports suggest that around 40% of autoantibodies are reactive to both GPIIb/IIIa and GPIb/IX. In terms of disease chronicity, GP-specific autoantibodies may be important in the pathogenesis of chronic ITP; from available data GPIIb/IIIa appears to play a major role in the development of chronic ITP in 30–40% of cases.

Which platelet epitopes are involved? Research has shown that the 33-kDa chymotryptic core fragment of GPIIIa is a frequent target of the autoantibodies. Fujisawa and colleagues, using synthetic peptides corresponding to GPIIIa sequences, showed that in 5 of 13 sera from patients with chronic ITP, binding was to residues 721–744 or 742–762, corresponding to the carboxy-terminal of GPIIIa. Recently, Nieswandt and colleagues have examined the pathogenic effects of IgG monoclonal antibodies of different IgG subclasses against murine GPIIb/IIIa, GPIbα, GPIb/IX, GPV and CD31. Their findings suggest that, at least in mice, the antigenic specificity of the antiplatelet antibodies determines the pathogenic effects rather than the IgG subclass. They also demonstrated that antibodies against GPIb/IX caused thrombocytopenia through an Fc-independent mechanism, while that from autoantibodies against GPIIb/IIIa involved the Fc system. 930

So far we have focused our attention on the B cells in ITP. However, at the T-cell level, it has been demonstrated that T cells from patients with chronic ITP will proliferate in vitro to disulphide-reduced GPIIb/IIIa or the molecule’s tryptic peptides. This suggested that autoreactive CD4+ Th cells in chronic ITP need to recognize a modified GPIIb/IIIa molecule, implying that antigen-processing mechanisms within recipient antigen-presenting cells may be required to present GPIIb/IIIa autoantigens in the context of self HLA-DR molecules. Subsequently, mapping studies using six large (200 amino acids) recombinant fragments encoding different portions of the GPIIbα and GPIIIa chains showed that the T cells from patients with ITP recognized primarily the amino-terminal portion of the two GP chains (GPIIbα 18–259 and GPIIIa 22– 262) and that these molecules also stimulated the production of antiplatelet autoantibodies. Ultimately, the GPIIIa molecule has been mapped for CD4+ T-cell specificities by using 15-mer peptides of the GPIIIa chain and this has revealed several immunodominant peptides spanning the entire breadth of the molecule.

The role of Helicobacter pylori in the development of ITP This Gram-negative microaerophilic bacterium is the main cause of gastritis and peptic ulcer disease. It has also been implicated in the development of gastric adenocarcinoma and mucosa-associated lymphoid tumours, and in some autoimmune disorders. A number of studies have shown improvement in platelet counts in ITP patients positive for H. pylori following eradication of the bacterium. However, the data from different studies are conflicting, with some centres showing a very high rate of response to eradication while others have a low rate. Further support for the involvement of H. pylori in ITP comes from studies which have shown that there is a reduction in the level of antiplatelet antibodies in plasma following eradication of the bacterium. How H. pylori may initiate or perpetuate ITP is not known. Possibilities include molecular mimicry, where there is crossreactivity between the antibody, bacterium and platelet antigens. The bacterial protein Cag-A may be the target antigen and research has shown that there is molecular mimicry between Cag-A and platelet-associated antibodies. Further evidence of involvement of bacterial antigens and ITP comes from the work of Semple and colleagues who showed that, in the presence of platelet autoantibodies, the lipopolysaccharide of Gramnegative bacteria can enhance platelet phagocytosis. Although the role of H. pylori is not fully elucidated, it is worth screening patients with ITP for the presence of the bacterium and eradicating with standard triple therapy to see whether the patient’s platelet count responds. In paediatric ITP, the underlying cause of thrombocytopenia is most likely molecular mimicry or immune complex deposi-

Primary immune thrombocytopenia

tion. In a typical case of molecular mimicry, the child generally suffers a trivial viral illness or receives an immunization. The antibody against the virus, by chance, recognizes an epitope on the platelet. This results in platelet opsonization followed by platelet destruction. An alternative mechanism involving immune complex formation occurs when immune complexes between antibody and viral antigen are adsorbed onto the platelet surface leading to the premature removal of the platelet. Immune complexes are transient phenomena and as far as we are aware do not cause ongoing platelet destruction.

and recently it was shown that ITP patients have a direct CD8+ T cell-mediated cytotoxicity that induces platelet destruction. At present, however, it is unknown whether cell-mediated platelet destruction contributes to the severity of disease or to the difficulty of treatment in some patients with ITP. However, recent studies by Wadenvik’s group in Sweden suggests that in some patients with ITP, T cells may interact directly with platelets inducing platelet lysis (Figure 49.2).

T cells may also be involved

Since thrombopoietin (TPO) is the principal growth factor involved in platelet production, it might be expected that TPO levels would be raised in patients with ITP. Plasma TPO levels are undoubtedly elevated in patients with aplastic anaemia, chemotherapy-induced thrombocytopenia and other marrow failure syndromes. However, in ITP, levels of TPO are normal or only modestly elevated (Figure 49.3). The reason for this has been poorly understood until fairly recently. The liver produces TPO at a constant rate and this binds to TPO receptors on platelets and megakaryocytes. The free (unbound) TPO is able to stimulate the bone marrow to generate sufficient platelets to counterbalance the natural daily platelet losses through platelet senescence. The key here appears to be the free TPO component. The greater this level, the more the bone marrow is driven to produce increased numbers of platelets. If the megakaryocyte mass and platelet numbers are increased, such as in ITP, TPO is bound to the receptors on these cells, leaving little free TPO to stimulate the bone marrow

Granzyme B Perforin CD8

TCR

Platelet antigen MHC I

400

1750 1500

300

1250 1000

200 750 500

100

TPO level (pg/mL)

CTL

Platelet count (× 109/L)

The role of T cells in the development of ITP is becoming clearer. That T cells are involved has been known for some time, since the autoantibodies in ITP are predominantly of the IgG subclass, and isotype switching from IgM to IgG requires T-cell help. However, some 40% of patients with chronic ITP have no detectible autoantibodies yet are thrombocytopenic and appear to have true ITP. CD8+ T cells have been linked to the pathogenesis of many autoimmune diseases such as type 1 diabetes,

Thrombopoietin levels in ITP

250

Platelet

0

Lysis

Figure 49.2 Model of cell-mediated cytotoxicity in chronic ITP. In the case of chronic ITP in the active phase, cytotoxic lymphocytes release toxic contents, such as granzyme B and perforin, and platelet lysis occurs.

Normal (n = 96)

Aplastic anaemia (n = 23)

ITP (n = 170)

0

Figure 49.3 In normal individuals serum thrombopoietin (TPO) levels are low. In aplastic states, due to the absence of megakaryocytes and platelets (which bind TPO), TPO levels are high. In patients with ITP, the platelet count is low and TPO levels are normal or modestly elevated. This is believed to be due to absorption of free TPO by the increased megakaryocyte and platelet mass which is found in ITP. (From Nichol 1998 with permission.) 931

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Table 49.2 Potential causes of thrombocytopenia in ITP.

Table 49.3 Investigation of suspected ITP.

Autoantibody opsonization of platelets leading to destruction by the reticuloendothelial system Autoantibody opsonization of megakaryocytes, with inhibition of megakaryocyte growth, differentiation and platelet release Autoantibody-induced megakaryocyte apoptosis Relative thrombopoietin deficiency Molecular mimicry and immune complex formation T-cell direct lysis of platelets

Basic evaluation of ITP Patient history Family history Physical examination Full blood count Peripheral blood film Blood group (Rh) and reticulocyte count Direct antiglobulin test Quantitative immunoglobulin measurement Bone marrow examination*

in patients with ITP. There is therefore a relative lack of TPO in patients with ITP, which compounds the platelet losses that occur through increased destruction of platelets by the reticuloendothelial system. In summary, ITP is much more complex than originally believed, with several mechanisms leading to thrombocytopenia (Table 49.2).

Natural history of ITP This has not been systematically studied. It is not clear which patients require treatment, whether treatment has any major beneficial effect on the patient, whether patients live longer, or whether treatment alters the natural history of the disease. For the majority of patients, ITP is a fairly minor disorder. Serious bleeding is not common and clinical sequelae of the disease are generally absent in patients with platelet counts above 30 × 109/L. There have been a few studies published looking at clinically relevant bleeding and platelet count. In normal individuals, there is a correlation between the platelet count and bleeding. The lower the platelet count, the more likely is bleeding. In ITP this is not the case, and most patients with ITP show little bleeding until the platelet count drops below 20 × 109/L. One study showed that no grade 4 bleeding occurred in patients with platelets greater than 10 × 109/L.

Diagnosis Despite significant advances in our understanding of ITP, the diagnosis remains one of exclusion in both paediatric and adult ITP. A thorough history should be obtained, looking for diseases that might cause thrombocytopenia. The patient should be asked about bleeding during previous surgery or dentistry. The sites of bleeding should be determined. In children, the possibility of child abuse should be considered, although abused children will not generally have petechial haemorrhages or purpura, though they may have bruising.

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Helicobacter pylori HIV HCV Tests that may be useful in selected cases Antiphospholipid antibody (including anticardiolipin and lupus anticoagulant) Antithyroid antibody and thyroid function Pregnancy test Antinuclear antibodies Viral PCR for parvovirus and cytomegalovirus Tests of unproven or uncertain benefit Glycoprotein-specific antibody Thrombopoietin assay Reticulated platelet count Indirect platelet-associated IgG Bleeding time Platelet survival study Serum complement levels *The need for bone marrow remains contentious and the indications for performing this test are discussed in Table 49.4.

A full physical examination should be normal apart from the expected clinical signs associated with thrombocytopenia. In addition to the ‘dry purpura’ of ITP, there should be an examination of the mucous membranes, including the mouth, and optic fundi looking for the presence of retinal haemorrhage. Enlargement of the liver, spleen or lymph nodes suggests an alternative diagnosis. The laboratory investigation of ITP is straightforward. The FBC should confirm the isolated thrombocytopenia. There may be a degree of iron deficiency anaemia but this should be in proportion to the clinical history. There should be no abnormal white blood cells or red cell fragments in ITP. If the latter are found, the underlying diagnosis may be thrombotic thrombocytopenic purpura, a disorder that is far more serious than ITP and which requires urgent treatment. Table 49.3 outlines the

Primary immune thrombocytopenia

most useful tests in the diagnosis of ITP, along with other tests that are either of limited value or no value.

Bone marrow examination Previously, one of the mainstays of diagnosis was assessment of the bone marrow. The rationale behind this was the possibility of a patient having a marrow disorder such as leukaemia, lymphoma or infiltration. It was believed that detection of these by performing a bone marrow aspirate and trephine biopsy would aid diagnosis and management. However, studies to date have shown quite clearly that in patients with isolated thrombocytopenia, and with no atypical symptoms or signs, no cases of bone marrow pathology were detected. For this reason, recent guidelines do not recommend performing a bone marrow examination in such individuals. If patients fail to respond to, or relapse following, first-line treatment, then a bone marrow examination should be carried out. Similarly, if there is any hepatomegaly, splenomegaly, lymphadenopathy or any clinical or laboratory feature suggesting the presence of a disease other than ITP, a bone marrow examination should be performed. Bone marrow examination should also be performed if the patient is above the age of 60 years, since myelodysplasia becomes more likely, and myelodysplastic syndrome may resemble ITP. Finally, a bone marrow examination should possibly be carried out if splenectomy is being contemplated (Table 49.4), but in patients who have previously responded well to first-line therapies this test is probably unnecessary. Cytologically, the bone marrow in ITP generally shows normal development and maturation of all cell lines. Megakaryocyte numbers are typically normal or increased. Increased numbers of small megakaryocytes may be a feature, suggesting increased proliferation of megakaryocytes required to compensate for the peripheral blood thrombocytopenia. What has not been appreciated until very recently is that the megakaryocytes may not be producing platelets at a sufficient rate, possibly through inhibition of platelet formation or release caused by the autoantibodies. In addition, MacMillan and others have shown that the binding of the glycoprotein autoantibodies may have other profound effects on thrombopoiesis, including the induction of apoptosis of megakaryocytes. The thrombocytopenia in ITP may therefore be due to peripheral destruction of platelets and a failure of production.

Table 49.4 Indications for bone marrow examination in ITP. Failure to respond to, or relapse following, first-line therapy Presence of atypical clinical or laboratory features Age > 60 years Before splenectomy (but see text)

Management Major haemorrhage is the most feared event in patients with ITP. Over the years, attempting to prevent this by treating all patients with a platelet count below 30 × 109/L has led to overtreatment. However, catastrophic bleeding is not commonly seen and the main aim of treatment today has shifted away from trying to normalize the platelet count towards finding a strategy that will allow the patient to achieve a ‘safe’ platelet count. What constitutes a safe platelet count will vary from patient to patient, and also each patient with ITP will have differing requirements throughout their disease history. For example, a young patient with platelet count of 20 × 109/L should be safe to work and carry out normal day-to-day activities but would not be deemed safe if he or she wished to ski or undergo surgery. The safe platelet count would therefore be higher during these haemostatic challenges. So the one-size-fitsall approach has failed to work over the years and has caused increased morbidity and mortality in patients due to the adverse effects associated with many of the treatments in current use. When considering ITP treatments, we need to separate these into two major approaches: short-term and long-term treatments. These are discussed later. There are many current treatments for ITP, although most have not been approved (licensed) for this indication. Those that do have a licence for ITP include corticosteroids, intravenous immunoglobulin (IVIg) and intravenous anti-D. All the others, of which there are many, have been used for many years but they have never undergone formal study and are not specifically approved for ITP. Even those that are licensed have variable licences, where the drugs are available in some European countries but not others. TPO receptor agonists are available in the USA and Australia but have not been fully approved in many other countries including the European Union. All current treatments are of relatively low efficacy when used alone (around 30%), and all treatments address solely platelet destruction. They do this through a variety of different mechanisms but the net result is immunosuppression. The original rationale behind this was that in order to reduce the degree of autoantibody production, platelet opsonization and destruction by the reticuloendothelial system, the entire immune response should be suppressed. To some extent this works, although the treatments are purely palliative and once the treatment stops the platelet count drops to baseline levels once again. The disadvantage of immunosuppression is that, as well as reducing the level of antiplatelet antibody or diminishing the effectiveness of the reticuloendothelial system, normal immunity is suppressed. This can lead to infections and other complications. One study from 2001 showed quite clearly that, of the patients with ITP who died, at least half succumbed to infection caused by the immunosuppressive agents.

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Traditionally, treatments have been grouped into first line, second line, third line, and so on depending on the disease stage in which they are used. These terms are not particularly useful clinically but do provide a guide as to which treatments to use initially and which should be used later. First-line therapies comprise corticosteroids, IVIg and antiD. These three treatments work fairly quickly (within 24–48 hours) and their efficacy rates are high at around 70–80%. The disadvantage of these and most of the other treatments is that the platelet count generally drops once the treatment is stopped. These treatments are seldom curative, and must be considered palliative therapies. In addition, IVIg and anti-D are pooled blood products. If patients fail to respond to first-line treatment they may then be given a second-line drug, such as azathioprine or mycophenolate mofetil. These agents are much slower-acting and the efficacy rates are much lower than with first-line therapies (Table 49.5). As with first-line drugs, second- and third-line treatments are palliative. Splenectomy is a surgical second-line option for patients who have relapsed after treatment with a first-line drug, although many clinicians prefer to defer splenectomy until later in the disease course. The rationale behind splenectomy is that the spleen is generally the main site of platelet destruction (and probably autoantibody production), so that removing the spleen prolongs the lifespan of the platelets and the platelet count rises. Although considered ‘curative’, splenectomy does not in fact cure ITP. Usually the autoantibodies remain and continue to opsonize the platelets. However, the main organ of platelet destruction has been removed and hence this is a form of phenotypic ‘cure’.

Table 49.5 Treatment options after first-line therapy. Non-approved second-line treatments Azathioprine Dexamethasone Methylprednisolone Ciclosporin Mycophenolate mofetil Cyclophosphamide Danazol Dapsone Vincristine Rituximab Surgical second-line treatment Splenectomy Approved second-line treatments Romiplostim Eltrombopag

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Short-term treatment This refers to treatments lasting 2 weeks or more, which can be used when patients have a sudden drop in their platelet count leading to an unacceptably low platelet count, or when patients face haemostatic challenges such as planned surgery or dentistry or other high-risk activity, where the main risk is excessive bleeding. Patients are generally started on treatment 1 or 2 weeks prior to the event, with regular monitoring of platelet counts. Providing the counts reach a satisfactory level, the surgery or other procedure is carried out and the treatment is stopped shortly afterwards. By about 2 weeks the platelet count will have drifted back to baseline levels. Table 49.6 shows the target platelet counts for the most common procedures.

Long-term treatment This is required when patients have an unsupported platelet count that is too low for normal day-to-day activities. Generally, these patients will have failed other therapies or, if they responded to corticosteroids, they require unacceptably high doses to maintain a safe count. In this setting the aim is not to achieve a specific count but rather to move the platelet count into a ‘safe’ zone. This will vary from patient to patient. In general, once platelets exceed 15–20 × 109/L the risk of bleeding is low. Older individuals, or those with comorbidities or previous bleeding, will generally require a higher platelet count. As we have already seen, most of the existing treatments used for long-term platelet control have known toxicities. The aim with long-term treatment is to use a treatment that has acceptable levels of toxicity but which achieves the desired platelet count.

Role of splenectomy for long-term control of the platelet count Splenectomy has been used as a treatment for ITP for many years. The procedure increases the lifespan of antibody-coated platelets and may reduce antibody production. Responses are seen in about two-thirds of patients who achieve a normal platelet count, and the response is often sustained with no additional therapy for at least 5 years. Even patients who do not have a complete response may still expect a partial or transient Table 49.6 Target platelet counts for procedures. Procedure

Target platelet count

Dentistry (non-invasive) Dentistry (fillings, local anaesthesia) Minor surgery Major surgery

Splenectomy ≥ 30 × 109/L ≥ 50 × 109/L ≥ 80 × 109/L

Primary immune thrombocytopenia

increase in platelet count. Around 14% of patients do not respond and approximately 20% of responders will relapse months or years later. Postoperative complications of splenectomy In a recent systematic analysis, the complication rates of splenectomy were 12.9% with laparotomy and 9.6% with laparoscopic splenectomy. The mortality rate is 1.0% with the open laparotomy procedure and 0.2% with laparoscopy. Venous thromboembolism (VTE) may occur following splenectomy, particularly if the platelet count rises to 1000 × 109/L or more. ITP itself carries a small increased risk of VTE, although the exact level of risk is not known, but there may be an additive VTE effect in patients with ITP who undergo splenectomy. ITP patients should be evaluated for this risk and appropriate thrombophylaxis should be provided. Predicting the response to splenectomy Clinicians have long sought possible predictors of response to splenectomy. Potential predictors have included response to oral corticosteroids, IVIg and others. Indium-labelled autologous platelet scanning appears to be the most sensitive predictor of response to splenectomy. However, indium scanning is not available in every hospital but is limited to only a few centres worldwide. Prevention of infection after splenectomy Splenectomized patients are at long-term risk for opportunistic post-splenectomy infection (OPSI) with encapsulated bacteria. OPSI is often rapidly progressive and has a poor outcome. The main organisms responsible for sepsis in splenectomized patients are pneumococci (50%), meningococci and Haemophilus influenzae. In order to prevent OPSI, patients should be given prophylactic polyvalent pneumococcal vaccine at least 4 weeks prior to, or 2 weeks after, splenectomy with revaccination according to local guidelines. H. influenzae b and meningococcal C conjugate vaccinations should also be administered prior to splenectomy. Revaccination in susceptible individuals for H. influenzae should be administered according to local guidelines. Vaccination status should be recorded in the case notes. In patients who have received rituximab in the past 6 months, vaccinations may not be effective due to B-cell depletion. In the UK the Chief Medical Officer advises the long-term use of oral antibiotics in the form of phenoxymethylpenicillin 250–500 mg twice daily, or equivalent, or erythromycin 500 mg twice daily if the patient is allergic to penicillin. However, the need for lifelong antibiotic prophylaxis remains unproven.

Patients with refractory ITP The term ‘refractory’ has caused confusion over the years due to a lack of agreement about whether the term should be used

only for patients who have failed splenectomy. The new Terminology Consensus document published in 2009 defines refractory ITP as that where the patient has undergone splenectomy which has failed or relapsed, and who has ‘severe’ ITP (clinically relevant bleeding) or a risk of bleeding. Some 20% or more of adult patients fall into this category. Clinically, patients with refractory ITP pose a major challenge since, by definition, they are resistant to many of the treatments in current use. They often have low platelet counts, in addition to bleeding that is difficult to control since the disease is unresponsive to conventional therapies.

Is drug treatment needed? Many adults with chronic refractory ITP are able to tolerate severe thrombocytopenia (platelet count as low as 10 × 109/L) relatively well, with near-normal quality of life. For patients who fail to respond to standard therapies and who require treatment, a limited number of options are available. The risks of continuing therapy should be discussed and evaluated with the patient and compared with the benefits that treatment may provide. Patients should be assessed for other possible causes of their thrombocytopenia, including drug-induced, infection, inherited thrombocytopenia and myelodysplastic syndromes. Instead of receiving further ineffective treatments, many patients choose to live with lower platelet counts and, providing they remain free of bleeding, a watching brief can be kept. If any bleeding does occur, the patient can be treated with rescue therapy such as IVIg in combination with intravenous corticosteroids or cyclophosphamide, aimed at raising the platelet count rapidly.

Rituximab This chimeric monoclonal antibody targets the CD20 antigen on B cells and is licensed for B-cell non-Hodgkin lymphoma. There have been several studies of its use in ITP and other autoimmune haematological disorders. However, there are no randomized controlled studies and the drug has not been approved for use in ITP. Nonetheless, rituximab does appear to be effective in ITP, even when patients have failed multiple therapies. The largest review of rituximab in ITP was conducted by Arnold and colleagues, whose data suggest that around 60% of patients will respond to rituximab and approximately 40% of all patients will achieve a complete response. The responses are bimodal, with an early phase within 1–2 weeks and a second peak at about 6–8 weeks. Response duration varies from 2 months in partial responders to 5 years or more in about 15– 20% of initially treated patients. There are safety concerns with rituximab. For example, it should be used with caution in any patient who has positive hepatitis B core antibody status since hepatitis B may be reactivated. Adverse events associated with rituximab are usually 935

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mild or moderate, with a low incidence of infections. However, B- and T-cell repertoires do not return to normal even when B-cell numbers have normalized. Severe infectious complications, including Mycoplasma pneumoniae and Aspergillus niger, echovirus, papovavirus and cytomegalovirus, have been reported. There are also reports of over 50 cases of progressive multifocal leucoencephalopathy (PML) associated with rituximab treatment in patients with lymphoma and connective tissue disorders. The use of cyclophosphamide, a potent immunosuppressant, appears to increase the risk of PML. In terms of dosing, instead of using standard lymphoma doses (375 mg/m2 weekly for 4 weeks), lower doses of rituximab (100 mg i.v. weekly for 4 weeks) have been shown to be equally effective. Finally, rituximab in combination with high-dose dexamethasone has recently been explored as a first-line treatment option, and appears to be highly effective in treatmentnaive patients.

ing antibodies to develop, pharmaceutical companies decided to create drugs with no sequence homology to the native TPO protein. Two TPO receptor agonists are now approved in the USA: one is a peptibody called romiplostim, and the other, eltrombopag, is a small non-peptide molecule. Both have been studied in patients with ITP both before and after splenectomy. The efficacy rates are encouraging in all patients, and since they do not involve immunosuppression and are not corticosteroidbased, they appear to offer many advantages over the drugs in current use. These drugs are likely to be used in place of the non-approved and relatively ineffective standard ITP therapies for non-pregnant adults, since the drugs have not been licensed for ITP in pregnancy or childhood. Studies in the latter have recently been approved in a number of countries in the European Union.

Combination chemotherapy

Children with ITP are treated based on clinical bleeding, quality of life and not just platelet count. Severe bleeding is uncommon in children with ITP, even when the platelet count is very low (< 10 × 109/L). The incidence of intracranial haemorrhage (ICH) in children with ITP is around 0.1–0.5%. As with adult ITP, there is no method to predict which children will develop ICH, and this complication has been reported even in children receiving treatment for ITP. In general, however, ICH is commoner in patients with refractory or resistant disease than at initial presentation. Investigation of childhood ITP is similar to that in adults, although the incidence of H. pylori infection is much lower and there is no need to screen for this.

This form of treatment may be effective for some patients with chronic refractory ITP. Recently, Tao and colleagues investigated a combination comprising intravenous cyclophosphamide on days 1–5 or 7 and prednisone on days 1–7, combined with vincristine on day 1, and one of the following: azathioprine on days 1 to 5 or 7 or etoposide on days 1–7. The overall response rate in the 31 patients treated was 67.9%, including a complete response in 41.9%; the therapy was well tolerated. Other combinations, such as CHOP and COP, have been used. However, the toxicities and side-effects of these therapies make them unpopular with both clinicians and patients. Other treatments that have been used with variable success include alemtuzumab, stem cell transplantation and interferon alfa. These modalities of treatment have low efficacy and high toxicities and are best avoided, unless they are used within a trial setting.

New treatments: TPO receptor agonists Because of the relative lack of TPO seen in ITP, as discussed earlier, there is the potential to use exogenous TPO in order to stimulate the bone marrow to generate more platelets. Early experience with recombinant human TPO (rhTPO) and recombinant human megakaryocyte growth and development factor (rhMGDF) yielded mixed results. The drugs appeared to be effective in raising the platelet count, but cross-reacting antibodies to the drugs neutralized endogenous TPO, making patients more thrombocytopenic than before the drug was given. For this reason development of rhTPO and rhMGDF ceased. More recently, rather than develop TPO receptor agonist drugs based on endogenous TPO, where there would be significant sequence homology and the potential for neutraliz936

ITP in children

‘Watch and wait’ policy Around 70% of children with acute ITP do not have significant bleeding symptoms and can be managed without drug therapy. Admission to hospital should be reserved for children with clinically significant bleeding, such as severe epistaxis or gastrointestinal or other bleeding. Most children with minor, mild or moderate symptoms can be safely managed in the outpatient setting. The emphasis is towards treating symptoms rather than platelet counts, since the platelet count is a surrogate marker and does not accurately predict clinical outcome.

General measures for persistent and chronic ITP in children The management of children with persistent/refractory ITP is much the same as for newly diagnosed ITP. Many children stabilize with an adequate platelet count (> 20–30 × 109/L) and have no symptoms unless injured. In children who are less than 10 years of age at diagnosis, spontaneous remission is likely to occur, and expectant management can continue depending on the risk of bleeding and the degree of activity restriction of the

Primary immune thrombocytopenia

child. The onset of menstruation may be problematic and can be managed with antifibrinolytic agents and the combined contraceptive pill. The optimal management of chronic ITP in childhood is not known. Treatment should be tailored to each individual child and situation, and therapy should be effective and it should not carry more risk than the untreated condition.

Treatment options in childhood ITP Children with severe bleeding symptoms should be treated. Treatment should also be considered in children with moderate bleeding or those at increased risk of bleeding. First-line treatment in children The majority of children require no medical treatment, and can be managed using a ‘watch-and-wait’ policy. If treatment is deemed to be required, options include IVIg, anti-D and corticosteroids. The other drugs used in adult ITP are not suitable for childhood disease due to their toxicities and long-term sequelae. IVIg raises the platelet count in more than 80% of children and does so more rapidly than steroids or no therapy. Transient side-effects are seen in 75% of children. Intravenous anti-D immunoglobulin can be given to Rh(D)-positive children as a short infusion and is useful in the outpatient setting. Mild extravascular haemolysis is common, with a mean drop in haemoglobin of 1 g/dL. Like IVIg, anti-D is a pooled blood product but it is derived from a smaller donor pool. The safety profile of anti-D is good with no reported transmission of infection to date. However, there have been reports of severe intravascular haemolysis leading to disseminated intravascular coagulation, most of which have been fatal. Prednisolone at a dose of 1–2 mg/ kg daily for a maximum of 14 days is effective in around 75% of children (platelet count > 50 × 109/L) within 72 hours. More prolonged high-dose corticosteroid regimens are associated with increased toxicity. There are data to suggest that higher doses of prednisolone (4 mg/kg) for shorter courses may be more effective and associated with fewer side-effects.

ITP in pregnancy Thrombocytopenia occurs in 5% of pregnancies, but most of these cases are gestational rather than immune-mediated. The diagnosis of ITP involves the exclusion of other causes of thrombocytopenia during pregnancy. Patient history, physical examination, blood count and blood film examination are used as in non-pregnant patients. The work-up of a pregnant patient with ITP is essentially the same as that of a non-pregnant patient. There are some conditions specific to pregnancy and these should be considered when investigating a pregnant patient with thrombocytopenia (Table 49.7).

Table 49.7 Causes of maternal thrombocytopenia in pregnancy. Gestational thrombocytopenia Pre-eclampsia, HELLP syndrome, disseminated intravascular coagulation Folate deficiency Massive obstetric haemorrhage Acute fatty liver HELLP, haemolysis, elevated liver enzymes, low platelets. Table 49.8 Investigation of suspected ITP in pregnancy. Coagulation screening (prothrombin time, PT) Activated partial thromboplastin time (APTT) Fibrinogen assay Liver function tests including bilirubin, albumin, total protein, transferases, γ-glutamyl transferase and alkaline phosphatase Antiphospholipid antibodies, including anticardiolipin antibodies and lupus anticoagulant SLE serology Review of the peripheral blood film Reticulocyte count Bone marrow examination: not required to make the diagnosis of ITP in pregnancy

Laboratory investigation of ITP in pregnancy ITP in pregnancy, as with other types of ITP, is a diagnosis of exclusion. All investigations carried out in pregnancy are aimed at excluding conditions that may result in thrombocytopenia. Some disorders such as thrombotic thrombocytopenic purpura and HELLP syndromes require urgent diagnosis and treatment since the mortality rates are high (Table 49.8).

Management of ITP in pregnancy There should be close collaboration between the obstetrician, haematologist, obstetric anaesthetist and neonatologist. Treatment is largely based on the risk of maternal haemorrhage. Throughout the first two trimesters, treatment is initiated when the patient is symptomatic and/or when platelet counts fall below 20 × 109/L, or when it is necessary to produce an increase in platelet count to a level considered safe for procedures such as obstetric delivery or epidural anaesthesia. Patients with platelet counts of 20–30 × 109/L or more do not require routine treatment.

Delivery A platelet count above 50 × 109/L is generally acceptable for standard vaginal or Caesarean delivery. For epidural anaesthesia 937

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a platelet count of 75 × 109/L is required, although this is not evidence-based.

Treatment options in pregnancy The primary treatment options for maternal ITP are similar to those of other adult ITP patients, namely corticosteroids and IVIg. There is limited evidence for the use of intravenous antiD, splenectomy and azathioprine. Other agents, such as vinca alkaloids, rituximab, danazol and most immunosuppressive drugs (apart from azathioprine), should not be used in pregnancy because of possible teratogenicity. Corticosteroids These are the most cost-effective option. Prednisolone is initially given at a dose of 10–20 mg/day and may then be adjusted to the minimum dose that produces a haemostatically effective platelet count. At least 90% of the administered dose of prednisone is metabolized in the placenta by 11β-hydroxlase, but high doses may have an effect on the fetus. Intravenous immunoglobulin If prolonged high-dose steroid therapy is required or significant side-effects occur, or a rapid platelet increase is required, IVIg should be considered. The conventional doses of IVIg and likely response rates are similar to those seen in non-pregnant patients. Options for maternal ITP refractory to first-line treatment As with non-pregnant adults, combining first-line treatments in the refractory patient may be appropriate in the weeks prior to delivery. High-dose methylprednisolone (1000 mg), possibly in combination with IVIg or azathioprine, has been suggested as a treatment for pregnant patients refractory to oral corticosteroids or IVIg. Azathioprine has been used for many years in the post-transplant setting and this drug appears to be safe in pregnancy though the response is generally slow. Splenectomy in pregnancy is rarely required but if essential is best carried out in the second trimester. Management of the neonate (of mothers with ITP) ITP in the neonate (from mothers with ITP) accounts for 3% of all cases of thrombocytopenia at delivery. The fetal or neonatal platelet count cannot be reliably predicted from the maternal platelet count. After delivery, a cord blood platelet count should be determined in all cases. Intramuscular injections (such as vitamin K) in the fetus should be avoided until the platelet count is known. Those infants with subnormal counts should be observed clinically and haematologically as the platelet count tends to fall further to a nadir between days 2 and 5 after birth. 938

Selected bibliography Megakaryopoiesis Broudy VC, Kaushansky K (1998) Biology of thrombopoietin. Current Opinion in Pediatrics 10: 60–4. Kaushansky K (1994) The mpl ligand: molecular and cellular biology of the critical regulator of megakaryocyte development. Stem Cells 12 (Suppl. 1): 91–6. Kaushansky K (1995) Thrombopoietin: basic biology, clinical promise. International Journal of Hematology 62: 7–15. Kaushansky K (1998) Thrombopoietin. New England Journal of Medicine 339: 746–54. Kaushansky K (2005) The molecular mechanisms that control thrombopoiesis. Journal of Clinical Investigation 115: 3339–47. Kaushansky K (2008) Historical review: megakaryopoiesis and thrombopoiesis. Blood 111: 981–6. Autoimmune disease Bao W, Bussel JB, Heck S et al. (2010) Improved regulatory T cell activity in patients with chronic immune thrombocytopenia treated with thrombopoietic agents. Blood [Epub ahead of print]. Davidson A, Diamond B (2001) Autoimmune diseases. New England Journal of Medicine 345: 340–50. Kamradt T, Mitchison NA (2001) Tolerance and autoimmunity. New England Journal of Medicine 344: 655–64. Mackay IR (2000) Science, medicine, and the future: tolerance and autoimmunity. British Medical Journal 321: 93–6. Maloy KJ, Powrie F (2001) Regulatory T cells in the control of immune pathology. Nature Immunology 2: 816–22. Sarzotti M (1997) Immunologic tolerance. Current Opinion in Hematology 4: 48–52. Sinha AA, Lopez MT, McDevitt HO (1990) Autoimmune diseases: the failure of self tolerance. Science 248: 1380–8. Taneja V, David CS (2001) Lessons from animal models for human autoimmune diseases. Nature Immunology 2: 781–4. ITP general British Committee for Standards in Haematology (2003) Guidelines for the investigation and management of idiopathic thrombocytopenic purpura in adults, children and in pregnancy. British Journal of Haematology 120: 574–96. Fujisawa K, Tani P, O’Toole TE, Ginsberg MH, McMillan R (1992) Different specificities of platelet-associated and plasma autoantibodies to platelet GPIIb-IIIa in patients with chronic immune thrombocytopenic purpura. Blood 79: 1441–6. Gernsheimer T (2008) Epidemiology and pathophysiology of immune thrombocytopenic purpura. European Journal of Haematology Supplementum 69: 3–8. Kuhne T, Michaels LA (2008) Helicobacter pylori in children with chronic idiopathic thrombocytopenic purpura: are the obstacles in the way typical in paediatric haematology? Journal of Pediatric Hematology/Oncology 30: 2–3. Nichol JL (1998) Endogenous TPO (eTPO) levels in health and disease: possible clues for therapeutic intervention. Stem Cells 16 (Suppl. 2): 165–75. Nieswandt B, Bergmeier W, Rackebrandt K, Gessner JE, Zirngibl H (2000) Identification of critical antigen-specific mechanisms

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in the development of immune thrombocytopenic purpura in mice. Blood 96: 2520–7. Provan D, Chapel HM, Sewell WA, O’Shaughnessy D (2008) Prescribing intravenous immunoglobulin: summary of Department of Health guidelines. British Medical Journal 337: a1831. Rodeghiero F, Ruggeri M (2008) Is splenectomy still the gold standard for the treatment of chronic ITP? American Journal of Hematology 83: 91. Rodeghiero F, Stasi R, Gernsheimer T et al. (2009) Standardization of terminology, definitions and outcome criteria in immune thrombocytopenic purpura of adults and children: report from an international working group. Blood 113: 2386–93. Stasi R, Provan D (2004) Management of immune thrombocytopenic purpura in adults. Mayo Clinic Proceedings 79: 504–22. Stasi R, Provan D (2008) Helicobacter pylori and chronic ITP. Hematology. American Society of Hematology Education Program 206–11. Stasi R, Sarpatwari A, Segal JB et al. (2009) Effects of eradication of Helicobacter pylori infection in patients with immune thrombocytopenic purpura: a systematic review. Blood 113: 1231–40.

Toxicities of current treatment Portielje JE, Westendorp RG, Kluin-Nelemans HC, Brand A (2001) Morbidity and mortality in adults with idiopathic thrombocytopenic purpura. Blood 97: 2549–54. Development of novel TPO receptor agonists Bussel JB, Cheng G, Saleh MN et al. (2007) Eltrombopag for the treatment of chronic idiopathic thrombocytopenic purpura. New England Journal of Medicine 357: 2237–47. Bussel JB, Provan D, Shamsi T et al. (2009) Effect of eltrombopag on platelet counts and bleeding during treatment of chronic

idiopathic thrombocytopenic purpura: a randomized, doubleblind, placebo-controlled trial. Lancet 373: 641–8. Bussel JB, Kuter DJ, Pullarkat V, Lyons RM, Guo M, Nichol JL (2009) Safety and efficacy of long-term treatment with romiplostim in thrombocytopenic patients with chronic ITP. Blood 113: 2161–71. Cines DB, Yasothan U, Kirkpatrick P (2008) Romiplostim. Nature Reviews. Drug Discovery 7: 887–8. George JN, Mathias SD, Go RS et al. (2009) Improved quality of life for romiplostim-treated patients with chronic immune thrombocytopenic purpura: results from two randomized, placebo-controlled trials. British Journal of Haematology 144: 409–15. Kuter DJ (2007) New thrombopoietic growth factors. Blood 109: 4607–16. Kuter DJ, Bussel JB, Lyons RM et al. (2008) Efficacy of romiplostim in patients with chronic immune thrombocytopenic purpura: a double-blind randomized controlled trial. Lancet 371: 395–403. Newland A (2008) Emerging strategies to treat chronic immune thrombocytopenic purpura. European Journal of Haematology Supplementum 69: 27–33. Newland A, Caulier MT, Kappers-Klunne M et al. (2006) An open-label, unit dose-finding study of AMG 531, a novel thrombopoiesis-stimulating peptibody, in patients with immune thrombocytopenic purpura. British Journal of Haematology 135: 547–53. Provan D, Newland A (2003) Idiopathic thrombocytopenic purpura in adults. Journal of Pediatric Hematology/Oncology 25 (Suppl. 1): S34–S38. Provan D, Butler T, Evangelista ML, Amadori S, Newland AC, Stasi R (2007) Activity and safety profile of low-dose rituximab for the treatment of autoimmune cytopenias in adults. Haematologica 92: 1695–8.

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CHAPTER 50

Haematological aspects of systemic disease

50

Atul B Mehta1 and A Victor Hoffbrand2 1

Royal Free Hospital, University College London School of Medicine London, UK Royal Free and University College Medical School, Royal Free Hospital, London, UK

2

Anaemia of chronic disease, 940 Pathogenesis, 940 Inhibition of erythropoiesis, 941 Iron metabolism, 941 Treatment, 941 Malignancy, 942 Anaemia, 942 Polycythaemia, 943 White cells, 943 Platelets, 944 Coagulation, 944 Connective tissue disorders, 945 Anaemia, 945 White cells, 946 Platelets, 946 Coagulation, 946

Other changes, 946 Renal disease, 946 Anaemia, 946 Polycythaemia, 947 Haemostatic abnormalities, 947 Endocrine disease, 948 Liver disease, 948 Anaemia, 948 Platelets and haemostasis, 949 Liver transplantation, 949 Infections, 949 Viruses, 949 Bacterial, fungal and protozoal infections, 950 Malaria, 952 Haemophagocytic lymphohistiocytosis (or haemophagocytic syndrome), 952

Anaemia of chronic disease The anaemia of chronic disease (ACD) is a common normochromic or mildly hypochromic anaemia that occurs in patients with a systemic disease (Table 50.1). It is characterized by a reduced serum iron and iron-binding capacity, and normal or raised serum ferritin with adequate iron stores (Table 50.2). It is not due to marrow replacement by tumour, bleeding, haemolysis or haematinic deficiency, although these often complicate it.

Pathogenesis The key factor in the pathogenesis of chronic anaemia in the setting of inflammation, infection and malignancy is an increased level of hepcidin. Hepcidin binds to ferroportin and the complex serves to prevent transmembrane iron transport. The body utilizes this mechanism to reduce the supply of iron

Postgraduate Haematology: 6th edition. Edited by A. Victor Hoffbrand, Daniel Catovsky, Edward G.D. Tuddenham, Anthony R. Green © 2011 Blackwell Publishing Ltd.

940

Haematological aspects of pregnancy, 953 Anaemia, 953 White cells, 953 Platelets, 953 Coagulation changes, 954 Haematological aspects of HIV infection, 954 Pathophysiology, 954 Anaemia, 954 White cells, 954 Platelets, 955 Coagulation, 955 Other changes, 955 Selected bibliography, 955

to microorganisms, but it also results in the reduction of iron absorption from the intestine and the sequestration of iron within macrophages, reducing supply of iron to developing erythrocytes. Additional mechanisms result from increased levels of inflammatory cytokines, including interleukin (IL)-1, IL-6, tumour necrosis factor (TNF) and transforming growth factor (TGF)-β. These interact with accessory marrow stromal cells and with the erythroid progenitors themselves to reduce their sensitivity to erythropoietin. In this way, the marrow is attempting to aid the recruitment of pluripotent stem cells to produce white blood cells in order to combat infection/ inflammation and possibly malignancy. These inflammatory cytokines have also been shown to reduce the circulating level of erythropoietin. However, the deficiency in erythropoietin is relative: although the plasma erythropoietin level in ACD remains inversely correlated with the haemoglobin, compared with patients with other types of anaemia and normal renal function it is inappropriately low. The plasma levels of TNF-α, IL-1 (IL-1α and IL-1β) and IL-6 are raised. TNF and IL-1 have been shown in experimental systems to reduce erythropoietin production by cultured hepatoma cells. In addition, IL-1α inhibits erythropoietin production by isolated serum-free perfused kidneys. A mild decrease in red cell lifespan occurs in

Haematological aspects of systemic disease

Table 50.1 Conditions associated with anaemia of chronic disorders. Chronic infections Especially osteomyelitis, bacterial endocarditis, tuberculosis, abscesses, bronchiectasis, chronic urinary tract infections Other chronic inflammatory disorders Rheumatoid arthritis, juvenile rheumatoid arthritis, polymyalgia rheumatica, systemic lupus erythematosus, scleroderma, inflammatory bowel diseases, thrombophlebitis Malignant diseases Carcinoma (especially metastatic or associated with infection), lymphoma, myeloma Others Congestive heart failure, ischaemic heart disease, AIDS

Table 50.2 Haematological features of anaemia of chronic disease. Haemoglobin Mean corpuscular volume Mean corpuscular haemoglobin Serum iron Total iron-binding capacity (transferrin) Transferrin saturation Serum ferritin Serum and urine hepcidin C-reactive protein Erythrocyte sedimentation rate

Not less than 9 g/dL Normal or mildly reduced (usually 77–82 fL) Usually normal; occasionally reduced Reduced Reduced Mildly reduced Normal or increased Raised Usually raised Usually raised

ACD but is at a level that could be compensated by a normal marrow. TNF has been shown to increase apoptosis in erythroid cells.

Inhibition of erythropoiesis Both TNF and IL-1 inhibit erythropoiesis in vitro and it is likely that TNF increases apoptosis of bone marrow erythroid cells. Anaemia is observed in humans treated with TNF and in animals receiving either cytokine. The effect of IL-1 is probably mediated by interferon (IFN)-γ secreted by T lymphocytes, whereas TNF action is probably mediated by IFN-γ produced by marrow stromal cells. IL-6 and TGF-β may also have roles as mediators of ACD through inhibition of erythropoiesis.

Iron metabolism A characteristic in ACD is the presence of a low serum iron with adequate reticuloendothelial iron stores, but with a reduction of iron granules in marrow erythroblasts. A fall in serum transferrin and a rise in serum ferritin occur as part of the acutephase response. The fall in serum iron can occur as early as 24 hours after the onset of a systemic illness, and will persist during the course of a prolonged illness. The fall in serum iron results from an impaired flow of iron from cells (including intestinal mucosal cells, hepatocytes and macrophages) to plasma. This is due to increased secretion of hepcidin by hepatocytes in response to inflammation. Hepcidin inhibits release of iron from macrophages and iron absorption. Inflammation can cause a 100-fold increase in urinary hepcidin excretion. Microbial products may act on Kupffer cells to secrete IL-6, which then stimulates hepcidin secretion from hepatocytes. Low serum iron inhibits proliferation of microorganisms. Hepcidin secretion is also increased in iron overload. This seems unlikely to be due to a direct effect on hepatocytes. It is possible that iron in Kupffer cells or sinusoidal cells, like infection, stimulates production of cytokines, which act on hepatocytes. In clinical specimens, there is a significant positive correlation between serum and urinary hepcidin and serum ferritin levels. The cytokines TNF, IL-1 and IFN-γ have all been shown to cause reduced serum iron and increased serum ferritin concentrations. The fall in serum iron is probably mainly, if not entirely, due to hepcidin. Anaemia usually reduces hepcidin secretion. In ACD, this effect is clearly abrogated by the effect of inflammation or malignancy increasing its secretion. Increased lactoferrin, occurring in response to inflammation and mediated by cytokines, competes with transferrin for iron and forms a complex that is taken up by macrophages in the liver and spleen. Increased intracellular apoferritin synthesis occurs in response to inflammation and malignancy and this too will bind iron. Both of these mechanisms reduce the amount of iron available for binding to serum transferrin.

Treatment The severity of the anaemia correlates with the activity and severity of the underlying chronic disease. Successful therapy of this leads to a reduction in the levels of the mediator cytokines, increased erythropoietin production and reduced inhibition of erythropoiesis. Correction of the anaemia may take weeks or months. Pharmacological doses of recombinant erythropoietin have been used successfully to improve anaemia in patients with rheumatoid arthritis, cancer and myeloma. This observation suggests that inadequate erythropoietin production and its reduced action are more important than disturbed iron metabolism in the pathogenesis of ACD. It further suggests that the suppressive action of various cytokines can be overcome by use 941

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of pharmacological doses of erythropoietin. Iron therapy should be reserved for patients who have genuine iron deficiency.

Malignancy Anaemia Anaemia is the most frequent haematological abnormality in cancer patients and may be due to many causes (Table 50.3). ACD (see above) will affect almost all cancer patients at some stage of their illness. The degree of anaemia reflects the extent of the malignancy and may be worsened by the myelotoxic effects of chemotherapy. Plasma erythropoietin levels tend to be inappropriately low and therapy with recombinant erythropoietin can reduce transfusion requirements by improving the haemoglobin level in cancer patients undergoing chemotherapy. Haemolysis Warm antibody autoimmune haemolytic anaemia (AIHA) is most frequently found in association with the following malignant diseases: chronic lymphocytic leukaemia, Hodgkin disease and non-Hodgkin lymphoma. However, it has also been reported in association with solid tumours (e.g. carcinoma of the ovary). Cold-antibody AIHA is less common, but occurs in association with monoclonal cold agglutinins in chronic cold agglutinin disease, Waldenström macroglobulinaemia and myeloma.

Table 50.3 Causes of anaemia in cancer patients. Type of anaemia

Associations

Anaemia of chronic disease Blood loss

All neoplasms Gastrointestinal neoplasms, gynaecological neoplasms

Haemolysis Immune Non-immune fragmentation syndrome Haemolysis: secondary to drugs Pure red cell aplasia Megaloblastic

Leucoerythroblastic Marrow hypoplasia Myelodysplasia

942

Ovarian carcinoma, lymphoma, others Mucin-secreting adenocarcinomas Mitomycin, ciclosporin, cisplatin Thymoma Chemotherapy, folate deficiency, cobalamin deficiency (gastric carcinoma) Metastatic disease in bone marrow Chemotherapy/radiotherapy Chemotherapy/radiotherapy

Microangiopathic haemolytic anaemia (MAHA) with intravascular haemolysis may occur in association with disseminated carcinoma. An abrupt onset of anaemia and thrombocytopenia often occurs, with a leucoerythroblastic blood picture, reticulocytosis and red cell fragmentation. Renal failure may occur as a complication. Mucin-secreting adenocarcinomas (especially gastric), breast cancer and lung cancer are the most common underlying malignancies; in about one-third of patients with MAHA, it is the presenting feature of the tumour. Abnormal blood vessels may be within the tumour itself (or within metastatic tumour thrombi, especially in the lungs) or fibrin deposition may occur at other sites because of disseminated intravascular coagulation (DIC). Widely disseminated disease with bone marrow infiltration is almost always present and the outlook is poor. A syndrome resembling MAHA and idiopathic haemolytic–uraemic syndrome (HUS) has been reported with a number of chemotherapeutic agents and following allogeneic bone marrow transplantation. Principal among these is mitomycin, although cisplatin, carboplatin, bleomycin and ciclosporin have also been reported to be responsible. Immune complex deposition has been implicated in the pathogenesis. Red cell aplasia Acquired pure red cell aplasia is associated with a thymoma in approximately 50% of patients, although it complicates only approximately 5% of thymomas. Antibodies to erythroid precursors have been demonstrated in some patients, and removal of the thymoma (which is usually benign) leads to resolution of the anaemia in about half of those affected. Immunosuppressive therapy with cyclophosphamide, ciclosporin, steroids or plasma exchange may be helpful in patients who relapse. Red cell aplasia may also occur in a minority of patients with chronic lymphocytic leukaemia (CLL) or non-Hodgkin lymphoma and with large granular lymphocytic (LGL) leukaemia and as part of general marrow aplasia due to chemotherapy or radiotherapy. Leucoerythroblastic anaemia The blood film (Figure 50.1) shows the presence of erythroblasts and granulocyte precursors (e.g. myelocytes and myeloblasts). It is seen in primary myelofibrosis but is also frequent when there is marrow infiltration by tumour. This disturbs the marrow microvasculature and allows early release of the precursors. Marrow infiltration is most commonly observed in breast (Figure 50.2), prostate and haematological malignancies, but also in tumours of lung, thyroid, kidney and gastrointestinal tract and melanoma. It can also occur as a reflection of active bone marrow response to peripheral consumption (acute haemolysis, DIC, septicaemia, hypersplenism) or of extramedullary haemopoiesis (e.g. myelofibrosis or megaloblastic anaemia).

Haematological aspects of systemic disease

cies (especially ovarian and gastrointestinal carcinoma) are also susceptible. The median latency prior to onset of MDS is 2–3 years and AML usually supervenes 6 months to 2 years later.

Figure 50.1 Nucleated red cells and an immature myeloid precursor in the peripheral blood film of a patient with a leucoerythroblastic anaemia.

Treatment Erythropoietin is effective in reversing anaemia in a proportion of patients with malignant disease, for example myeloma, lymphoproliferative disorders, MDS and carcinomas. It is recommended if the haemoglobin is less than 10 g/dL or in symptomatic patients with haemoglobin of 10–12 g/dL. Recombinant erythropoietin is not justified if the haemoglobin is above 12 g/dL as its use has been associated with an increased risk of thrombosis. The expression of both erythropoietin and its receptor is reported in a range of human tumours, including breast, prostate, colon, overy, uterine and head and neck squamous tumours. Caution is therefore advised in the use of recombinant erythropoietin to treat cancer-related and cancer treatmentrelated anaemia, and further studies are required. Dosage is 10 000 IU three times per week or 30 000 IU as a single weekly dose. The dose could be increased if there is no response (< 1 g/dL rise in 4 weeks). Patients most likely to respond are those who have low pretreatment erythropoietin levels (< 100 mU/mL), well-preserved marrow function and normal/low levels of serum ferritin (< 400 ng/mL). A number of studies have demonstrated that erythropoietin therapy can lead to a reduced need for blood transfusion, better quality of life for patients and possibly improved overall outcome of anticancer therapy.

Polycythaemia

Figure 50.2 Bone marrow aspirate showing infiltration by metastatic breast carcinoma.

Other causes Megaloblastic and dyserythropoietic anaemias are most commonly due to chemotherapy-induced disturbance of DNA synthesis within the bone marrow. Folate deficiency may also occur in patients with a poor diet and widespread disease. Cobalamin (vitamin B12) deficiency due to underlying pernicious anaemia may complicate cancer of the stomach. Both chemotherapy and non-ionizing radiotherapy may lead to the development of myelodysplastic syndrome (MDS), which may progress to acute myeloid leukaemia (AML). Alkylating agents, especially melphalan and chlorambucil, nitrosoureas and epipodophyllotoxins in particular have been implicated, and there is evidence of a synergistic effect of these agents with small chronic doses of radiotherapy. The principal categories of patients affected are those who have received therapy for a haematological malignancy, but patients treated for non-haematological malignan-

This is a rare complication of non-haematological malignancy. It usually arises through elaboration by tumour cells of erythropoietin and erythropoietin-like peptides. The tumours most commonly associated are renal cell carcinoma and hepatoma; others include uterine myoma, androgen-secreting ovarian tumours, phaeochromocytoma and cerebellar haemangioblastoma. Non-malignant conditions affecting these organs (e.g. renal cysts, viral hepatitis) may also rarely be associated.

White cells (Table 50.4) Granulocytosis Granulocytosis is a frequent manifestation of non-haematological malignancies. In part, the response is due to inflammation induced by the tumour. Interaction of tumour cells with host T lymphocytes and mononuclear phagocytic cells leads to the production of a range of cytokines, which induce white cell proliferation and differentiation. Tumour cells may also secrete specific agents that directly stimulate reactive proliferation. Cancer patients frequently have opportunistic infections, may bleed and are typically on a range of medications, all of which may influence the level of the white cell count. 943

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Table 50.4 White cell changes in malignancy. Neutrophils increased

Most, especially renal Hodgkin disease

Neutrophils decreased

Bone marrow infiltration Hypersplenism Treatment induced Chemotherapy/radiotherapy Large granular lymphocytic leukaemia

Basophils increased

Myeloproliferative disorders

Eosinophils increased

Hodgkin disease T-cell lymphomas Metastatic adenocarcinoma Other tumours (e.g. lung) Drug allergy Opportunistic infection

Monocytes increased

Carcinoma Hodgkin disease

Monocytes decreased

Treatment induced

Lymphocytes increased

Lymphoid malignancies Post splenectomy Opportunistic infection

Lymphocytes decreased

Treatment induced Radiotherapy/chemotherapy Lymphoma Opportunistic infection

Table 50.5 Thrombocytopenia in patients with malignant disease. Decreased production Chemotherapy/radiotherapy Marrow infiltration Accelerated destruction Hypersplenism Disseminated intravascular coagulation Drug-induced haemolytic–uraemic syndrome Autoimmune thrombocytopenia

Thrombocytosis A raised platelet count is frequently seen as a reactive phenomenon in patients with malignancy but is usually less than 1000 × 109/L and is rarely of clinical significance. Platelet function abnormalities Impaired platelet function leading to excessive bleeding is primarily seen in myeloproliferative disorders (e.g. essential thrombocythaemia) and MDS. Paraproteins, especially IgM in Waldenström macroglobulinaemia and IgG in myeloma, are frequent causes of impaired platelet aggregation and adhesion. Increased platelet aggregation occurs in cancer patients when tumour cells release adenosine diphosphate (ADP), prostaglandins or thrombin. Increased platelet adhesiveness has also been reported, but is probably secondary to other coagulation changes.

Coagulation (Table 50.6) Granulocytopenia This is most frequently due to chemotherapy or radiotherapy but may also occur with widespread marrow infiltration, for example by lymphoma or due to LGL leukaemia.

Platelets Thrombocytopenia (Table 50.5) This may arise through decreased production or accelerated peripheral destruction and/or hypersplenism. The former may result from extensive marrow infiltration or be secondary to chemotherapy or radiotherapy. Hypersplenism is usually due to splenic infiltration by a haematological malignancy (e.g. lymphoma, CLL) but is rarely due to obstruction of the splenic or portal vein by hepatic and pancreatic malignancies. Increased destruction occurs with DIC. Immune thrombocytopenia may occur in association with haematological malignancy (CLL, lymphoma, MDS) and rarely may complicate solid tumours (e.g. breast, lung, ovary).

944

A wide range of coagulation changes can occur in patients with malignant disease and can predispose to either haemorrhage or thrombosis. The distinction between activation of the coagulation part of the haemostatic pathway (i.e. DIC), which leads physiologically to activation of fibrinolysis (secondary fibrinolysis), and primary or direct activation of fibrinolysis may be difficult, and in some ways academic, in view of the intimate and dynamic relationship that exists between the coagulation and fibrinolytic pathways. These patients may also have other general medical problems, including infection; they may have undergone surgery, chemotherapy or radiotherapy and will typically be on a range of medications. All these factors may contribute to coagulation changes. Disseminated intravascular coagulation Chronic or compensated DIC is probably underdiagnosed in cancer patients. It occurs particularly in those with gastrointestinal, lung, pancreatic and breast neoplasms. Thrombosis, including migratory thrombophlebitis (Trousseau syndrome)

Haematological aspects of systemic disease

Table 50.6 Coagulation changes in malignancy.

Table 50.7 Circulating anticoagulants in malignancy.

Bleeding tendency Disseminated intravascular coagulation, acute or chronic Primary fibrinolysis Acquired platelet function defect Thrombocytopenia Circulating anticoagulants/inhibitors

Factor inhibitors Factor V Factor VII Factor VIII

Treatment-related bleeding disorders Thrombotic tendency Venous stasis: bed rest, venous compression/invasion by tumours Increased coagulation factors: FI, FV, FVII, FVIII, FIX, FXI Decreased inhibitors of coagulation: low antithrombin, proteins C and S Direct activation of coagulation by tumour cells: FVII, FX Indirect activation: via trypsin release, mucin secretion, monocytes or endothelial damage Increased platelet aggregability and adhesiveness Thrombocytosis

and non-bacterial thrombotic endocarditis, is a more frequent manifestation of DIC than haemorrhage. In contrast, acute or uncompensated DIC is uncommon with solid tumours but occurs frequently with acute promyelocytic leukaemia (APL, FAB M3) and is associated with excessive bleeding. The triggering event in APL is likely to be release from the malignant promyelocytes of procoagulants and proteases, which may directly activate both coagulation and fibrinolysis. Tumour cells may activate coagulation by release of tissue factor (TF), which activates factor (F)VII. Direct activation of FX through the action of a cancer procoagulant has been reported in lung, kidney, colon and breast cancer. The sialic acid moiety of secreted mucin can directly activate FX, whereas the systemic release of trypsin in pancreatic tumours can also activate coagulation. Tumour cells may also activate the monocyte/macrophage system to produce procoagulant materials including TF and FX activators. Primary fibrinolysis This is less common as a cause of increased bleeding than DIC, but can occur, for example, in patients with prostatic cancer who undergo surgery. The release of proteases from leukaemic cells in both APL and monocytic leukaemia has been reported to induce fibrinolysis. Platelet counts tend to be higher than those seen in DIC, and fibrinogen levels are low, with raised fibrin degradation products (FDPs). Acquired circulating anticoagulants (Table 50.7) The most frequent is an acquired von Willebrand syndrome (both type 1 and type 2 disease) in patients with a paraprotein

von Willebrand factor inhibitors Paraproteinaemic disorders Lymphoma Myeloproliferative disorders Chronic lymphocytic leukaemia Others Heparin-like anticoagulants: dysproteinaemias

or a B-lymphoid malignancy. A number of different mechanisms may operate. The paraprotein may be directed against an epitope within the FVIII–von Willebrand factor (VWF) molecule and inactivate it, or reduce its plasma half-life. Alternatively, immune complexes may form which bind non-specifically to FVIII–VWF and accelerate its clearance, or the malignant lymphoid cells may actually adsorb FVIII–VWF onto their surface. Paraproteins may also interfere with cross-linking of fibrin. Treatment-induced bleeding disorders Thrombocytopenia and MAHA may occur as a result of therapy (see below). Cancer patients with poor nutrition or who are on long-term antibiotics may develop vitamin K deficiency. lAsparaginase induces defective hepatic protein synthesis and can lead not only to impaired production of coagulation factors but also to low levels of antithrombin, plasminogen and proteins S and C, and so give rise to thrombosis, most seriously of the cerebral veins. Mithramycin, which is used in the treatment of malignant hypercalcaemia, causes thrombocytopenia as well as platelet function defects, coagulation factor deficiencies and increased fibrinolytic activity.

Connective tissue disorders (Table 50.8) Anaemia ACD (see above) is the most common haematological abnormality seen in patients with rheumatoid arthritis (RA). Iron deficiency may coexist, particularly in patients taking nonsteroidal anti-inflammatory agents. Folate deficiency may occur with severe disease and poor dietary intake. Warm-type AIHA with IgG and complement on the red cell surface is most frequently seen in systemic lupus erythematosus (SLE), although it can occur in the other connective tissue disorders, notably RA

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Table 50.8 Haematological changes in connective tissue disorders. Anaemia Anaemia of chronic disease Iron deficiency (drug-induced blood loss) Folate deficiency Sideroblastic anaemia Pure red cell aplasia (PRCA), especially systemic lupus erythematosus (SLE) Haemolytic anaemia: immune (especially SLE)/non-immune White cells Neutropenia (e.g. Felty syndrome) Neutrophilia Eosinophilia (e.g. Churg–Strauss syndrome, polyarteritis nodosa) Platelets Thrombocytopenia: immune/non-immune Platelet dysfunction Thrombotic thrombocytopenic purpura Thrombocytosis Pancytopenia SLE Coagulation Lupus inhibitor Specific factor deficiencies Disseminated intravascular coagulation Others Myelofibrosis Drug-related changes (e.g. aplastic anaemia due to gold, phenylbutazone; PRCA due to penicillamine) Cryoglobulinaemia Amyloidosis

and mixed connective tissue disorders. Sideroblastic anaemia has been reported in both SLE and RA, but MDS must be excluded. Pure red cell aplasia and dyserythropoietic anaemia with ineffective erythropoiesis are rare complications of SLE. Haemolysis can also occur as part of thrombotic thrombocytopenic purpura (TTP), complicating SLE.

to mature neutrophils have also been reported in SLE. Lymphopenia occurs in both SLE and RA, and may be a measure of disease activity. Eosinophilia may be seen in SLE, RA, polyarteritis nodosa and Churg–Strauss syndrome. The pathogenesis is unknown, but presumably involves release of cytokines by T lymphocytes. Functional defects in polymorph and lymphocyte function have been reported in SLE and RA.

Platelets Immune thrombocytopenia is a common manifestation of SLE and also occurs in mixed connective tissue disorders, scleroderma, RA and dermatomyositis. Autoantibodies to platelets may also impair platelet function. TTP is an association of SLE. Thrombocytosis is a non-specific reaction to inflammation and tissue damage in connective tissue disorders.

Coagulation A wide diversity of coagulation changes may occur in patients with connective tissue disorders. In part, this may be due to liver and renal disease or to drug therapy. DIC has been reported in SLE patients who have high levels of circulating immune complexes and resulting angiopathy. The lupus anticoagulant (see Chapter 47) occurs as a complication in about 10% of patients with SLE and is associated with a thrombotic tendency, thrombocytopenia, recurrent miscarriages and pulmonary hypertension. Specific coagulation factor inhibitors encountered in patients with connective tissue disorders (especially SLE) include antibodies to VWF and to FVIII, FVII and fibrinogen.

Other changes RA is one of the commonest causes of amyloidosis. An increased incidence of haematological malignancies (principally Hodgkin and non-Hodgkin lymphomas and B-lymphoproliferative disorders, including paraproteinaemias) has been noted in SLE, RA and, particularly, Sjögren syndrome. A wide range of haematological abnormalities also results from immunosuppressive therapy in these patients.

Renal disease (Table 50.9) White cells The inflammatory process in connective tissue disorders can lead to a neutrophilia. Neutropenia is a feature of Felty syndrome, which is associated with splenomegaly in patients with RA. The pathogenesis is multifactorial and involves increased margination of neutrophils, sequestration of neutrophils within the enlarged spleen, and immune complex-mediated and humoral inhibition of granulopoiesis in the marrow. Antibodies 946

Anaemia In acute renal failure, anaemia is commonly due to the drug or condition causing the renal failure, for example haemolysis due to sepsis or TTP. In chronic renal failure, anaemia is the most important haematological abnormality and its management has been revolutionized by the availability of recombinant human erythropoietin. Patients with acute or chronic renal failure

Haematological aspects of systemic disease

Table 50.9 Haematological changes in renal disease. Anaemia Failure of erythropoietin production Haemolysis: haemolytic–uraemic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP) Iron deficiency Folate deficiency Hyperparathyroidism Aluminium toxicity Polycythaemia Renal cell carcinoma Other renal diseases (e.g. cysts, hydronephrosis, nephritic syndrome, renal transplantation) Thrombocytopenia HUS TTP Disseminated intravascular coagulation Platelet function abnormalities Abnormal aggregation to ADP, adrenaline, collagen Decreased platelet adhesiveness Reduced platelet factor 3 availability Acquired storage pool defect Abnormal prostaglandin metabolism ↑ Prostacylin Defective platelet cyclooxygenase? Coagulation Hypocoagulability ↓ FXII, FXI, prothrombin ↓ FXII or inhibition Hypercoagulopathy ↓ Protein C ↓ Antithrombin ↓ Fibrinolysis

develop a normochromic normocytic anaemia, with the presence of ecchinocytes (burr cells) in the blood film. The reticulocyte count is normal or slightly low, and the bone marrow shows normoblastic erythropoiesis without the erythroid hyperplasia expected at that level of anaemia. Patients who have undergone nephrectomy tend to be more severely anaemic than patients with polycystic disease. Reduced erythropoietin levels occur in renal failure and this is the dominant cause of anaemia. An increase in serum creatinine above 133 µmol/L is associated with the loss of the normal inverse linear relation between plasma erythropoietin and haemoglobin concentration, but there is no direct correlation between reduction in glomerular filtration rate and impairment of renal erythropoietin production. Circulating inhibitors of erythropoiesis have also been

demonstrated. Chronic ambulatory peritoneal dialysis is more effective than haemodialysis in removing these inhibitors but, as recombinant erythropoietin can overcome these inhibitors, they are not of great clinical significance. Red cell survival is diminished in renal failure, but this is also a minor factor. Iron deficiency can arise through blood loss (exacerbated by haemodialysis). Folate deficiency arises in dialysed patients but is now prevented by prophylactic folic acid therapy. Renal failure is associated with elevated levels of 2,3-diphosphoglycerate and a right shift of the haemoglobin–oxygen dissociation curve. Recombinant erythropoietin therapy can fully correct anaemia in renal failure. It can be administered intravenously, subcutaneously or intraperitoneally. The subcutaneous route is effective at lower doses, and it is usual to commence at 5–75 units/kg per week, given in two or three divided doses. Anaemia is corrected up to a level of 10–12 g/dL at a rate of 1 g/ dL per month. Subclinical iron deficiency and impaired mobilization of storage iron are often present, so concomitant iron therapy is usually required. This can often be easily accomplished by the administration of intravenous iron, for example as iron dextran. An impaired response to recombinant erythropoietin should prompt a suspicion of iron, cobalamin or folate deficiency, haemolysis, infection, occult malignancy, aluminium toxicity or hyperparathyroidism. Hypertension occurs in about one-third of patients treated with recombinant erythropoietin and is dose dependent; the risk of thrombosis of an arteriovenous fistula is also increased. The optimum dose of erythropoietin is one that restores the haemoglobin level to the normal or near-normal range and improves symptoms without increasing the risk of thrombosis; for most patients, this is approximately 12–12.5 g/dL and should not exceed 14 g/dL.

Polycythaemia Secondary and inappropriate polycythaemia may result from either ectopic erythropoietin production by renal tumours or regional renal hypoxia (in benign disease and following renal transplantation), which disturbs physiological erythropoietin homeostasis. Up to 5% of patients with renal cell carcinoma have paraneoplastic polycythaemia.

Haemostatic abnormalities Abnormal platelet function is probably due to the accumulation of toxic metabolites (e.g. guanidinosuccinic and phenolic acids). DDAVP (1-deamino-8-d-arginine vasopressin) therapy, which leads to the appearance of large multimers of VWF, can shorten the bleeding time in anaemic patients. Dysfibrinogenaemia has been reported rarely, whereas FDPs are often elevated and may prolong the thrombin time. Hypercoagulopathy with a predisposition to thrombosis can also occur, especially after recombinant erythropoietin therapy. 947

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Haemodialysis with heparin anticoagulation can cause platelet activation. Fibrinolytic activity, antithrombin and protein C are all reduced in renal failure, and FV, FVII, FVIII:C and FX are increased. Thrombosis (particularly of the renal vein) is a particular feature of the nephrotic syndrome. Platelet hyperaggregability with increased plasma β-thromboglobulin is described and hypoalbuminaemia may enhance the synthesis of prostaglandins involved in platelet activation.

Endocrine disease (Table 50.10) Both hyperthyroidism and hypothyroidism are associated with mild anaemia, which is usually normochromic and normocytic, but may be macrocytic in hypothyroidism. A raised mean corpuscular volume (MCV) can occur without anaemia in hypothyroidism, and low MCV has been described in thyroTable 50.10 Haematological changes in endocrine disease. Red cells Anaemia Thyrotoxicosis (normochromic, normocytic or microcytic) Hypothyroidism (normochromic, normocytic, occasionally macrocytic) Diabetes mellitus (usually when complicated by infection, cardiac disease, renal failure, enteropathy) Hyperparathyroidism (normochromic, normocytic) Hypoadrenalism (normochromic, normocytic) Hypogonadism (normochromic, normocytic) Hypopituitarism (normochromic, normocytic) Polycythaemia (pseudo) Phaeochromocytoma Cushing syndrome White cells Cushing syndrome: neutrophil leucocytosis Phaeochromocytoma Hyperthyroidism: lymphocytosis Leucopenia: antithyroid drugs Diabetes mellitus: impaired polymorph function Platelets Diabetes mellitus: abnormal platelet function Hyperthyroidism Coagulopathy Diabetes mellitus: ↑ platelet aggregability, ↓ prostacyclin, ↑ FVIII, ↓ antithrombin Estrogen therapy: ↑ FVIII, ↑ VWF Cushing syndrome: ↑ FII, FIV, FIX, FXI, FXII

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toxicosis. Thyroid hormones stimulate erythropoiesis, and tissue oxygen demands are increased in hyperthyroidism, whereas in hypothyroidism oxygen utilization is reduced. However, plasma volume is also increased and part of the anaemia in hypothyroidism is dilutional and/or due to defective iron utilization. Coexistent deficiencies of iron (due to menorrhagia or achlorhydria), folate and cobalamin must be excluded. There is an increased incidence of pernicious anaemia in patients with hypothyroidism, hypoadrenalism and hypoparathyroidism. Antithyroid drugs (carbimazole, methimazole and propylthiouracil) can cause aplastic anaemia or agranulocytosis. Anaemia in patients with diabetes mellitus is usually due to complications of diabetes, although hyperglycaemia itself may lead to shortened red cell lifespan and decreased erythrocyte deformability. Polycythaemia (usually pseudo) can also occur with endocrine diseases. In anterior pituitary disease, androgen deficiency and adrenal insufficiency, a normochromic normocytic anaemia is common. Changes in leucocyte number and function are rarely of clinical significance, although many have been reported. Chemotaxis, phagocytosis and intracellular killing may all be disturbed in diabetes mellitus. Coagulation changes may contribute to a mild bleeding diathesis in hypothyroidism and to the thrombotic predisposition in diabetes mellitus.

Liver disease (Table 50.11) Liver disease causes a greater range of haematological change than does disease in any other organ, with the exception of the bone marrow. The liver is an important source of erythropoietin in the fetus, and serves as a haemopoietic organ in utero; extramedullary haemopoiesis occurs within the adult liver only in pathological states (e.g. myelofibrosis, severe haemolysis or megaloblastic anaemia).

Anaemia Anaemia occurs in up to 75% of patients with chronic liver disease. Portal hypertension often results in splenomegaly, which may cause haemodilution and pooling of red cells. Haemorrhage is a frequent complication, often due to oesophageal varices, and the red cell lifespan is shortened even in uncomplicated liver disease. Ferrokinetic studies suggest that the bone marrow response to anaemia is suboptimal, and many of the mechanisms that operate in ACD (see above) may be relevant. Macrocytosis occurs in approximately two-thirds of patients and erythropoiesis is macronormoblastic, indicating abnormal marrow function. Macrocytosis is particularly frequent in alcoholics, in whom reversible sideroblastic change may also occur. Target cells occur as the surface area of the cell increases, due to increased membrane lipid content without an increase in volume. Ecchinocytosis is fairly common because of

Haematological aspects of systemic disease

Table 50.11 Haematological changes in liver disease. Red cells Anaemia Anaemia of chronic disease Folate deficiency Iron deficiency (blood loss) Aplastic anaemia (viral hepatitis, rare) Sideroblastic anaemia (alcohol) Hypersplenism Microangiopathy/disseminated intravascular coagulation (DIC) (rare) Autoimmune (rare) Zieve syndrome (rare) Polycythaemia Hepatocellular carcinoma (rare) Infectious hepatitis (rare) White cells Neutrophilia Infection Haemorrhage Malignancy Haemolysis Neutrophil function Impaired chemotaxis (?due to lowered complement levels) Neutropenia Hypersplenism Eosinophilia Parasitic infestation Chronic active hepatitis (rare) Platelets Thrombocytopenia Hypersplenism, hepatic sequestration DIC Autoimmune (e.g. associated with viral hepatitis, primary biliary cirrhosis) Post liver transplantation Thrombocytosis Hepatoma (rare) Impaired platelet function Inhibitory factors (including high-density lipoprotein and apolipoprotein E) Other Benign monoclonal gammopathy (biliary + other cirrhosis) Cryoglobulinaemia (hepatitis B, hepatitis C, alcohol)

binding of the red cell membrane by abnormal high-density lipoproteins. In contrast, true acanthocytes are uncommon in uncomplicated liver disease, although they are a characteristic finding in ‘spur-cell anaemia’ (non-immune haemolytic anaemia in patients with alcoholic cirrhosis). Zieve syndrome, comprising haemolytic anaemia with hypertriglyceridaemia in patients with alcoholic liver disease, is also rare. Haemolysis due to the direct toxicity of copper ions on red cells is characteristically an early presentation of Wilson disease. Intracorpuscular changes are rare in liver disease. However, abnormal pyruvate kinase activity has been demonstrated in Zieve syndrome, and reduction of hepatocyte glucose-6-phosphate dehydrogenase (G6PD) levels in G6PD-deficient individuals, and in neonates, may exacerbate and prolong hyperbilirubinaemia with haemolysis. Viral hepatitis, including hepatitis A, B and C but most frequently hepatitis viruses yet to be fully characterized, may lead to a transient and mild pancytopenia or to severe aplastic anaemia.

Platelets and haemostasis These are discussed in Chapter 39.

Liver transplantation Orthotopic liver transplantation (OLT) is increasingly used for end-stage liver disease. Thrombocytopenia is frequently present prior to transplantation. The count tends to fall postoperatively despite platelet transfusions and this may be due to platelet sequestration in the transplanted liver. Immune thrombocytopenia has also been reported after OLT. Antibody-mediated haemolysis occurs in recipients of ABO-incompatible grafts, but the engrafted liver can also produce mild haemolysis due to anti-recipient ABO antibody. This is a form of humoral graftversus-host disease (GVHD), but T cell-mediated GVHD has also been reported after OLT. Although aplastic anaemia is a rare complication of viral hepatitis, there are reports that it may occur in as many as 30% of patients transplanted for fulminant non-A, non-B viral hepatitis.

Infections (Tables 50.12 and 50.13) Infection may produce a tremendous variety of haematological changes. Many of these are covered in other sections of this book.

Viruses Anaemia Haemolytic anaemia due to red cell autoantibody production, usually of the warm type, may occur, although cold-antibody syndromes have been reported in measles, influenza, infectious 949

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Table 50.12 Haematological changes in viral infection. Red cells Anaemia Autoimmune Measles Epstein–Barr virus (EBV) Hepatitis Cytomegalovirus (CMV) Human immunodeficiency virus (HIV) Others including herpesviruses, varicella, influenza Non-immune Microangiopathic haemolytic anaemia Reduced red cell production Marrow hypoplasia EBV (especially in X-linked lymphoproliferative syndrome) Hepatitis viruses HIV CMV (especially after renal or bone marrow transplantation) Others (rare) include togaviruses epidemic haemorrhagic fevers, dengue Red cell aplasia Parvovirus B19, especially with haemolytic anaemia White cells Neutrophilia Especially HIV, influenza, hepatitis, rubella, adenoviruses, measles, mumps, CMV and EBV as part of nearly all viral infections

mononucleosis and mumps. Paroxysmal cold haemoglobinuria is rare and occurs in children due to Donath–Landsteiner IgG anti-P antibodies. Non-immune MAHA may be associated with TTP or DIC, which may be the result of viral infections. Anaemia due to transient red cell aplasia is seen with parvovirus B19 infection in patients with haemolytic anaemias (‘aplastic crisis’). This virus may also cause erythema infectiosum, or fifth disease, in children. It invades and destroys red cell progenitors and the aplasia is terminated when neutralizing IgM and IgG antibodies develop. If the virus attacks pregnant women, it may cross the placenta and cause spontaneous abortion or hydrops fetalis. Intravenous immunoglobulin therapy has been used for severe cases (e.g. in pregnancy, HIV infection and after bone marrow transplantation). Bone marrow aspirate shows characteristic giant erythroblasts. Anaemia occurs with pancytopenia in virus-associated bone marrow aplasia, for example with hepatitis viruses. The presence of viruses, either within lymphocytes or on their cell surface, may lead to production of a range of cytokines (including TNF, IFN-α and IFN-γ), which inhibit haemopoietic cell proliferation in vitro and in vivo. This may cause a substantial reduction in erythropoiesis and is presumably the mechanism 950

Neutropenia Aplasia (see above) Complicating myalgic encephalitis (?Enteroviruses, EBV) Lymphocytosis Wide variety, especially early in course of infection Malignant transformation HTLV-I, EBV, HIV Platelets Thrombocytosis (e.g. Kawasaki) Thrombocytopenia Often history of viral prodrome in childhood immune thrombocytopenic purpura Autoimmune: EBV, hepatitis, rubella, CMV, HIV ↓ Production: aplasia (see above), measles, dengue, CMV, others ↑ Consumption: disseminated intravascular coagulation (DIC)/haemolytic–uraemic syndrome (see below) Coagulation changes DIC, especially varicella, vaccinia, rubella, arbovirus with/without microangiopathy, epidemic haemorrhagic fevers Haemolytic–uraemic syndrome: coxsackievirus, mumps, echoviruses Haemophagocytosis Herpesviruses, adenoviruses, CMV

underlying neutropenia and lymphopenia in viral infections. In infectious mononucleosis and other viral infections such as viral hepatitis, the virus infects B lymphocytes and the characteristic activated lymphocytes seen on the blood film are a reactive population of T cells. Platelets Thrombocytopenia may occur due to multiple mechanisms. Children with idiopathic thrombocytopenic purpura frequently give a history of a preceding viral illness, and autoantibody production is well described in infectious mononucleosis, rubella and cytomegalovirus infections. Reduction of bone marrow thrombopoiesis is frequently subclinical, but it is particularly important in virus-associated aplasia and dengue fever. Thrombocytosis can also occur in response to viral infections.

Bacterial, fungal and protozoal infections Anaemia ACD can occur in acute infections, overwhelming septicaemia and chronic or suppurative infection. Haemolytic anaemia is less common, but can occur through both immune (e.g. cold

Haematological aspects of systemic disease

Table 50.13 Haematological changes in bacterial, fungal and protozoal infections. Anaemia Anaemia of chronic disorders Haemolytic Immune: Mycoplasma, malaria, syphilis (PCH), listeriosis Non-immune: Clostridium perfringens (toxin related), Bartonella bacilliformis (Oroya fever) Malaria/trypanosomiasis with microangiopathy/disseminated intravascular coagulation (DIC), septicaemia Haemolytic–uraemic syndrome: verotoxin-producing Escherichia coli and Streptococcus pneumoniae Dilutional Splenomegaly (e.g. malaria, schistosomiasis) Blood loss Helicobacter pylori, Ancylostoma White cells Neutrophilia Virtually any bacterial/fungal infection Neutropenia Salmonella, Rickettsia, brucellosis, pertussis, disseminated tuberculosis (TB) Overwhelming septicaemia Neutrophil function defects Rare (e.g. Bacteroides, endocarditis) Lymphocytosis Whooping cough (Bordetella pertussis), Rickettsia Lymphopenia TB, acute bacterial infections, brucellosis Eosinophilia Aspergillosis, coccidioidomycosis, Chlamydia, streptococcal infections, Ancylostoma Eosinopenia Common in acute Bacteroides infections Monocytosis Subacute/chronic infections (e.g. disseminated TB, listeriosis) Pancytopenia Bone marrow suppression (e.g. disseminated TB, listeriosis) Haemophagocytosis: septicaemia Peripheral destruction (e.g. DIC)

haemolysins to cause severe intravascular haemolysis. DIC and MAHA can occur in any severe bacterial, fungal or protozoal infection. HUS has been associated with a range of bacterial infections, including Salmonella, Shigella and Campylobacter spp., but most frequently with verotoxin-producing strains of Escherichia coli. White cells Neutrophilia is the most common manifestation. Circulating neutrophils constitute less than 5% of the total body pool, and the neutrophil response shows great individual variation, with no clear relationship to the severity of the infection. The term leukaemoid reaction is used to describe marked leucocytosis (> 50 × 109/L), with circulating immature forms occurring in patients with non-leukaemic conditions, typically severe infection or haemolysis or with generalized malignancy. Such reactions are more common in children. Features that distinguish such a reactive leucocytosis from CML include the presence of toxic granulation, elevated leucocyte alkaline phosphatase, Döhle bodies, and the lack of twin peaks of neutrophils and myelocytes in the differential count. Neutropenia can also occur with virtually any bacterial infection, although it has been most frequently noted with Salmonella, Rickettsia and Brucella. Defects of neutrophil function may also occur.

PCH, paroxysmal cold haemoglobinuria.

Platelets Thrombocytosis is frequent in patients with chronic infections, and during the convalescent phase of acute infections. Thrombocytopenia also occurs during severe bacterial or fungal infection, particularly where there is bloodstream invasion or in intensive care patients. Certain rickettsial infections (e.g. Rocky Mountain spotted fever) are almost always associated with thrombocytopenia. Accelerated platelet destruction is the most frequent mechanism and can arise through DIC or microangiopathy with platelet attachment to damaged endothelium. Immune destruction can also occur and circulating immune complexes may lead to thrombocytopenia. Decreased platelet production is a less common mechanism, but may occur (e.g. in disseminated tuberculosis). The inflammatory and procoagulant responses to infections are clearly related. TNF-α, IL-1α and IL-6 may activate coagulation and inhibit fibrinolysis, whereas thrombin may stimulate inflammatory pathways. In severe infection, the end result may be endovascular injury, DIC, multiorgan failure and death.

antibodies with anti-I specificity in Mycoplasma infection) and non-immune mechanisms. Direct red cell invasion frequently results in severe haemolysis in infections caused by Bartonella baciliformis, with elements of intravascular haemolysis (due to increased red cell fragility) and extravascular haemolysis. Clostridium perfringens produces an α-toxin (a lecithinase) and a θ-toxin, and Staphylococcus aureus an α-toxin, which act as

Haemostasis DIC occurs frequently and may dominate the clinical picture in certain infections (e.g. bacterial meningitis). The acute-phase response that accompanies severe infection can lead to a rise in a range of coagulation factors, which may contribute to thrombotic manifestations. Suppurative thrombophlebitis, particularly in association with indwelling catheters, can occur in relation to both Gram-positive and Gram-negative infections. 951

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In patients with systemic inflammation and organ failure due to acute infection, plasma protein C levels are reduced, and recombinant human activated protein C given as an intravenous infusion over 96 hours reduces the death rate but may be associated with increased risk of bleeding. Activated protein C promotes fibrinolysis and inhibits thrombosis and inflammation and reduces circulating levels of D-dimers and IL-6.

Malaria Anaemia is the most prominent haematological manifestation of malarial infection. It is most marked with Plasmodium falciparum, which invades erythrocytes of all ages (P. vivax and P. ovale invade only reticulocytes, P. malariae only mature cells) and can give parasitaemia levels as high as 50%. Cellular disruption and haemoglobin digestion lead directly to haemolysis. Parasitized cells have an increased osmotic facility and lose deformability; they thereby become sequestered and destroyed within the spleen, which often becomes massively enlarged. Non-parasitized cells may then become sequestered within the spleen and a raised plasma volume contributes to the anaemia. In addition, malarial antigens may attach to non-parasitized red cells to give rise to a positive direct antiglobulin test and haemolysis via a complement-mediated immune response. Acute intravascular haemolysis with haemoglobinuria, often leading to renal failure (‘blackwater fever’), occurs rarely in P. falciparum infection. An inadequate bone marrow response to anaemia is seen with relative reticulocytopenia at times of active infection, with some recovery after effective therapy. TNF levels are typically elevated and ACD occurs. Leucocyte numbers may be slightly increased or normal, but leucopenia as a result of splenomegaly and impaired marrow function is characteristic. Eosinophilia is variable. Thrombocytopenia is seen in nearly 70% of P. falciparum infections and has multifactorial aetiology. Autoimmune mechanisms may operate as for red cells, splenic sequestration is a contributory factor, DIC (either acute as in blackwater fever, or low grade and chronic) is common, and ADP release from damaged red cells may lead to platelet activation and consumption.

Haemophagocytic lymphohistiocytosis (or haemophagocytic syndrome) (Table 50.14) (see also Chapters 10 and 17) This may occur in association with a wide range of systemic illness including malignancies (e.g. lymphoma), infections (particularly viral, e.g. Epstein–Barr virus, HIV) and autoimmune diseases. It typically manifests as fever, splenomegaly and jaundice with cytopenias. It is particularly common in patients 952

Table 50.14 Conditions associated with reactive haemophagocytosis. Infection Viral (e.g. herpesviruses, adenoviruses, cytomegalovirus) Bacterial, especially tuberculosis Fungal Tumours Haematological Others Drugs Phenytoin

Figure 50.3 Bone marrow aspirate showing active haemophagocytosis, which in this patient antedated the development of high-grade non-Hodgkin lymphoma by 6 months.

who are immunosuppressed or who are acutely ill (e.g. septicaemic). A familial form is recognized (familial haemophagocytic lymphohistiocytosis; see Chapter 17) Macrophage activation syndrome is a variant form of haemophagocytic lymphohistiocytosis that occurs in children with juvenile RA. Pancytopenia is usual, although cytopenias affecting an individual cell lineage also occur, and coagulopathy due to associated DIC is frequently present. Abnormal liver function commonly coexists. The serum ferritin is usually markedly elevated. The bone marrow (Figure 50.3) shows the presence of increased numbers of histiocytes displaying haemophagocytosis. Myelofibrosis and/or marrow hypocellularity are present in a minority of cases. The underlying mechanisms are poorly understood. Excessive production of cytokines (e.g. IFN-γ and TNF-α) by dysregulated T cells has been demonstrated. In macrophage activation syndrome, increased levels of IL-6, IL-8, IL-12, IL-18 and macrophage inflammatory protein (MIP)-1α

Haematological aspects of systemic disease

have also been reported. Treatment should be directed at the underlying disease process. Possible precipitating or complicating infection must be treated after appropriate cultures have been taken. Tuberculosis should be excluded. Immunosuppressive therapy (e.g. methylprednisolone) is appropriate if the condition occurs in the setting of an autoimmune condition. Anti-TNF antibody therapy has been successfully used in selected cases. Epstein–Barr virus-associated haemophagocytic lymphohistiocytosis may be indistinguishable from T-cell lymphoma and responds to chemotherapy, such as etoposide-containing regimens. The condition is usually of brief duration until recovery or, sometimes, death occurs.

Haematological aspects of pregnancy (Table 50.15) Pregnancy poses a major physiological challenge to the human body and a number of haematological changes accompany it.

Anaemia Maternal plasma volume increases by approximately 50% during the first and second trimesters of pregnancy, whereas the corresponding increase in red cell mass is only 20–30%. A dilutional anaemia results, so that the lower limit of normal haemoglobin concentration is approximately 10.5 g/dL between 16 and 40 weeks of pregnancy. The increase in maternal red cell mass, transfer of iron to the fetus (which takes place largely in the third trimester) and blood loss during labour together impose a requirement of about 800 mg of iron, so that iron deficiency frequently arises in mothers with normal or reduced iron stores. Folic acid requirements are also raised during pregnancy (increased folate breakdown due to increased nucleic acid synthesis in mother and fetus) and routine supplementation is advised even during early pregnancy to prevent megaloblastic anaemia and neural tube defects in the fetus. A physiological rise in MCV of 5–10 fL occurs during normal pregnancy with pre-existing aplasia. AIHA occurring during pregnancy is typically severe and refractory to therapy.

White cells Table 50.15 Haematological changes during pregnancy. Anaemia Dilutional Iron deficiency Folic acid deficiency Aplastic anaemia White cells Neutrophil leucocytosis Platelets Thrombocytopenia Immune thrombocytopenic purpura Eclampsia Haemolytic–uraemic syndrome Thrombotic thrombocytopenic purpura HELLP syndrome (haemolysis, elevated liver enzymes, low platelet count) Disseminated intravascular coagulation (DIC) Drug induced Coagulation Coagulation factors: vitamin K-dependent factors FII, FVII, FIX, FXI, FX increased, FVIII increased, von Willebrand factor increased, fibrinogen increased Coagulation inhibitors: protein C increased or no change, protein S decreased, antithrombin decreased or no change Fibrinolytic activity decreased DIC due to: abruptio placentae, intrauterine fetal death, amniotic fluid embolism, obstetric sepsis, eclampsia

A mild neutrophil leucocytosis with a left shift and occasional Döhle bodies occur during normal pregnancy.

Platelets The normal range for the platelet count (140–400 × 109/L) does not alter during pregnancy; thrombocytopenia occurring during pregnancy requires evaluation. Gestational thrombocytopenia complicates 8–10% of pregnancies and is characterized by mild thrombocytopenia occurring for the first time during pregnancy (platelets 80–150 × 109/L) and is usually not associated with neonatal thrombocytopenia or significant bleeding in the mother. Maternal immune thrombocytopenic purpura may antedate or present during pregnancy: it is often associated with increased levels of platelet-associated IgG, although this is a non-specific finding and the presence of serum platelet autoantibodies to platelet glycoproteins (GP)IIb/IIIa or GPIb/IX is more specific. The management of immune thrombocytopenic purpura is discussed in Chapter 49. Thrombocytopenia is regularly seen in pre-eclampsia. The mechanism is unknown, but increased aggregation is suggested, as low-dose aspirin therapy may reduce platelet consumption. TTP may occur at any time during pregnancy but typically it is before 24 weeks; the use of fresh-frozen plasma and plasma exchange has been shown to improve fetal outcome. HUS typically occurs within 48 hours of delivery in an otherwise normal pregnancy. The potentially fatal syndrome of haemolysis, elevated liver enzymes and low platelets (HELLP) occurs in up to 10% of pregnancies complicated by eclampsia. The existence of coagulation abnormalities with red cell fragmentation suggests that 953

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microangiopathy, DIC and endothelial damage all have a role in its pathogenesis. Fetal and maternal outcomes are characteristically poor. Basophilic stippling, crenated red cells and large platelets are characteristic peripheral blood findings in acute fatty liver of pregnancy.

Coagulation changes Normal pregnancy is associated with a range of alterations to haemostatic components (see Table 50.15), which combine to give an increased risk of haemorrhage, thrombosis and DIC, occurring in up to 40% of patients with abruptio placentae, leading to haemorrhage and shock. Amniotic fluid embolism typically occurs during the course of a difficult delivery in a multiparous woman and rapidly leads to a picture of chronic low-grade DIC, with onset over a period of 1–2 weeks. Venous stasis resulting from the gravid uterus combines with the coagulation changes to make pregnancy a hypercoagulable state; operative delivery imposes an additional risk.

Haematological aspects of HIV infection (Table 50.16) HIV infection and its treatment cause a range of haematological effects. The newer generation of treatments with highly active antiretroviral therapy (HAART) is changing the natural history of AIDS and increasingly turning it into a chronic disease. HAART is also less toxic, particularly to the marrow, than the treatments it has replaced in the developed world and the treatments that continue to be used to treat AIDS in the developing world.

Pathophysiology The major mechanism by which HIV infection suppresses haemopoiesis is through the action of cytokines (e.g. IFN-γ, TNFα). These are elaborated by activated lymphocytes in response to infection and have been shown to induce progenitor cell apoptosis and reduced growth of short-term marrow cultures. Direct infection of CD34 cells by HIV also occurs and crossculture experiments have demonstrated that HIV infection impairs proliferation of CD34 cells from AIDS patients more than its effect on bone marrow stromal cells. Morphological evidence of ineffective myelopoiesis is seen in the marrow, with evidence of myelodysplasia in all three lineages. An increased number of plasma cells is also frequently observed.

Table 50.16 Haematological complications of HIV. Pancytopenia, ineffective haemopoiesis, MDS Anaemia Drugs: anti-HIV, supportive care (e.g. ganciclovir) Nutritional Bleeding/phlebotomy Anaemia of chronic disease Parvovirus Thrombocytopenia Reduced platelet survival: antibodies, infection, splenomegaly, fever Reduced production: megakaryocyte differentiation reduced, CD34 reduction Neutropenia: antibody mediated, drug induced TTP/HUS/microangiopathy: usually treatment induced Coagulation abnormalities Thrombosis Antiphospholipid antibodies plus anticardiolipin antibodies Protein S deficiency Malignancy Lymphoma: non-Hodgkin/Burkitt-like, primary CNS, Hodgkin disease, others Myeloma Acute myeloid leukaemia HUS, haemolytic–uraemic syndrome; MDS, myelodysplastic syndrome; TTP, thrombotic thrombocytopenic purpura.

HIV infection itself is a prominent cause of ACD. There is a reduction in erythropoiesis as a result of the mechanisms described above. Infiltration of the marrow by tumour, such as Hodgkin lymphoma, is much commoner among HIV-positive subjects than among the normal population. There is also a relative reduction in erythropoietin levels. Treatment of HIV infection by HAART will lead to an improvement in anaemia. Other common causes include bleeding and infection. Certain infections, such as Mycobacterium avium and parvovirus B19, may directly involve the marrow and lead to decreased red cell production. Nutritional anaemia frequently arises from a defective diet and gastrointestinal disease (e.g. due to infection or drug toxicity) is common. Vitamin B12 defciency is seen in up to one-third of HIV-positive subjects and iron and folate deficiency are also common. Although treatment-induced anaemia is less common in the HAART era, treatment is a contributory factor in more than 50% of cases.

White cells Anaemia This is the commonest abnormality, occurring in up to 80% of patients; it may be due to a range of factors (see Table 50.16). 954

Leucopenia is frequently seen and predominantly due to lymphopenia. Neutropenia also occurs and is multifactorial. Granulocyte and monocyte production are reduced in HIV

Haematological aspects of systemic disease

infection; drug-induced changes are common and autoimmune neutropenia also occurs. Granulocyte colony-stimulating factor has been successfully used to improve the neutrophil count in infected HIV-positive subjects. Defects in neutrophil and monocyte function have also been demonstrated.

Platelets Thrombocytopenia is seen in up to 50% of HIV-infected patients. Reduced platelet production can occur for the reasons described above; however, it is usually due to increased platelet destruction. This is often antibody mediated (immune thrombocytopenia; see Chapter 49). HIV-specific antibodies have been shown to share a common epitope with antibodies against platelet GPIIb/IIIa. Non-specific absorption of immune complexes onto platelets also occurs and predisposes to immune thrombocytopenia. Reduced platelet production is common in HIV-positive subjects and direct infection of megakaryocytes by HIV has been described. Morphologically abnormal megakaryocytes are seen in the marrow. Other causes of thrombocytopenia include infection and microangiopathy. Both HUS and TTP are well described in the setting of HIV infection, particularly in the era before HAART. Treament of thrombocytopenia depends on the cause. Treatment of HIV infection using HAART frequently leads to improvement in the platelet count. Immune thrombocytopenia in HIV-infected subjects is treated in the same way as in the non-HIV population. However, immunosuppression carries particular risks and steroids should be used with extreme caution. Splenectomy is often effective, and anti-CD20 monoclonal antibody therapy may also be used.

Coagulation Coagulation abnormalities can occur in the setting of infection and acute illness. Circulating coagulation inhibitors have also been described. Protein S deficiency is well described and predisposes to an increased risk of thrombosis. DIC is common in the setting of infection and HIV infection.

Other changes Lymphoma is the commonest tumour in HIV-positive subjects (Chapter 35). Other haematological malignancies are also

reported to occur with increased frequency, for example myeloma and AML. HIV-positive subjects who develop these malignancies often display a prolonged premalignant phase, with smouldering myeloma and myelodysplasia frequently reported.

Selected bibliography Bernard GR, Vincent JL, Laterre P et al. (2001) Efficacy and safety of recombinant human activated protein C for severe sepsis. New England Journal of Medicine 344: 699–709. Bohlius J, Wilson J, Seidenfeld J et al. (2006) Recombinant human erythropoietins and cancer patients: updated meta-analysis of 57 studies including 9353 patients. Journal of the National Cancer Institute 98: 708–14. Dallalio G, Fleury T, Neans RT (2003) Serum hepicidin in clinical specimens. British Journal of Haematology 122: 996–1000. Ganz T (2003) Hepicidin, a key regulator of iron metabolism and mediation of anaemia of inflammation. Blood 102: 783–8. Guan X, Chen L (2008) Role of erythropoietin in cancer-related anaemia: a double-edged sword? Journal of International Medical Research 36: 1–8. Ludwig H, Rai KR, Blade J et al. (2002) Management of disease related anaemia in patients with multiple myeloma or chronic lymphocytic leukaemia: epoietin treatment recommendations. Haematology Journal 3: 121–30. McRae KR, Samuels P, Schreiber AD (1992) Pregnancy associated thrombocytopenia: pathogenesis and management. Blood 80: 2697–714. Malaria Working Party of the General Haematology Task Force of British Committee for Standards in Haematology (1997) The laboratory diagnosis of malaria. Clinical and Laboratory Haematology 19: 165–70. Nemeth E, Valore EV, Territo M et al. (2003) Hepicidin, a putative mediator of anaemia of inflammation, is a type II acute phase protein. Blood 101: 2461–3. Papadaki HA, Kritkos HD, Valatas V et al. (2002) Anemia of chronic disease in rheumatoid arthritis is associated with increased apoptosis of bone marrow erythroid cells: improvement following anti-tumour necrosis factor-α therapy. Blood 100: 474–82. Sloand E (2005) Hematologic complications of HIV infection. AIDS Reviews 7: 187–96. Weiss G, Goodnough LT (2005) Anemia of chronic disease. New England Journal of Medicine 352: 1011–23.

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CHAPTER 51

Haematological aspects of tropical diseases

51

Imelda Bates1 and Ivy Ekem2 1

Liverpool School of Tropical Medicine, Liverpool, UK University of Ghana Medical School, Accra, Ghana

2

Introduction, 956 Ethnic variations in reference ranges, 956 Tropical diseases with organisms in peripheral blood or bone marrow, 956 Malaria, 956 Babesiosis, 962 Filariasis, 962

African trypanosomiasis (sleeping sickness), 965 American trypanosomiasis (Chagas disease), 966 Leishmaniasis, 967 Non-specific haematological abnormalities associated with tropical diseases, 968 Hypersplenism, 968

Introduction For the purposes of this chapter, ‘tropical diseases’ refers to infectious diseases occurring predominantly in tropical areas. The chapter has been divided into two sections, covering tropical diseases in which organisms can be visualized in the blood or bone marrow and those that cause secondary haematological abnormalities. Around the world, 1 in 35 people (i.e. 2.9% of the population) are migrants, moving mostly from poorer to richer countries. In the UK, net international migration contributed to 68% of the population growth in 2005 and travelling migrants are responsible for a major component of imported tropical infections. Rapid increases in worldwide travel mean that haematologists need to know about the tropical diseases that can cause haematological abnormalities and be able to take relevant travel histories from patients. They also need to be aware of ethnic variations in reference ranges to avoid wasting resources on unnecessary investigations and to avoid causing undue anxiety for the patient.

Tropical diseases associated with changes in the full blood count, 968 Tropical infections with fever and haemorrhage, 969 Selected bibliography, 969

and Saudi Arabians than in white people. After the age of 1 year, Africans have lower counts than West Indians or black Americans (Table 51.1). This is due to the non-white populations having a greater number of neutrophils in the storage pool. Stimulation of a neutrophil response in these ethnic groups leads to rises in the neutrophil count to the same level as white populations irrespective of the baseline level. Indian, Chinese and Southeast Asian populations have the same white blood cell and neutrophil counts as white northern Europeans. The platelet counts in healthy West Indians and Africans may be 10–20% lower than in Europeans living in the same environment. It is not clear whether there are true ethnic variations in eosinophil counts but counts of up to 2 × 109/L have been described in healthy blood donors in Africa. Interpretation of variations in blood counts should therefore take account of the individual’s ethnic background.

Tropical diseases with organisms in peripheral blood or bone marrow Malaria

Ethnic variations in reference ranges The white blood cell count and relative and absolute neutrophil counts are lower in black people, Yemenite Jews, Palestinians

Postgraduate Haematology: 6th edition. Edited by A. Victor Hoffbrand, Daniel Catovsky, Edward G.D. Tuddenham, Anthony R. Green © 2011 Blackwell Publishing Ltd.

956

Epidemiology and biology Four species of plasmodia cause malaria in humans: Plasmodium falciparum, P. vivax, P. ovale and P. malariae. Plasmodium falciparum is by far the most dangerous and causes the greatest mortality and morbidity. Over 80% of P. falciparum infections in the UK are acquired in sub-Saharan Africa, while 85% of P. vivax infections are acquired in South Asia, especially India and Pakistan. In 2007, 1548 cases of malaria were reported in the

Haematological aspects of tropical diseases

Table 51.1 Automated white cell (WBC) and neutrophil counts in adults of different ethnic origins (95% ranges).

Male

White Afro-Caribbean African

Female

WBC (× 109/L)

Neutrophil count (× 109/L)

WBC (× 109/L)

Neutrophil count (× 109/L)

3.7–9.5 3.1–9.4 2.8–7.2

1.7–6.1 1.2–5.6 0.9–4.2

3.9–11.1 3.2–10.6 3.0–7.4

1.7–7.5 1.3–7.1 1.3–3.7

Sexual cycle in the gut of a female Anopheles

Skin

Sporozoites

Schizonts

To mosquito

Gametocytes

RBC Liver

Merozoites Schizonts

Merozoites Traphozoite Ring stages

Figure 51.1 Life cycle of the malarial parasite.

UK, of which 1139 (73%) were due to P. falciparum and 256 (16%) to P. vivax; there were five deaths. Malaria rates were highest in immigrants returning from visits to their countries of origin and accounted for one-third of all reports. The majority of travellers (81%) had not taken malaria prevention tablets. Malaria is transmitted by the bite of an infected female Anopheles mosquito. The infecting agent is the sporozoite and thousands of these spindle-shaped cells may be injected by a single bite. Infrequently, transmission may also occur through blood transfusion, bone marrow transplantation and transplacentally (0.5% of all UK cases). There have also been reports of

Blood vessels

malaria transmission in aircraft or near airports in temperate zones due to infected mosquitoes being brought to nonmalarious areas. Within a few hours of an infected bite, the sporozoites enter the hepatocytes, where they divide (Figure 51.1). Rupture of the hepatocyte releases the parasites into the blood where they attach to red cell membranes using specific receptors. The parasites feed on the red cell stroma, and the digestion products form the characteristic brown pigment (haemozoin) that accumulates in phagocytic cells. In the red cells asexual replication of the trophozoites (ring forms) occurs, giving rise to erythro957

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cyte schizonts. The schizonts mature into merozoites and are released into the circulation to reinfect other red cells. The periodicity of this release varies with the species and is responsible for the classical cyclical nature of malaria fevers. Relapses, which can occur months or years after the primary illness, are characteristic of infection with P. vivax and P. ovale, and are due to maturation of persistent hypnozoites in the liver. A few of the trophozoites develop into male and female banana-shaped gametocytes and are taken up by the mosquito at a blood meal. Inside the midgut of the mosquito, they undergo sexual reproduction and sporozoites migrate to the salivary glands, ready to infect another host when the mosquito bites. Unlike the schizonts of P. vivax, P. ovale and P. malariae, those of P. falciparum are not commonly seen in the peripheral blood of the human host. This is because P. falciparum-infected cells have surface cytoadherence molecules that enable them to be sequestered in the deep tissues. Therefore P. falciparum schizonts only appear in the blood in very severe infections or in splenectomized patients. Sequestration is responsible for some of the severe clinical consequences of P. falciparum malaria, such as cerebral malaria.

Clinical features The time between the infected bite and the appearance of clinical symptoms and parasites in the peripheral blood varies between species. It is 7–30 days (mean 10 days) in P. falciparum but can be months, or even years, with other species, particularly P. vivax and P. ovale, because of their hypnozoite stage. The dormancy time of hypnozoites varies with the different strains. Maximal immunity to malaria takes around 10 years to develop and is lost over the course of 1–5 years if the individual leaves a malarious area and is no longer exposed to infections. This is often not appreciated by students or immigrants from malaria-endemic countries, who are more prone to severe attacks of malaria when they return home after a prolonged period in a non-malarious area. All four Plasmodium species produce factors that cause release of tissue cytokines, especially from leucocytes. These cytokines produce fever and contribute to anaemia through marrow suppression. Splenomegaly is a common feature of

acute malaria and mild jaundice may also occur secondary to haemolysis. Other clinical features vary with different species. Plasmodium falciparum Plasmodium falciparum is the only species associated with complicated and severe disease. Death may occur after a single exposure to malaria, particularly in those with no immunity such as non-immune travellers or young children in endemic countries. Despite this, the majority of infections cause a self-limiting febrile illness. Recurrent fevers and other symptoms of malaria may be due to either recrudescence of blood forms that persist between attacks or to reinfection with a new strain or species. Recurrent attacks with different strains lead to the development of clinical immunity but not necessarily to complete clearance of parasites. Parasites may therefore be detected in a high proportion of adults in endemic areas who are clinically asymptomatic. In addition to fever with rigors, nausea and hot and cold phases, P. falciparum infection can also present with diarrhoea and cough (Table 51.2). Serious complications include severe anaemia, cerebral involvement and failure of major organs such as kidneys and liver. During pregnancy, immunity to malaria is reduced and parasite density increases. Even if parasites cannot be visualized in the peripheral blood, they may be sequestered in the placenta and compromise fetal development. Plasmodium falciparum is therefore an important cause of low birth weight in neonates and anaemia in pregnant women. Both of these factors have a detrimental effect on the later development of the infant. Plasmodium malariae The incubation period of P. malariae may be several weeks. It is associated with recurrent fever, anaemia and enlargement of the liver and spleen. Without treatment, recrudescences may occur with decreasing severity over many years. Clinical symptoms of malaria have been reported up to 30 years after the initial infection. Plasmodium vivax and Plasmodium ovale These species cause a similar clinical picture with bouts of fever occurring periodically up to 5 years after the initial infection. These are relapses, due to reinvasion of red cells by hypnozoites,

Target organ

Clinical features

Potential common misdiagnosis

Gastrointestinal Respiratory Neurological

Diarrhoea, vomiting Cough, pulmonary oedema Delirium, coma, convulsions, focal neurological signs Oliguria, haemoglobinuria Jaundice, hypoglycaemia Anaemia, splenomegaly

Traveller’s diarrhoea Pneumonia, cardiac failure Encephalitis, meningoencephalitis

Renal Hepatic Haematological

958

Nephritis Hepatitis Viral infection, lymphoma

Table 51.2 Clinical features of P. falciparum malaria infection.

Haematological aspects of tropical diseases

rather than recrudescences, which are due to incomplete treatment of the primary infection. The trigger for hypnozoites to reactivate after dormancy is unknown.

Haematological abnormalities Normochromic normocytic anaemia is common, particularly in children, but the degree and rapidity of onset are very variable. The haemoglobin may fall by up to 2 g/dL each day. In malaria-endemic regions, chronic anaemia due to nutritional deficiencies, intestinal helminths, HIV and haemoglobinopathies may be compounded by the effects of malaria. In chronically anaemic patients, the oxygen dissociation curve is shifted to the right so they are better able to tolerate further falls in haemoglobin. The clinical effects of anaemia in malaria are therefore due to a combination of the degree and rate of fall of haemoglobin. The anaemia associated with malaria has multiple aetiologies. Red cells containing parasites are removed from the circulation by the reticuloendothelial system. There is also accelerated destruction of non-parasitized cells and dyserythropoiesis in the bone marrow. Both parasitized and non-parasitized red cells lose deformability and the high shear rates in the spleen enhance their removal by that organ. In acute malaria reticulocyte response is suppressed. Erythropoietin levels are usually elevated, although occasionally they are less than anticipated for the degree of anaemia. Uncommon complications of malaria that can exacerbate the anaemia are hyperreactive malarial splenomegaly and ‘blackwater fever’. Hyperreactive malarial splenomegaly is characterized by massive splenomegaly with hypersplenism (see later) and occurs as a result of a disordered immune response to malaria. Blackwater fever is associated with severe intravascular haemolysis with haemoglobinuria, and can lead to acute renal failure. It has been associated with antimalarial drugs, particularly quinine, and may be more common in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. The high prevalence of severe anaemia in areas with intense malaria transmission has generally been ascribed to malaria, but recent studies in Malawian children have suggested that the anaemia is multifactorial, with bacteraemia, malaria, hookworm, HIV and deficiencies of G6PD, vitamin A and vitamin B12, but not iron and folate deficiency, being important. Case fatality rates of children with severe anaemia in Africa are 9–18% and mortality rises steeply at haemoglobin concentrations of less than 4 g/dL. Severe decompensated malarial anaemia can be accompanied by hypovolaemia and acidosis and therefore requires urgent intravenous rehydration and blood transfusion. The high risk of transfusion-transmissible infections in poorer countries makes it especially important to prevent and adequately treat the milder forms of anaemia so that transfusions can be avoided. Antimalarial prophylaxis, presumptive malarial treatment and insecticide-treated bednets are valuable in reducing malaria infections and anaemia in specific

groups, such as pregnant women and young children, in endemic countries. The white cell count in malaria is usually normal but may be raised in severe disease. Other white cell changes that have been described in malaria include a leucoerythroblastic response, monocytosis, eosinopenia and a reactive eosinophilia during the recovery phase. Neutrophil activation, indicated by raised leucocyte elastase levels, may be apparent in severe malaria. Mild thrombocytopenia with counts around 100 × 109/L is common. It is due to increased splenic clearance and is associated with increased platelet turnover and raised thrombopoietin levels. Pancytopenia without massive splenomegaly has also been described. The bone marrow of patients with acute malaria due to any of the four species shows prominent dyserythropoiesis. This may persist for weeks after the acute infection and is caused by intramedullary cytokines produced by the infection. Erythrophagocytosis and macrophages containing malaria pigment are frequently seen in marrow samples from malaria patients. Although malaria is associated with thrombocytopenia and activation of the coagulation cascade and fibrinolytic system, bleeding and haemorrhage are uncommon even though the prothrombin and partial thromboplastin times may be prolonged. The trigger for this activation is unknown but there is evidence that it may be a combination of procoagulant cytokines and parasitized erythrocytes, which can directly activate coagulation pathways. Disseminated intravascular coagulation (DIC) is not important in the pathogenesis of severe malaria. Fibrinogen levels are often increased and there is rapid fibrinogen turnover with consumption of antithrombin and factor (F)XIII, and increased fibrin degradation products (FDPs). Microparticle formation from platelets, red cells and macrophages also causes widespread activation of blood coagulation. Malaria has recently been found to result in increased levels of circulating active von Willebrand factor (VWF). Haematological indicators of a poor prognosis in severe malaria include: • leucocytosis > 12 × 109/L; • severe anaemia (packed cell volume < 15%); • thrombocytopenia < 50 × 109/L; • prolonged prothrombin time (> 16 s); • prolonged partial thromboplastin time (> 40 s); • reduced fibrinogen (< 2 g/L); • hyperparasitaemia > 100 000/µL (high mortality > 500 000/µL); • > 20% of parasites are pigment-containing trophozoites and schizonts; • > 5% of neutrophils contain visible malaria pigment.

Genetic haematological protection mechanisms Plasmdium vivax needs Duffy blood group antigen as a receptor to enter red cells. This antigen is absent in at least two-thirds of black races who consequently have a natural resistance to infec959

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tion with P. vivax. The protective effect of HbAS against the life-threatening complications of malaria is well recognized. Similar but less marked protection is associated with other red cell genetic abnormalities such as G6PD deficiency, α thalassaemia trait and HbC trait.

Diagnosis Microscopy Direct visualization of parasites by light microscopy using a combination of thick and thin blood films is the gold-standard diagnostic technique for malaria (Table 51.3 and Figure 51.2). A Romanowsky stain (e.g. Field, Giemsa, Leishman) pH 7.2 is used so that the parasite cytoplasm stains blue and the nuclear chromatin red. A thick blood film should be used as the first screening tool as it allows larger volumes of blood to be examined than the thin film. However, the parasites appear distorted due to the process of lysing the red cells so this method cannot be used for parasite morphology and speciation. A thin blood film allows visualization of undistorted parasites and of the size and shape of the red cells but has low sensitivity because of the small amount of blood that can be examined. The disadvantages of basing a diagnosis of malaria on blood film examination include the following. • A negative film does not exclude malaria: at least three films taken during episodes of fever should be examined and even if these are negative it does not entirely exclude the diagnosis especially in the presence of antimalarial drugs.

• A positive film does not prove that symptoms are due to malaria: asymptomatic parasitaemia is common in adults from endemic areas. • Parasites, particularly P. falciparum gametocytes, may be washed off the slide during staining; bulk staining of slides may result in transfer of parasites between slides. • Parasite density does not necessarily correlate with disease severity, although heavy parasitaemia (> 5% of red cells infected) indicates a poor prognosis. Malaria pigment may persist in phagocytic cells for several weeks after an acute attack and may be helpful in retrospective diagnosis of malaria. Automated haematology analysers may produce an abnormal pattern on the white cell differential count histogram. Debris below the white cell threshold may be due to malaria parasites and manual examination of blood films is indicated if this pattern is flagged up by the analyser. Antigen detection Rapid diagnostic tests for malaria are based on detection of the malaria antigen histidine-rich protein (HRP)2 or parasite lactate dehydrogenase (pLDH). They have been incorporated into immunochromatographic antigen-capture kits for rapid diagnosis. The sensitivity of these dipstick strip tests approaches that of thick film microscopy (i.e. 0.002% parasitaemia equivalent to 100–200 parasites/µL of blood). HRP2 protein may remain positive for 14 days after successful treatment and false positives due to rheumatoid factor have been reported. pLDH

Table 51.3 Differentiating features of human Plasmodium/Babesia species in stained thin blood films.

Appearance of infected red blood cells (size and shape) Red cells with multiple parasites/cell Stages present in peripheral blood Ring form (young trophozoite)

Gametocyte

960

P. falciparum

P. malariae

P. vivax

P. ovale

Babesia sp.

Both normal

Normal shape; size normal or smaller

As for P. vivax, but some have irregular edges

Both normal

Common

Rare

1.5–2 times larger than normal; shape normal or oval Occasional

As for P. vivax

Common

Rings and gametocytes; occasionally schizonts Delicate small ring; scanty cytoplasm; sometimes at the edge of red cell (‘accolé form’)

All stages

All stages

As for P. vivax

Ring one-third of the diameter of cell; heavy chromatin dot; vacuole sometimes ‘filled in’ Round or oval; dark coarse pigment

Ring one-third to half of the diameter of cell; heavy chromatin dot

As for P. vivax

Only rings and rare pear-shaped forms (‘Maltese cross’); no gametocytes Resembles ring of P. falciparum; look for pear-shaped structure

Round or oval

Round or oval (smaller than P. vivax)

‘Crescent’ or ‘sausage’ shape is characteristic

No gametocyte

Haematological aspects of tropical diseases

(a)

(c)

is only produced by viable parasites so it becomes negative 2–3 days after successful treatment. None of these kits are able to provide quantitative information about parasitaemia but some are able to distinguish between P. falciparum and other species. Potential uses of malaria antigen detection tests include: • confirmation of malaria diagnosis on a blood film; • detection of P. falciparum when the microscopist is inexperienced (e.g. on-call or emergency situations); • determination of species when there is a possibility of mixed infection; • monitoring response to treatment (not HRP2-based tests). Antibody detection Malarial antibodies can remain in the blood after the eradication of parasites, so their detection is not useful for diagnosis in the acute attack. The main uses of malarial antibody detection are for excluding malaria as a cause of recurrent fever, for

(b)

Figure 51.2 Stages in the life cycle of Plasmodium falciparum in Giemsa-stained thin films; the cells are not enlarged or decolorized: (a) delicate early ring forms; (b) ring forms with prominent Maurer’s clefts; and (c) ring forms and early and late schizonts (schizonts are not commonly seen in the peripheral blood).

population surveys and as a screening test for blood transfusion purposes. DNA-based methods DNA probes have been developed for malaria diagnosis but their use is generally restricted to research and epidemiological surveys.

Haematological implications of treatment for falciparum malaria Chloroquine was the first-line treatment for malaria in many countries for decades and was generally well tolerated. However, widespread parasite resistance has now seriously restricted its use and it is therefore being superseded by newer drugs, often in combination, to reduce the development of resistance. Some antimalarial drugs have significant haematological side-effects, as described below. 961

Postgraduate Haematology

Pyrimethamine is used in combination with a long-acting sulphonamide, such as sulfadoxine (as in Fansidar). It is a dihydrofolate reductase inhibitor and may therefore induce megaloblastic anaemia and other cytopenias. The sulphur component of these combinations may rarely cause cytopenias and methaemoglobinaemia. Dapsone acts by inhibiting the synthesis of dihydrofolic acid and is used as part of a fixed combination with proguanil or chlorproguanil. It may be associated with haemolytic anaemia, methaemoglobinaemia and eosinophilia. Primaquine, an inhibitor of protein synthesis, is active against the hypnozoites of P. vivax and the gametocytes of P. falciparum. It causes oxidant haemolysis in patients with G6PD deficiency and, rarely, methaemoglobinaemia. Quinine is usually reserved for life-threatening infections especially in endemic countries. It acts by disrupting the food vacuole of the parasite. Rarely, it is associated with immune thrombocytopenia and severe intravascular haemolysis. Mefloquine, halofantrine and artemisinin-related compounds do not commonly cause significant haematological side-effects. A combination of artemether and lumefantrine (Coartem or Riamet) is commonly used as first-line treatment in endemic areas. It has no major haematological side-effects. Some antibacterial drugs, such as doxycycline, clindamycin, trimethoprim and sulphonamides, have also been used for their antimalarial effect and may be associated with haematological side-effects. For malaria due to P. vivax, P. ovale and P. malariae, chloroquine is still widely used for treatment as resistance is generally low; primaquine is added to prevent relapses in vivax and ovale malaria.

Babesiosis Epidemiology and biology Babesiosis is not a tropical disease but is briefly described here as it can be confused with malaria. Babesiosis is endemic in the northeastern and upper midwestern regions of the USA and is found sporadically in other parts of the USA, Europe, Asia, Africa and South America. It is primarily a disease of animals and rarely infects humans. It is due to a protozoan parasite transmitted by the bite of the ixodid tick. Following the bite, the organisms penetrate red cells, where they take on an oval, round or pear shape and multiply by budding. The erythrocytic ring forms of Babesia microti and B. divergens may be confused with malaria P. falciparum rings, but they do not produce pigment or cause alterations in red cell morphology. A minority of organisms take on a folded shape and are thought to be gametocytes. Babesia bovis, B. microti and B. divergens are responsible for the majority of human infections, which range from asymptomatic to severe and occasionally are fatal. Most of the cases reported from Europe have been due to B. divergens and 962

occurred in patients without a functioning spleen. In North America almost all the cases have been due to B. microti, which usually produces a subclinical infection. Specific laboratory diagnosis of babesial infection is made by morphological examination of Giemsa-stained blood smears, serology and amplification of babesial DNA using polymerase chain reaction (PCR).

Clinical features The incubation period varies from 1 to 4 weeks, and the severity and progression of the clinical features vary with the infecting species. Most patients have no recollection of a tick bite. The disease presents with fever, prostration, mild hepatosplenomegaly and haemolytic anaemia with jaundice and haemoglobinuria. Severe complications, including acute tubular necrosis, respiratory distress and DIC, and a fulminant fatal course has been described in patients who are splenectomized, immunosuppressed, infected with HIV or elderly. Haematological abnormalities The anaemia may be mild to moderately severe and is due to parasite-induced abnormalities in the red cell membrane. Haemolytic anaemia, which may last for several weeks, is a prominent feature of babesiosis, particularly in splenectomized individuals. Although the haemolysis is due to complement or antibody coating of the red cells, the direct antiglobulin test (DAT) is usually negative. Haptoglobin levels are reduced and the reticulocyte count is increased. The presence of parasitaemia needs to be interpreted with caution as parasites may persist for months after the resolution of symptoms and the level of parasitaemia does not parallel the severity of disease (see Table 51.3). Babesia parasites may be confused with Pappenheimer bodies in splenectomized patients with active haemolysis. Thrombocytopenia may occur in severe cases. Total white cell counts are usually normal or low. Haematological implications of treatment for babesiosis The combination of atovaquone and azithromycin is the treatment of choice for mild or moderate babesiosis, whereas clindamycin and quinine and exchange transfusion are indicated for severe disease. Atovaquone has been associated with anaemia and neutropenia; azithromycin and clindamycin have been associated with neutropenia and thrombocytopenia.

Filariasis Epidemiology and biology There are two groups of human filariasis: those that occur in the blood (lymphatic filariasis) and those that occur in the skin (onchocerciasis). Only lymphatic filariasis is considered in this chapter, as it is associated with detectable organisms in the peripheral blood. Two species of filarial worms cause lymphatic filariasis in humans and are relevant for haematologists, Wuchereria ban-

Haematological aspects of tropical diseases

Endemic Uncertain Non-endemic

Figure 51.3 Global distribution of lymphatic filariasis.

crofti and Brugia malayi. They have different geographical distributions, with W. bancrofti being the most widespread. More than 90% of infections due to W. bancrofti are found in Asia, although it also occurs in Africa, America and the Pacific Islands. Filariasis due to B. malayi occurs in China, Indo-China, Thailand, Malaysia, Indonesia, the Philippines and south-west India (Figure 51.3). The worms are 4 cm (male) to 10 cm (female) in length and can live for over 10 years in the lymphatics. Microfilariae, which are 250–300 µm long, are produced by the female worm and released into the blood after 3–8 months, where they may live for up to 1 year. Microfilariae densities can reach 10 000/mL but are usually much lower. They exhibit daily periodicity in the blood and this timing is designed to match the biting habits of their mosquito vectors, culicine and anopheline mosquitoes. The microfilariae develop in the mosquito and pass into the proboscis, ready to be injected into another human.

Clinical presentation There is wide variation in the presenting features of lymphatic filariasis, which may occur 6 months or more after the infective bite. The symptoms and signs are due to lymphangitis. There

are recurrent bouts of fever with heat, redness and pain over lymphatic vessels. In fair-skinned people, the lymphangitis can be seen to spread distally (i.e. the opposite direction to septic lymphangitis). In W. bancrofti infection, these repeated episodes of inflammation eventually result in the typical chronic picture of filariasis, including hydrocele, lymphoedema and elephantiasis, chyluria and tropical pulmonary eosinophilia. The clinical picture in B. malayi infection is similar but it does not cause hydrocele or chylous urine.

Other filariae with blood-inhabiting larvae Loa loa This occurs in the rain-forest belt of Africa, especially West Africa. The adult worms migrate through the subcutaneous tissues, including the conjunctiva, and occasionally can be seen passing across the eye. Mansonella perstans This is a non-pathogenic and common infection of people in Africa. These organisms may therefore coexist in the blood with W. bancrofti but can be distinguished by their smaller size and absence of a sheath. 963

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Mansonella ozzardi This is also probably non-pathogenic and occurs in the West Indies and South America.

Haematological abnormalities Eosinophilia is the major and most frequent haematological abnormality produced by lymphatic filariasis. Tropical pulmonary eosinophilia is an unusual complication of filariasis and is due to an immunological hyperresponsiveness to microfilariae in the lungs. It is more common in men than women. Although microfilariae are absent from the blood in this syndrome, they may be seen in lung biopsies and adult worms can be visualized in lymphatics on ultrasound. There is an extreme eosinophilia, with eosinophil counts of greater than 10 × 109/L; the level of eosinophilia is not related to the severity of symptoms. In tropical pulmonary eosinophilia, diethylcarbamazine treatment reduces the eosinophil count and produces resolution of symptoms. This rapid response to treatment distinguishes filariasis from other causes of marked eosinophilia, such as helminths that affect the lungs (Ascaris, Strongyloides, Schistosoma subsp. trichinosis and Toxocara). Diagnosis of filariasis The adult worms residing in the lymphatics are inaccessible, so diagnosis is based on finding microfilariae in the peripheral blood. The level of filaraemia is inversely related to the clinical signs because much of the damage is due to immunological responses to the microfilariae rather than to the organisms themselves. Furthermore, the presence of microfilariae does not necessarily mean that they are causing clinical problems and, conversely, a lack of microfilariae in the blood does not exclude a diagnosis of filariasis. The peripheral blood findings must therefore be assessed in the context of the clinical picture.

(a)

To optimize the chances of finding scanty microfilariae in the blood, the sample should be taken at the appropriate time for the expected peak concentration of microfilariae (i.e. around midnight or midday for nocturnally and diurnally periodic forms respectively). There are many techniques for demonstrating microfilariae in the laboratory. The simplest method is a wet preparation of fresh blood. Microfilariae will survive in venous blood collected into EDTA for 2 days at room temperature. Motile microfilariae can be seen on a slide under low power and can be counted in a counting chamber. Numbers of microfilariae may be low, requiring concentration techniques such as blood filtering. For species identification, thick and thin blood films should be stained with Giemsa or haematoxylin and the microfilariae differentiated according to the pattern of their sheaths, nuclei distribution and size (Figure 51.4). The edges of the film should be examined carefully as microfilariae tend to be concentrated at the periphery and are easily missed if the microscopist immediately uses high power in the centre of the film. Detection of circulating antigen by enzyme-linked immunosorbent assay (ELISA) or immunochromatography (ICT) has replaced microscopy for the diagnosis of bancroftian, but not brugian, filariasis. An antigen ICT card test is available for the detection of W. bancrofti, which does not react with other filariae and is highly sensitive (100%) and specific (92%). Filarial DNA can be detected by PCR, and ultrasound scans can help identify adult worms within the lymphatic system. Serological tests are not very helpful as most individuals from endemic areas have antibodies to crude filarial antigens and there is cross-reactivity with other filariae and nematodes.

Haematological implications of treatment for filariasis Oral diethylcarbamazine is the drug of choice in all forms of lymphatic filariasis, including subclinical infection. Alternative

(b)

Figure 51.4 Microfilariae of W. bancrofti in thick film: (a) microfiliaria showing the negative impression of the sheath (× 365); (b) tail of the microfilaria showing that the nuclei do not extend into the tail (× 912). 964

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treatments include combinations of albendazole and ivermectin. None of these drugs has common, serious haematological side-effects. It has recently been discovered that depletion of Wolbachia endobacteria, a symbiont of Onchocerca, by tetracycline antibiotics leads to long-lasting sterility of adult female worms.

Republic, Chad, Democratic Republic of Congo, Côte d’Ivoire, Guinea, Malawi, Uganda and United Republic of Tanzania. During recent epidemic periods the prevalence reached 50% in several villages in the Democratic Republic of Congo, Angola and southern Sudan. Improved surveillance has reduced the number of new cases over the last decade.

African trypanosomiasis (sleeping sickness)

Clinical features The bite of a tsetse fly is very painful and causes a small indurated lesion that may persist for some days. The local multiplication of the trypanosomes may cause a marked inflammatory reaction (a chancre) that regresses after 2–3 weeks. Entry of the trypanosomes into the bloodstream is associated with fever, which tends to be less marked in West African trypanosomiasis than in the East African variety. East African trypanosomiasis is primarily a disease of cattle and only enters human hosts by accident. It is therefore less well tolerated than West African sleeping sickness, having a more aggressive course and intense symptoms. The early stages of sleeping sickness can be associated with prominent lymphadenopathy, particularly of the posterior cervical nodes, and mild splenomegaly. These features may be suggestive of infectious mononucleosis, tuberculous lymphadenitis or a lymphoproliferative disorder. Severe anaemia, haemorrhages and petechiae may occur at this stage.

Epidemiology and biology African sleeping sickness is caused by the haemoflagellate protozoa Trypanosoma brucei gambiense in West and Central Africa, and T. brucei rhodesiense in eastern Africa (Figure 51.5). These parasites are fusiform in shape, 12–35 µm long and morphologically indistinguishable from each other. The disease is transmitted by the bite of the tsetse fly, which is only found in Africa. The trypanosomes multiply by fission in the vicinity of the infected bite and are then disseminated by the bloodstream. Congenital transmission has also been described. The distribution of African sleeping sickness is determined by the ecological limits of the tsetse fly vector and lies in the region between Senegal and Somalia in the north and the Kalahari and Namibian deserts in the south. Sleeping sickness is an important public health problem in Central African

Human African trypanosomiasis Epidemic High endemic Endemic At risk

Figure 51.5 Global distribution of African trypanosomiasis. 965

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Both types of African sleeping sickness cause a protracted febrile illness which, despite the name, is not always associated with drowsiness. Death is inevitable if the disease is left untreated. As the disease progresses parasitaemia decreases, trypanosomes invade the central nervous system (CNS) and neurological disturbances due to inflammatory chronic meningoencephalitis supervene. In West African trypanosomiasis, the disease runs its course over several years but in East African trypanosomiasis infection CNS involvement may occur within weeks.

Haematological abnormalities The aetiology of the anaemia in sleeping sickness is multifactorial but primarily due to phagocytic removal of immune complex-coated red cells from the circulation. Trypanosomes liberate haemolytic factors that contribute to this process, and increases in plasma volume cause a dilutional anaemia. There is a failure to incorporate iron into red cell precursors and the resulting dyserythropoiesis means that the bone marrow is unable to compensate for the fall in haemoglobin. There may be a moderate leucocytosis with increased monocytes, lymphocytes and plasma cells. Mott morular cells have also been described in sleeping sickness. The bone marrow is hypercellular, with areas of gelatinous degeneration. As the disease advances, a bleeding tendency may develop due to thrombocytopenia, vascular injury and coagulopathy. Platelet dysfunction has also been described and is manifest as clumping and abnormal aggregation responses. DIC with raised FDPs may occur in the later stages. Although some of these haematological changes can be linked to the non-specific polyclonal activation of B cells, overall the mechanisms underlying these are not well understood. Diagnosis of sleeping sickness Trypanosomes can be seen on stained thin blood films but the number of trypanosomes in the circulation can vary considerably and is often low, so concentration techniques are usually required. Quantitative buffy coat method is the technique of choice for diagnosis of African sleeping sickness. This involves concentrating the trypanosomes at the plasma–platelet interface in a special microhaematocrit tube using differential centrifugation. Parasites are identified by labelling with the fluorescent marker acridine orange. Wet preparations of fluid aspirated from the lymph nodes, bone marrow or cerebrospinal fluid (CSF) may also reveal live motile organisms. This technique is more likely to be productive in the case of infection with T. brucei rhodesiense than T. brucei gambiense. The organisms are fragile so care must be taken not to damage them when making the smears. The highly specific and sensitive serological card agglutination test for trypanosomiasis (CATT) may be used in conjunction with a direct visualization method. If these tests are positive, 966

then CSF examination is mandatory to determine the stage of the illness.

Haematological implications of treatment for African sleeping sickness Pentamidine and suramin are the drugs of choice for the early stages of West and East African trypanosomiasis respectively. They have a cure rate of around 90% but are only able to achieve modest CSF concentrations so they cannot be used for later stages of the disease. The most common haematological sideeffects of pentamidine are leucopenia, thrombocytopenia and anaemia. Suramin has serious side-effects, including haemolytic anaemia and bone marrow toxicity. Melarsoprol, an arsenic-based compound, has been the drug of choice for late-stage sleeping sickness but is highly toxic, with a mortality of 4–12%. Its main adverse effect is a fatal encephalopathic syndrome; haematological toxicity is not a particular problem. Eflornithine is expensive but is of benefit in late-stage sleeping sickness, particularly West African disease; 25–50% of patients treated with this drug exhibit bone marrow toxicity with pancytopenia.

American trypanosomiasis (Chagas disease) Epidemiology and biology Chagas disease is caused by a haemoflagellate protozoa, T. cruzi, which is transmitted by triatomine bugs that infest poor-quality housing. It can also be transmitted through blood transfusions and congenitally. It is restricted to a region in the Americas between Argentina and the southern states of the USA. Clinical features The incubation period is usually a couple of weeks but may be up to several months if transmission was through blood transfusion. In the acute phase, swelling at the site of entry of the organism, a chagoma, may be accompanied by fever, mild hepatosplenomegaly and local or generalized lymphadenopathy. The trypanosomes multiply intracellularly in muscle tissue, particularly the heart, colon and oesophagus. Once infection has occurred, the organisms will be present for life unless treatment is given. The chronic phase of the disease is associated with heart disease in 30% of infected individuals, which is manifest as arrhythmias and cardiomegaly. A small proportion of individuals also have clinical involvement of the gastrointestinal tract and other hollow organs resulting in loss of peristalsis, organomegaly and organ failure. Asymptomatic infection is common and poses a problem for blood transfusion services in endemic areas, so some countries routinely screen blood for American trypanosomiasis.

Haematological aspects of tropical diseases

Diagnosis of Chagas disease Although similar methods to those used for African trypanosomiasis can be helpful for diagnosis, serological tests are more commonly used as the primary diagnostic tool. They are based on enzyme immunoassay or immunofluorescent antibody test and have good sensitivity. PCR may also be useful but is not in routine use. Haematological implications of treatment for American trypanosomiasis There are only two effective drugs for the treatment of T. cruzi infection, nifurtimox and benznidazole. Major haematological side-effects are not common with either drug, although agranulocytosis has been reported with benznidazole.

Clinical features The clinical expression of leishmaniasis depends on both the genotypic potential of the parasite and the immunological response of the patient. Incubation period varies from days to years but is generally 2–6 months. Onset can be sudden with high fever, or gradual with intermittent fever. Diarrhoea, joint pains, weight loss and bleeding gums occur in the acute phase. This is followed by progressive muscle wasting, protuberant abdomen, fever, weight loss, anaemia and hepatosplenomegaly. The splenomegaly appears early and the spleen increases in size in relation to the duration of the disease, so that eventually it may reach into the left hyopchondrium. In immunocompromised patients, such as transplant recipients and those with advanced HIV disease, kala-azar behaves like an opportunistic infection.

Leishmaniasis Epidemiology and biology Visceral and cutaneous leishmaniases are caused by protozoan flagellates that are transmitted through the infective bite of a phlebotomine sandfly. Following an infected bite, parasites spread from the inoculation site to the mononuclear phagocytic system. Only the visceral form (kala-azar) is associated with organisms in haemopoietic tissues and thus is considered here. Visceral leishmaniasis is due to the species Leishmania donovani and L. infantum and is found in 47 countries throughout the world, with extension limits from 45° N to 32° S; 90% of cases are in Bangladesh, India, Nepal, Sudan and Brazil (Figure 51.6). The number of cases of visceral leishmaniasis, particularly around the Mediterranean basin in southern Europe, has increased in association with HIV-related immunosuppression.

Figure 51.6 Global distribution of visceral leishmaniasis.

Haematological abnormalities Normochromic normocytic anaemia is a frequent and clinically significant feature of visceral leishmaniasis and haemoglobin levels of 7–10 g/dL are common. The massive splenic enlargement results in hypersplenism with consequent pancytopenia. Liver dysfunction with jaundice, ascites and deranged coagulation may occur in the late stages and has a poor prognosis. The bleeding tendency may be exacerbated by thrombocytopenia. In all patients with unexplained splenomegaly, pancytopenia or fever, a high index of suspicion of leishmaniasis needs to be maintained to prevent fatalities. Diagnosis of leishmaniasis Definitive diagnosis is based on detection of the parasites, or their DNA, in smears of bone marrow, splenic aspirate or fluid aspirated from enlarged lymph nodes.

Highly endemic countries

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Figure 51.7 Bone marrow aspirate of leishmaniasis infection showing a macrophage containing numerous organisms. The presence of both a nucleus and a small paranuclear kinetoplast gives the organisms their characteristic ‘double-dot’ appearance (MCG ×940).

On microscopy Leishmania are usually seen as intracellular amastigotes in mononuclear cells but can also be seen extracellularly (Figure 51.7). They are 2–6 µm in diameter and contain a nucleus lying close to the rod-shaped kinetoplast, and an internal flagellum. Using a Romanowsky stain, the nucleus and kinetoplast stain purple and can be clearly distinguished. Amastigotes can be seen in both bone marrow aspirates and in trephine-impression smears. They are rarely seen in peripheral blood and then only in buffy coat preparations. Direct microscopic visualization is less sensitive than molecular diagnosis, particularly when there is co-infection with HIV. PCR can be performed on lesion aspirate, marrow, blood and biopsy material. The indirect fluorescent antibody tests ELISA and DAT are useful for detecting antibodies to visceral leishmaniasis, but results may be inconclusive in immunosuppressed patients.

Haematological implications of treatment for leishmaniasis For the last 80 years, the treatment of leishmaniasis has been based on pentavalent antimonials, although their mode of action is still unclear. Sodium stibogluconate is the most commonly used antimonial. It can be associated with worsening anaemia and thrombocytopenia, although its most serious detrimental effects are on cardiac function. Resistance levels to antimonials are high in some countries including India, Bangladesh, Brazil and Sudan, so other options such as amphotericin, paromomycin and miltefosine need to be considered. HIV co-infected patients do not respond well to antimonials so for these individuals amphotericin is the drug of choice. Amphotericin can produce haematological side-effects, commonly normocytic normochromic anaemia, but its most serious 968

Figure 51.8 Patient with massive splenomegaly due to hyperreactive malarial splenomegaly.

toxicity is related to renal, cardiac, neurological and hepatic dysfunction. Side-effects can be reduced by using the liposomal preparation. Immunocompromised patients may require prophylaxis to prevent relapses.

Non-specific haematological abnormalities associated with tropical diseases Hypersplenism Hypersplenism is a syndrome characterized by splenomegaly and cytopenias. Tropical infections associated with massive splenomegaly include hyperreactive malarial splenomegaly, visceral leishmaniasis, schistosomiasis and trypanosomiasis (Figure 51.8). The cytopenias in hypersplenism are due to a combination of sequestration and haemodilution. The degree of expansion of plasma volume is proportional to the size of the spleen, and can be improved by splenectomy. The thrombocytopenia and neutropenia are rarely severe enough to cause clinical problems. Most of the thrombocytopenia is due to pooling in the spleen which, when massively enlarged, can hold up to 90% of the platelet mass. Neutropenia is the result of increased marginalization of granulocytes. Treatment of the underlying disorder generally leads to regression of the splenomegaly with resolution of the haematological abnormalities.

Tropical diseases associated with changes in the full blood count Anaemia Anaemia of chronic disease is a common and non-specific finding in many types of tropical infections. Some infections are responsible for specific types of anaemia. For example, hook-

Haematological aspects of tropical diseases

worm infection contributes to iron deficiency anaemia, and megaloblastic anaemia secondary to parasite consumption of vitamin B12 is a feature of infection with the tapeworm Diphyllobothrium latum. Intraerythrocytic parasites, such as those that cause malaria and babesiosis, may be associated with significant intravascular haemolysis. White cell abnormalities Severe infections particularly due to bacteria may cause a neutrophilia and a leukaemoid reaction with circulating myeloid precursors and neutrophils with toxic granulation, vacuolation and Döhle body formation. Lymphocytosis with neutropenia, splenomegaly, nose bleeds, rash and neurological complications is a feature of rickettsial diseases (e.g. typhus, Q fever, trench fever). Some, such as trench fever, may persist for many years and are transmissible in blood transfusions. The absence of neutrophilia in the presence of marked fever is a particular feature of typhoid. Lymphocytosis or monocytosis may also be present in typhoid; other clinical features include hepatosplenomegaly and, in severe disease, haemorrhage from ileal ulcers exacerbated by DIC. Helminths and other predominantly tropical organisms that invade tissues may be associated with a significant eosinophilia (> 0.5 × 109/L). Such diseases include loiasis, lymphatic filariasis, schistosomiasis, trichinosis, toxocariasis, strongyloidiasis, hydatid disease, oriental liver flukes and guinea worm.

Tropical infections with fever and haemorrhage Relapsing fever Relapsing fevers are borne by either lice or ticks. Louse-borne relapsing fever is endemic in the horn of Africa and Rwanda. Tick-borne relapsing fever has a wider distribution through Africa, the Mediterranean basin and the Middle East. They have a relapsing course and severe disease is characterized by fever with jaundice, neutrophilia, thrombocytopenia and DIC. There is a marked bleeding tendency, with petechial haemorrhage and epistaxis. Spiral organisms (Borrelia spp.) can be seen in the blood. Relapsing fevers respond well to tetracycline but this must be given with care as it can generate a severe lifethreatening Jarisch–Herxheimer reaction. Viral haemorrhagic fevers These are caused by arenaviruses, filoviruses, bunyaviruses and flaviviruses and are classified according to their reservoir hosts and their primary means of transmission. They are divided into: • rodent-associated viruses (e.g. Lassa fever, hantaviruses); • arthropod-borne viruses (e.g. dengue, yellow fever and Chikungunya viruses); • unknown vectors or hosts (e.g. Marburg, Ebola). They often occur in epidemics, have human-to-human transmission and may only be suspected if a relevant travel history

is elicited from the patient. Dengue and yellow fever are becoming increasingly important imported infections.

Dengue The four types of dengue virus belong to a group of flaviviruses and are transmitted by Aedes mosquitoes. Dengue is endemic in tropical areas of Asia, Africa, South America and the Caribbean and is particularly virulent in Southeast Asia, including Thailand and Vietnam. There has been a resurgence of the disease as a result of urbanization, poverty, the demise of Aedes eradication programmes and increasing travel. Primary infection occurs in young children and is usually asymptomatic. Older children and adults develop acute fever, headache and myalgia (‘breakbone fever’). Leucopenia may accompany this stage of the illness. Severe complications may arise in those who have had previous dengue infection. These include hypotensive shock, marked thrombocytopenia with spontaneous bleeding, neutropenia, bone marrow hypoplasia and abnormal megakaryocytopoiesis. Lassa fever, Ebola virus and Marburg virus These are endemic in equatorial Africa and are important because they cause potentially fatal infections and have the ability to spread from person to person. Only about 10% of infected individuals become ill. Of these 1–2% develop fatal disease. The clinical features of these three haemorrhagic fevers are similar and are characterized by headache, fever and oesophagitis. Spontaneous bleeding occurs in 25% of hospitalized patients and is thought to be due to abnormal platelet function. Case reports suggest that treatment with ribavirin may be helpful. Yellow fever Yellow fever virus is transmitted by Aedes mosquitoes and exists throughout equatorial Africa, and northern and central southen America. It invades hepatocytes, causing hepatocellular dysfunction and jaundice. Fever, myalgia and back pain may be followed by jaundice, bleeding and, in the most severe cases, renal failure.

Selected bibliography Malaria Francischetti IM, Seydel KB, Monteiro RQ (2008) Blood coagulation, inflammation and malaria. Microcirculation 15: 81–107. Ghosh K, Shetty S (2008) Blood coagulation in falciparum malaria: a review. Parasitology Research 102: 571–6. Groot E, de Groot PG, Fijnheer R, Lenting PJ (2007) The presence of active von Willebrand factor under various pathological conditions. Current Opinion in Hematology 14: 284–9. Health Protection Agency Advisory Committee on Malaria Prevention guidelines. Available at http://www.nathnac.org/pro/ clinical_updates/importedmalaria_250408.htm 969

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WHO malaria treatment guidelines. Available at http://www.who. int/malaria/docs/TreatmentGuidelines2006.pdf

Babesiosis Vannier E, Gewurz BE, Krause PJ (2008) Human babesiosis. Infectious Disease Clinics of North America 22: 469–88, viii–ix. Filariasis Hoerauf A (2006) New strategies to combat filariasis. Expert Review of Antiinfective Therapy 4: 211–22. African trypanosomiasis (sleeping sickness) World Health Organization. Information on African trypanosomiasis (sleeping sickness). Available at http://www.who.int/ mediacentre/factsheets/fs259/en/ (accessed 4 March 2009). Viral haemorrhagic fevers Centers for Disease Control Special Pathogens Branch. Information on viral hemorrhagic fevers. Available at http://www.cdc.gov/

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ncidod/dvrd/Spb/mnpages/dispages/vhf.htm (accessed 4 March 2009). World Health Organization. Information on viral hemorrhagic fevers. Available at http://www.who.int/topics/haemorrhagic_ fevers_viral/en/ (accessed 4 March 2009).

General Bain BJ (2006) Blood Cells: A Practical Guide, 4th edn. WileyBlackwell, Oxford. Calis JC, Phiri KS, Faragher EB et al. (2008) Severe anaemia in Malawian children. New England Journal of Medicine 358: 888–99. Cheesbrough M (2005) District Laboratory Practice in Tropical Countries. Parts 1 and 2. Tropical Health Technology, 2nd edn. Cambridge University Press, Cambridge. Cook GC, Zumla A (eds) (2009) Manson’s Tropical Diseases, 22nd edn. Saunders/Elsevier, Edinburgh. Lewis SM, Bain BJ, Bates I (eds) (2006) Dacie and Lewis Practical Haematology, 10th edn. Churchill Livingstone, London.

CHAPTER 52

52

Neonatal haematology Irene AG Roberts Centre for Haematology, Imperial College London, London, UK

Developmental haemopoiesis, 971 Neonatal anaemia, 971 Definition and pathophysiology, 971 Causes of neonatal anaemia, 972 Anaemia of prematurity, 977 A simple diagnostic approach to neonatal anaemia, 978 Neonatal polycythaemia, 978 White cell disorders, 979 Normal values, 979

Neutropenia, 979 Haemostasis and thrombosis in the newborn, 979 Developmental haemostasis, 980 Screening tests for bleeding disorders, 980 Inherited coagulation disorders, 980 Acquired disorders of coagulation, 981 Neonatal thrombocytopenia, 981

Developmental haemopoiesis Haemopoiesis begins in the yolk sac in the third week of gestation and moves sequentially to the aorta–gonad–mesonephros (AGM) by 5 weeks’ gestation, the liver by 6–8 weeks and the bone marrow around the 11th week of gestation. The AGM involutes early in the first trimester and the liver is the principal site of haemopoiesis until the end of the third trimester. The predominant lineage during fetal life is erythropoiesis but platelets and all types of leucocyte found in adult blood are also seen from as early as 4–5 weeks’ gestation. There are a number of differences between erythropoiesis in neonates and adults: red cell morphology is distinctive with large numbers of crenated red cells, particularly in preterm neonates (Figure 52.1); red cell lifespan is reduced (35–50 days in preterm infants, 60–70 days in term infants); susceptibility to oxidant-induced injury is increased because of differences in the glycolytic and pentose phosphate pathways; the erythropoietin response to anaemia is blunted; and specific embryonic and fetal globin chains are synthesized (Table 52.1). The first globin chain produced is ε-globin, followed by α- and γ-globin chains. Haemoglobin F (HbF, α2γ2) is produced from 4–5 weeks’ gestation and is the predominant haemoglobin until after birth. Adult haemoglobin (HbA, α2β2) is produced from 6–8 weeks’ gestation, but remains at low levels until after birth. In term babies, the average HbF at birth is 70–80%, the HbA is 25–30%,

Postgraduate Haematology: 6th edition. Edited by A. Victor Hoffbrand, Daniel Catovsky, Edward G.D. Tuddenham, Anthony R. Green © 2011 Blackwell Publishing Ltd.

Neonatal thrombosis: physiology and developmental aspects, 982 Screening tests for thrombophilia in neonates, 982 Inherited thrombotic disorders in neonates, 982 Acquired thrombotic problems in neonates, 983 Selected bibliography, 983

there are small amounts of HbA2 and sometimes a trace of Hb Barts (γ4). Immediately after birth, rates of haemoglobin synthesis and red cell production fall in response to the sudden increase in tissue oxygenation at birth. In term babies, the haemoglobin reaches a mean of 13–14 g/dL at 4 weeks and 9.5–11 g/dL at 7–9 weeks of age. Studies of well preterm infants show a steeper fall in haemoglobin, reaching a mean of 6.5–9 g/dL at 4–8 weeks of age. The reticulocyte count falls after birth as erythropoiesis is suppressed and increases to normal values at 6–8 weeks of age. The blood volume at birth varies with gestational age and the timing of clamping of the cord. In term infants, the average blood volume is 80 mL/kg and in preterm infants 106 mL/kg (range 85–143 mL/kg). Term and preterm babies have adequate stores of iron, folic acid and vitamin B12 at birth. However, stores of both iron and folic acid are lower in preterm infants and are depleted more quickly, leading to deficiency after 2–4 months if the recommended daily intakes are not maintained.

Neonatal anaemia Definition and pathophysiology Any neonate with a haemoglobin of less than 13 g/dL at birth should be considered anaemic and may require investigation (Figure 52.2). However, it is important to be aware that the haemoglobin concentration is affected by the site of sampling (it is up to 4 g/dL lower in venous than in heel-prick samples in 971

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Table 52.2 Common causes of neonatal anaemia. Reduced red cell production Diamond–Blackfan anaemia Congenital viral infections, e.g. parvovirus, cytomegalovirus Congenital dyserythropoietic anaemia Pearson syndrome Increased red cell destruction (haemolysis) Alloimmune: haemolytic disease of the newborn (Rh, ABO, Kell, other) Red cell membrane disorders, e.g. hereditary spherocytosis Red cell enzyme deficiencies, e.g. pyruvate kinase deficiency Some haemoglobinopathies, e.g. α-thalassaemia major, HbH disease Figure 52.1 Typical erythrocyte morphology in a preterm neonate. Crenated red cells are a normal feature of the blood film of preterm neonates during the first few weeks of life. This film is from a neonate born at 26 weeks’ gestation and shows the number of crenated cells present in neonates under 28 weeks’ gestation. The numbers of these cells is inversely proportional to gestational age at birth.

Blood loss Occult haemorrhage before or around birth, e.g. twin–twin, fetomaternal Internal haemorrhage, e.g. intracranial, cephalhaematoma Iatrogenic, due to frequent blood sampling Anaemia of prematurity Impaired red cell production plus reduced red cell lifespan

Table 52.1 Composition of haemoglobins in the human embryo, fetus and neonate. Haemoglobin

Globin chains

Gestation

α-Globin gene cluster*

β-Globin gene cluster*

Embryonic Hb Gower 1 Hb Gower 2 Hb Portland

ξ2 α2 ξ2

ε2 ε2 γ2

From 3–4 weeks

Fetal HbF

α2

γ2

From 4 weeks

Adult HbA HbA2

α2 α2

β2 δ2

From 6–8 weeks From 30 weeks

From 4 weeks

*The α-globin gene cluster is situated on chromosome 16 and the β-globin gene cluster on chromosome 11. Note that fetuses and neonates with α-thalassaemia major, who are unable to synthesize α-globin chains, will have Hb Portland as well as Hb Barts (β4), detectable by haemoglobin electrophoresis or HPLC.

the first few days of life) and the timing of the clamping of the cord (around 3 g/dL higher after late clamping). The clinical significance of neonatal anaemia depends on whether the baby is able to maintain adequate tissue oxygenation. This in turn depends on the position of the haemoglobin–oxygen dissocia972

tion curve, which is principally determined by the concentrations of HbF and 2,3-diphosphoglycerate (2,3-DPG); a high HbF and low 2,3-DPG both shift the curve to the left, i.e. the affinity of haemoglobin for oxygen is increased and less oxygen is released to the tissues. This is the situation just after birth and may be more of a problem for very preterm babies as their HbF levels are greater than 90%. Over the first few months of life, 2,3-DPG levels rise and HbF levels fall so that the haemoglobin– oxygen dissociation curve gradually shifts to the right, the oxygen affinity of haemoglobin falls and oxygen delivery to the tissues increases, ameliorating the effects of the falling haemoglobin.

Causes of neonatal anaemia Anaemia may be caused by reduced red cell production, increased red cell destruction (haemolysis) or blood loss (Table 52.2). Neonatal anaemia due to reduced red cell production The main diagnostic clues to reduced red cell production are the combination of a low reticulocyte count (< 20 × 109/L) with a negative direct antiglobulin test (Coombs test). The most common causes are congenital parvovirus infection and genetic red cell aplasias, particularly Diamond–Blackfan anaemia (DBA).

Neonatal haematology

Anaemia

Reticulocyte count

Low reticulocytes

Raised reticulocytes Check Coombs' test

Red cell aplasia

Coombs' test positive

Coombs' test negative

Parvovirus B19 DBA rare, e.g. Pearson's

Diagnosis probably HDN Identify antibody, check blood film and bilirubin

HDN excluded Check Kleihauer

Kleihauer negative

Kleihauer positive

Check MCV

Fetomaternal bleed

MCV normal or high Review blood film Abnormal Inherited red cell membrane disorder, e.g. HS Inherited red cell enzyme disorder, e.g. G6PD or pyruvate kinase deficiency Normal G6PD deficiency (film may show oxidative haemolysis but is more often normal)

MCV low α-Thalassaemias α-Thalassaemia major or HbH disease Check Hb electrophoresis on baby and parents (all will have low MCV) Hereditary pyropoikilocytosis Check FBC and film on parents (one or both will usually have elliptocytes)

Figure 52.2 A diagnostic algorithm for neonatal anaemia. The most useful screening tests for investigating unexplained neonatal anaemia are the reticulocyte count, the Coombs test and the mean corpuscular volume (MCV) of the red cells. DBA,

Diamond–Blackfan anaemia; G6PD, glucose-6-phosphate dehydrogenase; HDN, haemolytic disease of the newborn; HS, hereditary spherocytosis.

Parvovirus B19 and fetal/neonatal anaemia Maternal infection with parvovirus B19 can cause severe fetal anaemia and in 9% of cases leads to intrauterine death. The baby has marked reticulocytopenia (often < 10 × 109/L) and thrombocytopenia may also occur. The diagnosis is made by maternal serology and demonstration of B19 DNA in the fetus or neonate by dot-blot hybridization or polymerase chain reaction (PCR) of peripheral blood (bone marrow aspiration for morphology and parvovirus B19 PCR may be necessary in difficult cases). Severe cases require intrauterine transfusion but have a good long-term outcome if they survive to delivery.

almost never present at birth. DBA, which occurs in five to seven babies per million live births, has a clear family history in 20% of cases (autosomal dominant or recessive) and appears to be sporadic in the remaining 80%. It usually presents as increasing anaemia over the first few weeks or months of life but more severe cases manifest as second-trimester anaemia or hydrops fetalis. Around 40% of infants have associated congenital anomalies, particularly craniofacial dysmorphism, neck anomalies and thumb malformations similar to those seen in Fanconi anaemia. The blood film shows normochromic red cells with an absence of polychromasia and nucleated red cells despite severe anaemia (Figure 52.3). Reticulocytopenia is usually severe but automated reticulocyte counts of 20–30 × 109/L are sometimes seen. Erythroid precursors are absent in the bone marrow aspirate. These features are diagnostic of DBA if parvovirus infection is excluded. Raised red cell levels of adenosine deaminase (ADA) in the patient and/or parents may be useful

Genetic red cell aplasia Apart from DBA, the genetic causes of congenital red cell aplasia are extremely rare. They include congenital dyserythropoietic anaemia (CDA) and Pearson syndrome; the other inherited bone marrow failure syndromes, such as Fanconi anaemia, are

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Figure 52.3 Blood film from a neonate with Diamond–Blackfan anaemia. This baby presented with fetal anaemia at 20 weeks’ gestation and received intrauterine transfusion. At birth, the baby had normochromic anaemia and the blood film showed a complete absence of polychromasia and nucleated red cells, despite a haemoglobin of 7 g/dL.

for confirming the diagnosis, although normal red cell ADA levels do not exclude DBA. Recent studies indicate that DBA is due to defects in structural ribosomal proteins (RP). At present mutations in six genes (RPS19, RPS24, RPS17, RPL5, RPL11 and RPL35a) have been identified and account for about 50% of DBA cases, RPS19 mutations being the commonest (25% of all DBA). In the neonatal period, the only treatment of DBA is red cell transfusion, although steroids are used in older infants and children. The other genetic causes of congenital red cell aplasia, CDA and Pearson syndrome, can be distinguished from DBA by marrow morphology. In Pearson syndrome, which is caused by mutations in mitochondrial DNA and presents with normochromic anaemia, neutropenia, thrombocytopenia and failure to thrive in the first few weeks of life, there is highly characteristic vacuolation of early erythroid cells on the marrow aspirate (Figure 52.4). Unfortunately, the prognosis for children with Pearson syndrome is very poor, with few surviving beyond the second year of life. Mutations in codanin-1 and SEC23B have recently been shown to be responsible for many, but not all, cases of type I and II CDA respectively. Neonatal anaemia due to increased red cell destruction (haemolysis) The main diagnostic clues suggesting a haemolytic anaemia are increased numbers of reticulocytes and/or circulating nucleated red cells (Figure 52.5), unconjugated hyperbilirubinaemia, a positive Coombs test (if immune) and characteristic changes in

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Figure 52.4 Pearson syndrome. Bone marrow aspirate from a neonate with Pearson syndrome showing typical vacuolation of erythroblasts and a dysplastic megakaryocyte.

the morphology of the red cells on a blood film (e.g. hereditary spherocytosis; Figure 52.5). The main cause of immune haemolytic anaemia is haemolytic disease of the newborn. The main causes of non-immune neonatal haemolysis are red cell membrane disorders, red cell enzymopathies and, occasionally, haemoglobinopathies (Table 52.2).

Immune haemolysis, including haemolytic disease of the newborn The principal alloantibodies causing haemolytic disease of the newborn are those against rhesus antigens (anti-D, anti-c and anti-E), anti-Kell, anti-Kidd (Jk), anti-Duffy (Fy) and antibodies of the MNS blood group system, including anti-U. Anti-D is the most frequent alloantibody causing significant haemolytic anaemia, affecting 1 in 1200 pregnancies. Anti-Kell antibodies are less common but can cause severe fetal and neonatal anaemia as they inhibit erythropoiesis as well as causing haemolysis. Most babies with haemolytic disease of the newborn present with jaundice and/or anaemia; in severe cases there is hepatosplenomegaly and/or skin deposits due to extramedullary haemopoiesis. ABO haemolytic disease occurs only in offspring of women of blood group O and is confined to the 1% of such women who have high-titre IgG antibodies. Haemolysis due to anti-A is more common (1 in 150 births) than that due to antiB. In contrast with anti-rhesus antibodies, both anti-A and anti-B usually cause hyperbilirubinaemia without significant anaemia; however, hydrops has occasionally been described. The blood film in ABO haemolytic disease characteristically shows very large numbers of spherocytes with little or no increase in nucleated red cells (see Figure 52.5a); this contrasts with rhesus haemolytic disease of the newborn, in which there are few spherocytes and large numbers of circulating nucleated red cells (see Figure 52.5b).

Neonatal haematology

Figure 52.5 Haemolytic disease of the newborn. (a) Blood film from a baby with rhesus haemolytic disease of the newborn due to anti-D showing polychromasia and large numbers of nucleated red cells but relatively few spherocytes. (b) Blood film from a neonate with ABO haemolytic disease of the newborn due to anti-A showing very large numbers of spherocytes, polychromasia and no nucleated red cells.

(a)

Management of haemolytic disease of the newborn All neonates at risk should have cord blood taken for measurement of haemoglobin, bilirubin and a Coombs test and should remain in hospital until hyperbilirubinaemia and/or anaemia have been properly managed. Rhesus-alloimmunized infants with haemolysis should receive phototherapy from birth, as the bilirubin can rise steeply; this prevents the need for exchange transfusion in some infants. In haemolytic disease due to anti-Kell, anaemia is usually more prominent than jaundice and minimal phototherapy may be necessary despite severe anaemia. ABO haemolytic disease of the newborn usually just requires phototherapy as anaemia is uncommon. The indications for exchange transfusion in haemolytic disease of the newborn are: • severe anaemia (haemoglobin < 10 g/dL at birth); and/or • severe or rapidly increasing hyperbilirubinaemia. The British Committee for Standards in Haematology (BCSH) have published useful guidelines for neonatal exchange transfusion. Intravenous immunoglobulin has been used to reduce the need for exchange transfusion but the value of this approach remains to be proven in clinical trials. Neonates with haemolytic disease due to anti-rhesus antibodies may develop ‘late’ anaemia at a few weeks of age, requiring ‘top-up’ transfusion; irradiated blood must be used for infants previously receiving intrauterine transfusion to prevent the risk of transfusion-associated graft-versus-host disease. Recombinant erythropoietin sometimes prevents the need for top-up transfusion for late anaemia but is not effective when haemolysis is brisk. Folic acid (500 µg/kg daily) should be given to all babies with haemolysis until they reach 3 months of age.

Neonatal haemolytic anaemia due to red cell membrane disorders In neonates, these disorders present with jaundice and moderate anaemia (usually due to hereditary spherocytosis), as an incidental finding on routine blood films in the absence of

(b)

jaundice or anaemia (usually hereditary elliptocytosis) or, occasionally, as severe transfusion-dependent haemolytic anaemia with a characteristic low mean corpuscular volume (MCV) of 50–60 fL (usually due to hereditary pyropoikilocytosis). The main clues are a family history and an abnormal blood film, as red cell membrane disorders can nearly always be recognized by the characteristic shape of the red cells (Figure 52.6a). Identification of the exact type of membrane abnormality may require specialized investigations. The osmotic fragility test is of limited value in neonates and recent data indicate that the dye binding test is more useful. If the clinical phenotype is severe and family history or family studies are unhelpful, red cell membrane electrophoresis is indicated to clarify the diagnosis (on pretransfusion blood samples to minimize diagnostic confusion due to transfused cells). Hereditary spherocytosis occurs in 1 in 5000 live births to parents of northern European extraction. It is caused by mutations in the genes for spectrin, ankyrin, protein 4.1 or protein 3 and is usually inherited in an autosomal dominant fashion. The blood film shows spherocytes and is identical to that of ABO haemolytic disease (Figure 52.6b). Neonatal anaemia due to hereditary spherocytosis is usually moderate (haemoglobin 7–10 g/dL); it is not uncommon for affected neonates to require one or two transfusions during the neonatal period before a transfusion-free plateau haemoglobin of 8–10 g/dL is achieved after a few months. Hereditary elliptocytosis, which is caused by different mutations in the genes for spectrin, ankyrin or protein 4.1, usually has no clinical manifestations in the neonate apart from elliptocytes on the blood film. However, neonates who are homozygous or compound heterozygous for hereditary elliptocytosis mutations have severe haemolytic anaemia. The most common form is hereditary pyropoikilocytosis, which causes severe transfusion-dependent haemolytic anaemia. The diagnosis is easily made from the low MCV and blood films of the baby (which show bizarre fragmented red cells and microspherocytes; see Figure 52.6a), and both parents (one or both of whom

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(a)

(b)

usually has elliptocytosis). Red cell transfusion is necessary until the child is old enough to undergo splenectomy, to which there is an excellent response.

Neonatal haemolysis due to red cell enzymopathies The commonest inherited red cell enzymopathies presenting in the neonatal period are glucose-6-phosphate dehydrogenase (G6PD) deficiency and pyruvate kinase deficiency. G6PD deficiency has a high prevalence in individuals from Central Africa (20%) and the Mediterranean (10%). In neonates, G6PD deficiency nearly always presents with jaundice within the first few days of life; the vast majority of affected neonates are boys, as the G6PD gene is on the X chromosome. The jaundice is often severe, whereas anaemia is uncommon. The blood film is usually completely normal but in the small number of neonates with anaemia due to G6PD deficiency the blood film shows typical changes of oxidative haemolysis. The diagnosis is made by assaying G6PD on a peripheral blood sample. The pathogenesis of the jaundice is unclear as most babies with G6PD deficiency have no evidence of haemolysis. Management of neonatal G6PD deficiency requires close monitoring of the bilirubin to prevent kernicterus, particularly when interactions with other risk factors for neonatal hyperbilirubinaemia are present, such as Gilbert syndrome or hereditary spherocytosis, and also the counselling of the babies’ parents regarding which medicines, chemicals and foods may precipitate haemolysis. For the vast majority of patients there is no chronic haemolysis and no anaemia and therefore folic acid supplements are not indicated. Pyruvate kinase deficiency is the second most common inherited red cell enzymopathy in neonates and is transmitted in an autosomal recessive fashion. It is clinically heterogeneous, varying from anaemia severe enough to cause hydrops fetalis to a mild unconjugated hyperbilirubinaemia. In severe cases, the jaundice has a rapid onset within 24 hours of birth and exchange transfusion may be required. The diagnosis is made by measur976

Figure 52.6 Red cell membrane disorders. (a) Blood film from a baby with hereditary pyropoikilocytosis showing microspherocytes, red cell fragments and polychromasia. (b) Blood film from a neonate with hereditary spherocytosis showing large numbers of spherocytes and polychromasia.

ing pretransfusion red cell pyruvate kinase activity; in mild cases enzyme activity may be relatively modestly reduced making the diagnosis difficult and it is often useful to assay levels in the parents for confirmation. The blood film is sometimes distinctive but more often shows non-specific changes of nonspherocytic haemolysis. Management in the neonatal period depends on the severity of the jaundice and anaemia; some, but not all, children are transfusion dependent and folic acid supplements should be given to prevent deficiency due to chronic haemolysis. The other red cell enzymopathies are rare. The most important in the neonatal period is triosephosphate isomerase deficiency, as one-third of patients present with neonatal haemolytic anaemia, often many months before the devastating neurological features of this disorder become apparent. Another cause of acute haemolysis confined to the neonatal period is infantile pyknocytosis. This poorly understood condition, which is probably due to transient glutathione peroxidase deficiency, usually presents at 1–6 weeks of age, produces moderately severe anaemia requiring one to two red cell transfusions, shows changes of oxidative haemolysis on the blood film (Figure 52.7) and resolves by 6–12 weeks of age. Measurement of glutathione peroxidase levels in affected neonates may be useful (parental levels are normal) but the diagnosis is usually easily made from the blood film.

Neonatal haemolysis due to haemoglobinopathies The only haemoglobinopathy that presents typically in the neonatal period is α-thalassaemia major, as all four α-globin genes are deleted. Occasional non-thalassaemic structural α- and γglobin gene mutations, which are clinically completely silent in adults and children, cause transient neonatal haemolytic anaemia in the neonate because they are unstable (e.g. Hb Hasharon, Hb Poole), whereas the major β-globin haemoglobinopathies (sickle cell disease and β-thalassaemia major) rarely manifest clinically in neonates.

Neonatal haematology

Figure 52.7 Infantile pyknocytosis. Blood film from a neonate with infantile pyknocytosis and a low level of erythrocyte glutathione peroxidase showing pyknocytes, fragmented red cells, polychromasia and occasional spherocytes.

Figure 52.8 α-Thalassaemia major. Blood film from a neonate with α-thalassaemia major, born at 28 weeks’ gestation, showing severe hypochromia, microcytosis, target cells, polychromasia and nucleated red cells.

α-Thalassaemia major predominantly affects families of Southeast Asian, Mediterranean or Middle Eastern origin, and presents with second-trimester fetal anaemia or hydrops fetalis, which is fatal within hours of delivery. The only long-term survivors of α-thalassaemia major are those who have received intrauterine transfusions followed by regular postnatal transfusions and/or a bone marrow transplant. There is also a high incidence of hypospadias and limb defects in survivors and others have severe neurological problems. If intrauterine transfusions are delayed until anaemia is severe, neonatal pulmonary hypoplasia is a cause of early mortality. The diagnosis of αthalassaemia major should be suspected in any case of severe fetal anaemia that presents in the second trimester, and any case of hydrops fetalis with severe anaemia in which the parents come from high-prevalence areas, particularly Southeast Asia. Checking the blood counts of the parents will immediately identify whether they are at risk of having a child with αthalassaemia major: both parents will have hypochromic microcytic red cell indices, with MCV usually below 74 fL and mean corpuscular haemoglobin (MCH) usually less than 24 pg. The diagnosis of α-thalassaemia major is confirmed by haemoglobin electrophoresis or HPLC (which shows Hb Barts, Hb Portland and sometimes HbH; HbF and HbA are absent); the blood film shows hypochromic microcytic red cells with vast numbers of circulating nucleated red cells (Figure 52.8).

in monochorionic twins with monochorial placentas. If the bleeding is chronic, there may be a marked difference in birth weight between twins: the donor twin is smaller, pale, lethargic and may have overt cardiac failure; the recipient twin may be plethoric, with hyperviscosity and hyperbilirubinaemia and polycythaemia. Fetomaternal haemorrhage occurs spontaneously or secondary to trauma usually in the third trimester. Most episodes involve very small quantities of blood (0.5 mL or less) but acute loss of more than 20% of the blood volume may cause intrauterine death, circulatory shock or hydrops. Diagnostic clues are anaemia at birth in an otherwise well baby with no or minimal jaundice. The most useful diagnostic tests are a Coombs test to exclude immune haemolysis, a reticulocyte count to exclude red cell aplasia, a Kleihauer test on maternal blood to quantify the number of HbF-containing fetal red cells in the maternal circulation and a blood film (Figure 52.9). Blood loss around the time of delivery is usually due to obstetric complications (e.g. placenta praevia, placental abruption); in these circumstances, the babies are often extremely ill, with circulatory shock, anaemia worsening rapidly after birth, large numbers of circulating nucleated red cells and disseminated intravascular coagulation (DIC).

Neonatal anaemia due to blood loss Blood loss as a cause of neonatal anaemia may be very obvious (e.g. a cephalhaematoma or rupture of the cord) or be concealed and easy to miss unless specifically sought (e.g. fetomaternal bleeds). Usually, the most serious blood loss occurs prior to delivery and important causes of this are twin–twin transfusion and fetomaternal blood loss. Twin–twin transfusion occurs

The normal fall in haemoglobin in preterm neonates has been termed ‘physiological anaemia of prematurity’, as it does not appear to be associated with any abnormalities in the baby. The pathogenesis is not fully elucidated but contributory factors include the reduced lifespan of fetal erythrocytes, the relatively low erythropoietin concentration, the rapid growth rate and iatrogenic blood loss. Routine supplementation of preterm

Anaemia of prematurity

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Figure 52.9 Fetomaternal haemorrhage. (a) Blood film from a neonate with a haemoglobin of 5 g/dL at birth due to a large fetomaternal haemorrhage; the main features are the marked polychromasia, large numbers of nucleated red cells and normal red cell morphology. (b) Kleihauer test showing HbA-containing maternal ‘ghost’ cells and pink-staining HbF-containing fetal cells.

(a)

neonates with folic acid and iron means that nutritional deficiency rarely plays a role. The diagnosis is usually straightforward: a well preterm baby with a slowly falling haemoglobin, unremarkable blood film showing normochromic/normocytic red cells, slightly low reticulocytes (20 × 109/L) and no nucleated red cells. Management of anaemia of prematurity and the role of erythropoietin The severity of anaemia of prematurity and thereby the need for red cell transfusion can be reduced by (i) limiting iatrogenic blood loss by appropriate use of blood tests; (ii) administering iron and folate supplements to all preterm infants (iron 3 mg/ kg daily from 4–6 weeks of age or iron-fortified formula with 0.5–0.9 mg/dL of iron and folic acid 50 µg daily or 500 µg once weekly); and (iii) judicious use of erythropoietin. The many controlled trials of recombinant erythropoietin for prevention of neonatal anaemia have been reviewed extensively. Erythropoietin reduces the number of transfusions in relatively well infants with low transfusion requirements, but not in sick preterm infants who have a need for frequent phlebotomy and multiple transfusions. The main roles for recombinant erythropoietin in neonates are in ameliorating the anaemia in infants who have received intrauterine transfusions for alloantibodymediated anaemia and in preterm babies of Jehovah’s Witnesses. The usual dose is 300 µg/kg of epoetin beta by subcutaneous injection three times per week, starting in the first week of life, together with oral iron supplements. Indications for red cell transfusion in neonatal anaemia The BCSH recently revised their guidelines for transfusion of fetuses, neonates and older children; the guidelines contain recommendations about the products and indications for red cell transfusion in neonates. These are consensus guidelines and need to be adapted for use in each individual neonatal intensive 978

(b)

Table 52.3 Causes of neonatal polycythaemia. Intrauterine growth restriction Maternal hypertension Maternal diabetes Chromosomal disorders: trisomy 21, 18 or 13 Twin–twin transfusion Delayed clamping of the cord Endocrine disorders: thyrotoxicosis, congenital adrenal hyperplasia

care unit, depending on the case mix of babies, as adherence to strict neonatal transfusion guidelines reduces both the number of transfusions and donor exposure.

A simple diagnostic approach to neonatal anaemia Red cell disorders associated with neonatal or fetal anaemia present in three main ways: with a low haemoglobin, with jaundice due to haemolysis or with hydrops. A diagnostic algorithm to help identify which of these causes is most likely, which can be excluded and what further investigations are most appropriate is shown in Figure 52.2.

Neonatal polycythaemia For both term and preterm infants, polycythaemia can be defined as a central venous haematocrit of greater than 0.65, as there is an exponential rise in blood viscosity above this level. However, even at haematocrits greater than 0.70, only a minority of neonates have clinical signs of hyperviscosity, such as lethargy, hypotonia, hyperbilirubinaemia and hypoglycaemia. Causes of polycythaemia are shown in Table 52.3. Treatment is

Neonatal haematology

controversial and is not necessary in infants with very minor symptoms (e.g. borderline hypoglycaemia or poor peripheral perfusion). Infants with neurological signs and a haematocrit greater than 0.65 should have a partial exchange transfusion (using a crystalloid solution such as normal saline) to reduce the haematocrit to 0.55.

White cell disorders Normal values In the neonate, normal values for leucocytes, particularly neutrophils, are affected by a number of factors including gestational age, postnatal age, antenatal history, perinatal history and ethnic origin. Neutrophil counts in healthy babies increase for the first 12 hours then fall to a nadir at 4 days of age. The neutrophil count is higher in capillary samples and after vigorous crying; it is lower in neonates of African origin. Healthy preterm babies often have circulating myeloblasts and lymphoblasts, although these usually form less than 5% of the white cell differential count.

Neutropenia A pragmatic approach is to consider a neutrophil count at birth of less than 2 × 109/L as abnormal and worth monitoring, and a neutrophil count during the first month of life of less than 0.7 × 109/L as significant enough to merit further investigation. Causes of neutropenia The commonest cause of neutropenia at birth in preterm neonates is reduced neutrophil production, secondary to intrauterine growth restriction or maternal hypertension. Most affected neonates also have thrombocytopenia and increased erythropoiesis (polycythaemia and/or increased circulating nucleated red cells), secondary to fetal tissue hypoxia. The neutropenia resolves spontaneously usually within a few days of birth. The commonest cause of neutropenia in term infants is bacterial or viral infection. Other important causes of neutropenia are alloimmune neutropenia and severe congenital neutropenia (SCN) due to failure of neutrophil production (e.g. Kostmann syndrome), both of which predispose to severe neonatal infection. Alloimmune neutropenia is the neutrophil equivalent of haemolytic disease of the newborn and alloimmune thrombocytopenia, and may affect 3% of all deliveries. It occurs when fetal neutrophils express paternally derived neutrophil-specific antigens absent on maternal neutrophils and against which the mother produces IgG neutrophil alloantibodies. The causative antibodies are usually anti-NA1 or -NA2. It presents in the first few days of life with fever and infections of the respiratory tract,

urinary tract and skin, particularly due to Staphylococcus aureus, and the mainstay of treatment is antibiotics. The diagnosis is made by demonstrating antineutrophil antibodies in the mother and baby, which react against paternal, but not maternal, neutrophil antigens. The neutropenia is self-limiting, usually resolving within 1–2 months. Congenital and inherited neonatal neutropenias should be sought when the neutropenia is prolonged, if there is a relevant family history or consanguinity or if the baby has typical dysmorphic features (e.g. thumb/radial abnormalities in Fanconi anaemia). SCN is the most likely cause in the neonatal period. Infants usually present with severe infections and a marked neutropenia (< 0.2 × 109/L), often with a compensatory monocytosis. The diagnosis is made by the severity of the neutropenia, the bone marrow appearance (arrest of differentiation at the myelocyte/promyelocyte stage) and the absence of antineutrophil antibodies. The inheritance of SCN can be autosomal recessive or dominant or X-linked. Mutations in ELA2 (neutrophil elastase) are the most common cause of SCN, but other causes include mutations in GFI1 (growth factor-independent protein 1) and WAS (Wiskott–Aldrich syndrome protein, WASp). Recently, homozygous mutations in the HAX1 gene were found to explain the SCN in the original Kostmann family but this appears to be a rare cause of the disease. Congenital leukaemias The most common forms of congenital leukaemia are acute monoblastic leukaemia and acute megakaryoblastic leukaemia; acute megakaryoblastic leukaemia is particularly common in babies with Down syndrome. The babies present with signs of anaemia, thrombocytopenia and/or skin lesions caused by leukaemic infiltration. The blood film and bone marrow aspirate show large numbers of primitive blast cells. The prognosis is extremely poor; few are cured by chemotherapy and bone marrow transplantation may be the best option. Around 5–10% of neonates with Down syndrome have a transient megakaryoblastic leukaemia also known as transient abnormal myelopoiesis or transient myeloproliferative disorder (TMD), characterized by leucocytosis and circulating blast cells (Figure 52.10). TMD develops in fetal life and nearly always presents in the first week of life. Most cases spontaneously resolve within 2–3 months and no treatment is indicated, but 20–30% subsequently develop acute megakaryoblastic leukaemia within the first 5 years of life. Mutations in the GATA1 gene are found in virtually all cases of TMD and acute megakaryoblastic leukaemia associated with Down syndrome.

Haemostasis and thrombosis in the newborn Bleeding and thrombotic problems are relatively common in neonates, particularly in those who are preterm and/or sick, and 979

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inherited disorders as there is considerable overlap between the deficiency states and the lower limit of normal. The most useful screening tests in neonates are the prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen and platelet count. Bleeding times are generally unhelpful in neonates and investigation of platelet function abnormalities is often deferred until a few months of age when platelet aggregometry and PFA-100 become practical.

Inherited coagulation disorders The commonest inherited disorders presenting in the neonatal period are FVIII deficiency (haemophilia A), which has a frequency of 1 in 5000 male births, and FIX deficiency (haemophilia B), which occurs in 1 in 30 000 male births. Figure 52.10 Transient leukaemia in a neonate with Down syndrome. Leucoerythoblastic blood film showing increased numbers of blast cells, which spontaneously returned to normal by 2 months of age.

the number of genetic and acquired causes of thrombophilia that can be identified in neonates and their families is rising.

Developmental haemostasis Coagulation proteins are present at measurable levels from the 10th week of gestation and gradually rise during fetal life. They do not cross the placenta, or do so in very small amounts, and therefore need to be independently synthesized by the fetus. ‘Normal values’ in the neonate vary not only with gestational but also with postnatal age. Data for babies at less than 30 weeks’ gestation derive from fetoscopy samples: levels of the vitamin K-dependent factors (FII, FVII, FIX and FX) and of FXI and FXII are all low (< 40% of adult values) and remain so during the first month of life. In contrast, even in preterm babies (> 30 weeks), levels of FV, FXIII and fibrinogen are normal at birth and levels of F VIII and von Willebrand factor (VWF) are normal or increased. Platelet counts at birth in term and preterm neonates are within the normal adult range. Many studies have found impaired function of neonatal platelets in vitro in term and preterm infants; the most consistent abnormalities are reduced aggregation in response to adrenaline, ADP and thrombin. Their significance in clinical practice is unclear as the bleeding time is normal in term and preterm infants (≤ 135 s).

Screening tests for bleeding disorders Nearly all significant bleeding disorders in neonates can be identified using simple screening tests; exceptions are FXIII deficiency and platelet function disorders. It is often helpful to test both parents for coagulation abnormalities to help identify 980

Factor VIII deficiency Almost 40% of patients with inherited FVIII deficiency present in the neonatal period. The clinical signs include intracranial haemorrhage, cephalohaematomas and bleeding after circumcision or from venous or arterial puncture sites. As in adults, the diagnosis is made by finding an isolated prolonged APTT and reduced FVIII clotting activity. Acute management of the bleeding neonate with haemophilia requires intravenous administration of recombinant FVIII (50–100 units/kg twice daily) to achieve FVIII levels of 1.0 unit/mL. As the half-life of FVIII is shorter than in adults, more frequent dosing or a continuous FVIII infusion may be required. For neonates with intracranial bleeding treatment with FVIII should continue for at least 2 weeks. Fibrin glue may be useful in circumcisionassociated bleeds. For patients diagnosed prenatally, vaginal delivery is safe provided that no difficulties are anticipated and vacuum extraction is avoided. The role of prophylactic FVIII administration to haemophiliac newborns following difficult delivery to reduce the risk of intracranial bleeding (1–4%) is controversial but is increasingly used. Prophylactic FVIII should also be used for a newborn haemophiliac when a previous sibling has had a major intracranial bleed. Factor IX deficiency Deficiency of FIX is clinically indistinguishable from FVIII deficiency. As FIX levels are also low in liver disease and vitamin K deficiency, it is important to recheck FIX levels at 6 weeks and 6 months of age if the diagnosis is in doubt. Neonates with bleeding are treated with recombinant FIX concentrate (100 units/kg i.v. once daily, monitored to achieve a FIX level of 1.0 IU/mL). von Willebrand disease in neonates The two forms of von Willebrand disease (vWD) that can present in neonates are type 2B vWD, which presents with thrombocytopenia and bleeding is uncommon, and type 3 vWD, which has a clinical phenotype similar to haemophilia

Neonatal haematology

as levels of both VWF and FVIII are low. The inheritance in type 3 vWD is autosomal recessive. The diagnosis is made by measuring VWF, FVIII and the pattern of VWF multimers. At present, type 3 vWD is treated with intermediate purity FVIII (Haemate-P is the most commonly used product). Factor XIII deficiency This rare autosomal recessive disorder usually presents with delayed bleeding from the umbilical cord during the first 3 weeks of life. The diagnosis is made by measuring clot solubility in 5 mol/L urea solution as a screening test followed by a specific FXIII assay; molecular tests for the common mutations are also available. The routine diagnostic coagulation screen is normal. Bleeding is treated with FXIII concentrate; cryoprecipitate (10 mL/kg) can also be used.

Acquired disorders of coagulation Causes of acquired coagulopathy in neonates include vitamin K deficiency, DIC, liver disease, metabolic disorders, extracorporeal membrane oxygenation and giant haemangioendotheliomas (Kasabach–Merritt syndrome). Vitamin K deficiency Levels of vitamin K-dependent procoagulant factors (FII, FVII, FIX and FX), protein C and protein S are low at birth because of poor placental transfer of vitamin K, low vitamin K stores at birth, low vitamin K in breast milk and the lack of bacterial vitamin K synthesis in the sterile neonatal gut. Vitamin K deficiency can lead to haemorrhagic disease of the newborn, also known as vitamin K deficiency bleeding (VKDB). Early VKDB presents in the first 24 hours of life with severe gastrointestinal and intracranial haemorrhage. It is usually due to maternal medication that interferes with vitamin K, for example anticonvulsants (phenobarbital, phenytoin), antituberculous therapy and oral anticoagulants. Classical VKDB presents at 2–7 days in 0.25–1.7% of babies who have not received prophylactic vitamin K at birth, especially if breast-fed or with poor oral intake. Late VKDB occurs between 2 and 8 weeks after birth and presents with sudden intracranial haemorrhage in an otherwise well breast-fed term baby or in babies with liver disease. The diagnosis of VKDB is usually made by an isolated prolonged PT, which corrects following vitamin K administration. If doubt remains, assays of the inactive form of FII (decarboxyprothrombin; PIVKA II) can be used to confirm the diagnosis. Treatment of VKDB is vitamin K 1 mg intravenously or subcutaneously with fresh-frozen plasma only in severe haemorrhage. Classic and late VKDB can be prevented by a single intramuscular dose of vitamin K or, slightly less effectively, by weekly 1-mg oral doses of vitamin K over the first 12 weeks of life (in exclusively breast-fed infants neither single oral dose vitamin K nor daily very low dose vitamin K 25 µg are effective in preventing classic VKDB). Some studies, but not others, suggest a link

between intramuscular vitamin K at birth and later childhood malignancies. In healthy babies, the choice of which route of administration is increasingly being left to parents. However, infants of mothers taking drugs that inhibit vitamin K (e.g. warfarin) are at risk of early VKDB and these babies should receive intramuscular vitamin K 1 mg as soon as possible after birth. Disseminated intravascular coagulation The main triggers of DIC in neonates are severe hypoxia and/ or acidosis and sepsis. It occurs in sick neonates and presents with generalized bleeding, including pulmonary haemorrhage and oozing from venepuncture sites. The usual pattern of coagulation abnormalities in neonatal DIC is prolongation of the PT, APTT and TT, together with low platelets and fibrinogen. D-dimers are increased but are not specific and can be found in healthy neonates with no evidence of coagulopathy. The most important aspect of management of DIC is treatment of the underlying cause. Blood product replacement is indicated for clinical bleeding, aiming to maintain the platelet count above 30 × 109/L and fibrinogen greater than 1 g/L.

Neonatal thrombocytopenia Thrombocytopenia occurs in 1–5% of all neonates and up to 50% of neonates who are preterm and sick. It usually presents in one of two clinical patterns: early thrombocytopenia (within 72 hours of birth) and late thrombocytopenia (after 72 hours of life) (Table 52.4). The most frequent causes of early thrombocytopenia in preterm infants are intrauterine growth restriction and maternal hypertension or diabetes; the most frequent causes in term infants are neonatal alloimmune thrombocytopenia (NAITP) and thrombocytopenia secondary to maternal immune thrombocytopenic purpura (ITP). The most common causes of late thrombocytopenia are sepsis and necrotizing enterocolitis. Neonatal alloimmune thrombocytopenia This is the platelet equivalent of haemolytic disease of the newborn and affects around 1 in 1000 pregnancies. It is frequently severe (platelet count < 30 × 109/L) and occurs in the first pregnancy in almost 50% of cases. Thrombocytopenia results from transplacental passage of maternal platelet-specific antibodies to human platelet antigens (HPA), which the mother lacks but which the fetus inherits from the father. In 80% of cases, these are anti-HPA-1a antibodies and in 10–15% anti-HPA-5b; occasional cases are due to anti-HPA-3a. HLADRB3*0101-positive women are 140 times more likely to make anti-HPA-1a than HLA-DRB3*0101-negative women. Intracranial haemorrhage occurs in 10% of cases, with longterm neurodevelopmental sequelae in 20% of survivors. The diagnosis is made by demonstrating platelet antigen incompatibility between mother and baby and anti-HPA anti981

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Table 52.4 Causes of neonatal thrombocytopenia. Early (< 72 hours) Placental insufficiency (PET, IUGR, diabetes) Neonatal alloimmune thrombocytopenia Birth asphyxia Perinatal infection (group B Streptococcus, Escherichia coli, Listeria) Congenital infection (CMV, toxoplasmosis, rubella) Maternal autoimmune (ITP, SLE) Severe rhesus HDN Thrombosis (renal vein, aortic) Aneuploidy (trisomy 21, 18, 13) Congenital/inherited (TAR, Wiskott–Aldrich) Late (> 72 hours) Late-onset sepsis and necrotizing enterocolitis Congenital infection (CMV, toxoplasmosis, rubella) Maternal autoimmune (ITP, SLE) Congenital/inherited (TAR, Wiskott–Aldrich) CMV, cytomegalovirus; HDN, haemolytic disease of the newborn; ITP, idiopathic thrombocytopenic purpura; IUGR, intrauterine growth restriction; PET, pre-eclampsia; SLE, systemic lupus erythematosus; TAR, thrombocytopenia with absent radii.

bodies in the mother. Transfusion with HPA-compatible platelets is recommended for neonates with platelet counts below 30 × 109/L and/or those with bleeding. Intravenous IgG (total dose 2 g/kg, over 2–5 days) may be useful if thrombocytopenia is prolonged. Prenatal management of NAITP is controversial. Most centres have abandoned the invasive approach, except in very high risk cases, as this relies on fetal blood sampling plus fetal transfusion with HPA-compatible platelets, which carries a risk of preterm delivery and fetal death. Instead, most centres now use a non-invasive approach relying on maternal intravenous IgG therapy during the second half of pregnancy. Neonatal autoimmune thrombocytopenia This is secondary to transplacental passage of maternal platelet autoantibodies in maternal ITP and systemic lupus erythematosus (SLE), which affects 1–5 in 10 000 pregnancies. Around 10% of infants develop thrombocytopenia, which is severe in less than 1%. In babies with severe thrombocytopenia, intravenous IgG is usually effective. In some cases neonatal thrombocytopenia is prolonged and may last 6–8 weeks before spontaneously resolving. Indications for platelet transfusion A number of countries have published consensus guidelines to help decide the indications for platelet transfusion in term and

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preterm neonates. In general, platelet transfusion is not considered necessary in well babies if the platelet count is above 20– 30 × 109/L. Most groups agree that the threshold for transfusing sick babies, particularly preterm babies in the first few weeks of life, should be higher (30–50 × 109/L).

Neonatal thrombosis: physiology and developmental aspects Neonates have an increased risk of thrombosis (2.4 per 1000 hospital admissions) compared with older infants and children. This is largely due to the physiologically low levels of many of the inhibitors of coagulation and the frequent use of indwelling vascular catheters. Concentrations of antithrombin, heparin cofactor II and protein C are decreased at birth; protein S levels are also low but overall protein S activity is normal as it exists mainly in its free active form due to the virtual absence of its binding protein (C4b-BP) in neonates. Levels of plasminogen at birth are only 50% of adult values so neonates have a reduced ability to generate plasmin in response to fibrinolytic agents.

Screening tests for thrombophilia in neonates The only inherited deficiencies for which there is a proven role in neonatal thrombosis are deficiencies in proteins C and S, which cause purpura fulminans. FV Leiden and the prothrombin 20210A promoter mutation (prothrombin20210A) have not yet been reported to cause neonatal thrombotic problems in isolation. Recent guidelines from the Haemostasis and Thrombosis Task Force of the BCSH state that congenital thrombophilia should be considered and screened for in any child with: • clinically significant thrombosis, including spontaneous thrombotic events, unanticipated or extensive venous thrombosis, ischaemic skin lesions or purpura fulminans; and • a positive family history of neonatal purpura fulminans. The screening tests that should be performed in all suspected cases of thrombophilia include protein C activity, protein S, antithrombin, FV Leiden and prothrombin20210A. In addition, babies with thrombosis born to mothers with SLE and/or antiphospholipid syndrome should be tested for lupus anticoagulant, as antiphospholipid antibodies may cross the placenta and are a rare cause of neonatal thrombosis in such babies. The relevance of serum liporotein a and the MTHFR genotype to neonatal management is unclear at present.

Inherited thrombotic disorders in neonates Proteins C and S deficiency Protein C deficiency occurs in 1 in 160 000–360 000 births. Affected babies usually present within hours or days of birth

Neonatal haematology

with purpura fulminans, in which there is DIC and rapidly progressive life-threatening haemorrhagic necrosis due to dermal vessel thrombosis or cerebral, renal vein or ophthalmic thrombosis. The diagnosis of protein C deficiency is made by the clinical picture and undetectable levels of protein C (< 0.01 units/mL) in the patient, together with heterozygote levels in the parents. Protein C-deficient heterozygotes rarely present as neonates and have low protein C levels, which may overlap with the lower limit of normal in the first few months of life, delaying diagnosis until 6 months or later. Treatment is with protein C concentrate (40 units/kg, aiming to maintain a plasma level > 0.25 units/mL). Protein S deficiency presents with identical features; levels of protein S are undetectable (< 0.01 units/mL) and treatment is with fresh-frozen plasma (10–20 mL/kg) to maintain a plasma protein S in excess of 0.25 units/mL. Antithrombin deficiency Homozygous antithrombin deficiency usually presents later in childhood but neonatal deep venous thrombosis and inferior vena cava thrombosis have been reported. Heterozygous antithrombin deficiency is more common (1 in 2000–5000 births); it usually presents in the second decade of life but neonatal presentation with aortic thrombosis, myocardial infarction and cerebral dural sinus thrombosis may occur.

Acquired thrombotic problems in neonates The most common risk factors for neonatal thrombosis are an intravascular catheter and shock, secondary to sepsis, hypoxaemia or hypovolaemia. Thrombosis particularly affects the renal veins, the aorta, aortic arch or cerebral vessels. Catheter-related thrombosis causes more than 80% of venous thromboses and more than 90% of arterial thromboses. The diagnosis is made by Doppler ultrasound or contrast angiography. Treatment of catheter-related thrombosis depends on the severity and extent of thrombosis. The first step is prompt removal where possible. If signs progress despite catheter removal, unfractionated heparin or low-molecular-weight heparin should be started using a dosing regimen adapted for neonates. Thrombolytic therapy with urokinase or tissue plasminogen activator has been used successfully for catheter-related thrombosis in neonates, but experience in preterm neonates is very limited. It is important to maintain fibrinogen at levels less than 1 g/L and the platelet count greater than 50 × 109/L in neonates receiving thrombolytic therapy, and heparin (starting dose 28 units/kg per hour) is often given to maintain vessel patency after thrombolytic therapy, although there is no evidence that this is beneficial. Excellent guidelines for antithrombotic therapy in neonates are regularly updated by the American College of Chest Physicians.

Selected bibliography Andrew M (1997) The relevance of developmental haemostasis to haemorrhagic disorders of newborns. Seminars in Perinatology 21: 70–85. Anwar R, Minford A, Gallivan L et al. (2002) Delayed umbilical bleeding. A presenting feature for factor XIII deficiency: clinical features, genetics, and management. Pediatrics 109: E32143. Bishara N, Ohls RK (2009) Current controversies in the management of anaemia of prematurity. Seminars in Perinatology 33: 29–34. British Committee for Standards in Haematology Haemostasis and Thrombosis Task Force (2002) The investigation and management of neonatal haemostasis and thrombosis. British Journal of Haematology 119: 295–309. British Committee for Standards in Haematology Transfusion Task Force. Transfusion guidelines for neonates and older children. Available at www.bcshguidelines.com/ Brown K (2000) Haematological consequences of parvovirus B19 infection. Baillière’s Best Practice and Research. Clinical Haematology 13: 245–59. Bussel JB, Sola-Visner M (2001) Current approaches to the evaluation and management of the fetus and neonate with imune thrombocytopenia. Seminars in Perinatology 33: 35–42. Chalmers EA, Williams MD, Richards M et al. (2005) Management of neonates with inherited bleeding disorders: a survey of current UK practice. Haemophilia 11: 186–7. Dzierzak E, Speck NA (2009) Of lineage and legacy: the development of mammalian haematopoietic stem cells. Nature Immunology 9: 129–36. Israels SJ, Cheang T, McMillan-Ward EM et al. (2001) Evaluation of primary hemostasis in neonates with a new in vitro platelet function analyzer. Journal of Pediatrics 138: 116–19. Kulkarni R, Ponder KP, James AH et al. (2006) Unresolved issues in diagnosis and management of inherited bleeding disorders in the perinatal period: a White Paper of the Perinatal Task Force of the Medical and Scientific Advisory Council of the National Hemophilia Foundation, USA. Haemophilia 12: 205–11. Lallemand AV, Doco-Fenzy M, Gaillard DA (1999) Investigation of nonimmune hydrops fetalis: multidisciplinary studies are necessary for diagnosis. Review of 94 cases. Pediatric and Developmental Pathology 2: 432–9. Monagle P, Chalmers E, Chan A et al. (2009) Antithrombotic therapy in neonates and children. American College of Chest Physicians Evidence-Based Clinical Practice Guidelines, 8th edition. Chest 133 (6 Suppl.): 887S–968S. New H, Stanworth SJ, Engelfriet CP et al. (2009) Neonatal transfusions. Vox Sangunis 96: 62–85. Puckett RM, Offringa M (2000) Prophylactic vitamin K for vitamin K deficiency bleeding in neonates. Cochrane Database of Systematic Reviews 4: CD002776. Roberts IAG (2008) The changing face of haemolytic disease of the newborn. Early Human Development 84: 515–23. Roberts I, Stanworth S, Murray NA (2008) Thrombocytopenia in the neonate. Blood Reviews 22: 173–86.

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Shearer MJ (2009) Vitamin K deficiency bleeding (VKDB) in early infancy. Blood Reviews 23: 49–59. Sohan K, Billington M, Pamphilon D, Goulden N, Kyle P (2002) Normal growth and development following in utero diagnosis and treatment of homozygous alpha-thalassaemia. BJOG: an International Journal of Obstetrics and Gynaecology 109: 1308–10.

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Watts TL, Roberts IAG (1999) Haematological abnormalities in the growth-restricted infant. Seminars in Neonatology 4: 41– 54. Webb D, Roberts I, Vyas P (2007) Haematology of Down syndrome. Archives of Disease in Childhood 92: F503–F507. Wee LY, Fisk NM (2002) The twin–twin transfusion syndrome. Seminars in Neonatology 7: 187–202.

APPENDIX 1

Normal values

Haemoglobin Red cells (erythrocytes) PCV (haematocrit) MCV MCH MCHC White cells (leucocytes) Total Neutrophils Lymphocytes Monocytes Eosinophils Basophils Platelets Red cell mass Plasma volume Serum iron Total iron-binding capacity Serum ferritin* Serum vitamin B12* Serum folate* Red cell folate*

Males

Females

13.5–17.5 g/dL 4.5–6.5 × 1012/L 40–52%

11.5–15.5 g/dL 3.9–5.6 × 1012/L 36–48%

Males and females

80–95 fL 27–34 pg 20–35 g/dL 4.0–11.0 × 109/L 2.5–7.5 × 109/L 1.5–3.5 × 109/L 0.2–0.8 × 109/L 0.04–0.44 × 109/L 0.01–0.1 × 109/L 150–400 × 109/L 30 ± 5 mL/kg 45 ± 5 mL/kg

27 ± 5 mL/kg 45 ± 5 mL/kg 10–30 µmol/L 40–75 µmol/L (2.0–4.0 g/L as transferrin)

40–340 µg/L

14–150 µg/L 160–925 ng/L 3.0–15.0 µg/L 160–640 µg/L

*Normal ranges differ with different commercial kits. MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; MCV, mean corpuscular volume; PCV, packed cell volume.

Postgraduate Haematology: 6th edition. Edited by A. Victor Hoffbrand, Daniel Catovsky, Edward G.D. Tuddenham, Anthony R. Green © 2011 Blackwell Publishing.

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APPENDIX 2

World Health Organization classification of tumours of the haematopoietic and lymphoid tissues

Myeloproliferative neoplasms

Myelodysplastic syndromes

Chronic myelogenous leukaemia, BCR–ABL1 positive Chronic neutrophilic leukaemia Polycythemia vera Primary myelofibrosis Essential thrombocythaemia Chronic eosinophilic leukaemia, not otherwise specified Mastocytosis Cutaneous mastocytosis Systemic mastocytosis Mast cell leukaemia Mast cell sarcoma Extracutaneous mastocytoma Myeloproliferative neoplasms, unclassifiable Myeloid and lymphoid neoplasms associated with eosinophilia and abnormalities of PDGFRA, PDGFRB or FGFR1 Myeloid and lymphoid neoplasms associated with PDGFRA rearrangement Myeloid neoplasms associated with PDGFRB rearrangement Myeloid and lymphoid neoplasms with FGFR1 abnormalities

Refractory cytopenia with unilineage dysplasia Refractory anaemia Refractory neutropenia Refractory thrombocytopenia Refractory anaemia with ring sideroblasts Refractory cytopenia with multilineage dysplasia Refractory anaemia with excess blasts Myelodysplastic syndromes associated with isolated del(5q) Myelodysplastic syndromes, unclassifiable Myelodysplastic syndromes in children

Myelodysplastic/myeloproliferative neoplasms Chronic myelomonocytic leukaemia Atypical chronic myeloid leukaemia, BCR–ABL1 negative Juvenile myelomonocytic leukaemia Myelodysplastic/myeloproliferative neoplasms, unclassifiable Refractory anaemia with ringed sideroblasts (RARS) associated with marked thrombocytosis*

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Acute myeloid leukaemia Acute myeloid leukaemia (AML) with recurrent genetic abnormalities AML with t(8;21)(q22;q22), RUNX1–RUNX1T1 AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22), CBFB–MYH11 Acute promyelocytic leukaemia with t(15;17)(q22;q11–12), PML–RARA AML with t(9;11)(p22;q23), MLLT3–MLL AML with t(6;9)(p23;q34), DEK–NUP214 AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2), RPN1– EVI1 AML (megakaryoblastic) with t(1;22)(p13;q13), RBM15– MKL1 AML with mutated NPM1* AML with mutated CEBPA* Acute myeloid leukaemia with myelodysplasia-related changes Therapy-related myeloid neoplasms Acute myeloid leukaemia, not otherwise categorized AML with minimal differentiation AML without maturation AML with maturation Acute myelomonocytic leukaemia Acute monoblastic and monocytic leukaemia

World Health Organization classification of tumours of the haematopoietic and lymphoid tissues

Acute erythroid leukaemia Acute erythroid leukaemia, erythroid/myeloid Acute pure erythroid leukaemia Acute megakaryoblastic leukaemia Acute basophilic leukaemia Acute panmyelosis with myelofibrosis Myeloid sarcoma Myeloid proliferations related to Down syndrome Transient abnormal myelopoiesis Acute myeloid leukaemia associated with Down syndrome Blastic plasmacytoid dendritic cell neoplasm Acute leukaemias of ambiguous lineage Acute undifferentiated leukaemia Acute biphenotypic leukaemia

Precursor lymphoid neoplasms B lymphoblastic leukaemia/lymphoma B lymphoblastic leukaemia/lymphoma, not otherwise specified B lymphoblastic leukaemia/lymphoma with recurrent cytogenetic/molecular genetic abnormalities B lymphoblastic leukaemia/lymphoma with t(9:22) (q34;q11.2), BCR–ABL1 B lymphoblastic leukaemia/lymphoma with t(11q23), MLL rearranged B lymphoblastic leukaemia/lymphoma with t(12;21) (p13;q22), TEL–AML1 (ETV6–RUNX1) B lymphoblastic leukaemia/lymphoma with hyperdiploidy B lymphoblastic leukaemia/lymphoma with hypodiploidy (hypodiploid ALL) B lymphoblastic leukaemia/lymphoma with t(5;14)(q31;q32), IL3–IGH B lymphoblastic leukaemia/lymphoma with t(1;19) (q23;p13.3), E2A–PBX1 (TCF3–PBX1) T-lymphoblastic leukaemia/lymphoma

Mature B-cell neoplasms Chronic lymphocytic leukaemia/small lymphocytic lymphoma B-cell prolymphocytic leukaemia Splenic marginal zone lymphoma Hairy cell leukaemia Splenic lymphoma/leukaemia, unclassifiable Splenic diffuse red pulp small B-cell lymphoma* Hairy cell leukaemia variant* Lymphoplasmacytic lymphoma Waldenström macroglobulinaemia Heavy chain diseases Alpha heavy chain disease Gamma heavy chain disease Mu heavy chain disease Plasma cell myeloma

Solitary plasmacytoma of bone Extraosseous plasmacytoma Extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) Nodal marginal zone B-cell lymphoma Paediatric type nodal marginal zone lymphoma Follicular lymphoma Paediatric type follicular lymphoma Primary cutaneous follicle centre lymphoma Mantle cell lymphoma Diffuse large B-cell lymphoma (DLBCL), not otherwise specified T-cell/histiocyte-rich large B-cell lymphoma DLBCL associated with chronic inflammation EBV-positive DLBCL of the elderly Lymphomatoid granulomatosis Primary mediastinal (thymic) large B-cell lymphoma Intravascular large B-cell lymphoma Primary cutaneous DLBCL, leg type ALK-positive large B-cell lymphoma Plasmablastic lymphoma Primary effusion lymphoma Large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease Burkitt lymphoma B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and Burkitt lymphoma B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and classical Hodgkin lymphoma

Mature T-cell and NK-cell neoplasms T-cell prolymphocytic leukaemia T-cell large granular lymphocytic leukaemia Aggressive NK-cell leukaemia Systemic EBV-positive T-cell lymphoproliferative disease of childhood (associated with chronic active EBV infection) Hydroa vaccineforme-like lymphoma Adult T-cell leukaemia/lymphoma Extranodal NK/T-cell lymphoma, nasal type Enteropathy-associated T-cell lymphoma Hepatosplenic T-cell lymphoma Subcutaneous panniculitis-like T-cell lymphoma Mycosis fungoides Sézary syndrome Primary cutaneous anaplastic large-cell lymphoma Primary cutaneous aggressive epidermotropic CD8-positive cytotoxic T-cell lymphoma* Primary cutaneous γδ T-cell lymphoma Primary cutaneous small/medium CD4-positive T-cell lymphoma* Peripheral T-cell lymphoma, not otherwise specified Angioimmunoblastic T-cell lymphoma 987

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Anaplastic large-cell lymphoma, ALK positive Anaplastic large-cell lymphoma, ALK negative*

Hodgkin lymphoma Nodular lymphocyte-predominant Hodgkin lymphoma Classical Hodgkin lymphoma Nodular sclerosis classical Hodgkin lymphoma Lymphocyte-rich classical Hodgkin lymphoma Mixed cellularity classical Hodgkin lymphoma Lymphocyte depleted classical Hodgkin lymphoma

Histiocytic and dendritic cell neoplasms Histiocytic sarcoma Langerhans cell histiocytosis Langerhans cell sarcoma Interdigitating dendritic cell sarcoma Follicular dendritic cell sarcoma Dendritic cell tumour, not otherwise specified Indeterminate dendritic cell tumour Fibroblastic reticular cell tumour

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Post-transplant lymphoproliferative disorders Early lesions Reactive plasmacytic hyperplasia Infectious mononucleosis-like Polymorphic post-transplant lymphoproliferative disorder Monomorphic post-transplant lymphoproliferative disorder (B- and T/NK-cell types)* Classical Hodgkin lymphoma-type post-transplant lymphoproliferative disorder *These represent provisional entities or provisional subtypes of other neoplasms. They are provisional because there are insufficient data to support their being a definite entity, significant controversies about their defining features and/or uncertainty about whether they are unique or closely related to other definite entities. Further classify according to the lymphoma they resemble.

Index

Notes Page numbers in italics represent figures, those in bold represent tables. vs. indicates a comparison or differential diagnosis. The following abbreviations have been used: AIHA autoimmune haemolytic anaemia ALL acute lymphoblastic leukaemia AML acute myeloid leukaemia ATG antithymocyte globulin CLL chronic lymphocytic leukaemia CML chronic myeloid leukaemia DIC disseminated intravascular coagulation DLBCL diffuse large B-cell lymphoma FISH fluorescence in situ hybridization GVHD graft-versus host disease HLA human leucocyte antigen HSC haematopoietic stem cells Ig immunoglobulin MHC major histocompatibility complex NHL non-Hodgkin lymphoma PCR polymerase chain reaction SCT stem cell transplantation TTP thrombotic thrombocytopenic purpura A A1 subgroup, ABO system, 246 A2 subgroup, ABO system, 246 AA see aplastic anaemia (AA) AA amyloidosis, 611 A antigen see ABO blood group ABCB7 gene mutations, X-linked sideroblastic anaemia, 44 abdominal pain allogeneic SCT complications, 738 leukaemias, 471

abetalipoproteinaemia, 136 acquired haemolytic anaemias, 174 ABH antigens see ABO blood group ABH secretor status, 248, 251 ABL1–BCR gene, CML, 486, 486 ABO blood group, 244–250, 245 A1 subgroup, 246 A2 subgroup, 246 ABH antigens, 248–250 glycoproteins, 248, 249 ABH secretor status, 248, 251 antibodies, 247–248 A antigen, 247 antigens, 245–247 see also specific antigens B antigen, 247 blood transfusion recipient testing, 280 genes, 249–250 grouping, 246 H antigen, 246–247, 250 H genes, 250 immediate haemolytic transfusion reactions, 283 incidence, 245, 245–246 incompatibilities, haemolytic disease of the fetus and newborn, 293, 297 independent gene segregation, 247 rare variants, 247 testing, blood donation, 275 von Willebrand disease, 808 ABO haemolytic disease, 974 abortion anti-D antibodies development, 258 spontaneous see spontaneous abortion absorption tests, agglutination reactions, 239–240 ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine) classical Hodgkin lymphoma treatment, 647, 648, 650 Hodgkin lymphoma treatment, 646

Postgraduate Haematology: 6th edition. Edited by A. Victor Hoffbrand, Daniel Catovsky, Edward G.D. Tuddenham, Anthony R. Green © 2011 Blackwell Publishing Ltd.

acaeruloplasminaemia, 53 ACAMPS (apoptotic cell-associated molecular patterns), 304 acanthocytosis, 136 ACD see anaemia of chronic disease (ACD) ACE inhibitors high-attitude erythrocytosis, 692 renal amyloidosis, 610 acetylcholinesterase, 228 aciclovir, allogeneic SCT infections, 736 acidaemia, congenital methylmalonic, 74 acidosis, massive transfusions, 289 aciduria congenital methylmalonic, 74 orotic, megaloblastic anaemia causing, 81 aclarubicin, AML treatment, 421 acquired aplastic anaemia aetiology, 207–208 clinical features, 211–213 mucosal haemorrhage, 211 skin haemorrhage, 211 TERT/TERC mutations, 212–213 diagnosis, 213–214 differential diagnosis, 214 immune thrombocytopenic purpura, 213 immunosuppressive therapy, 216, 218–220 ATG, 215, 216, 218–220 haematopoietic SCT, 220–224, 221 inherited vs., 207 management, 214–220 algorithms, 217 immunosuppressive therapy see above indications, 216 infections, 215 iron chelation therapy, 215 stabilization, 215–216 supportive care, 214–215 transfusions, 214–215 pathogenesis, 208–211, 210 genetic predisposition, 211 haematopoietic defects, 208–209 immune system, 209 mouse models, 209, 211 989

Index

acquired circulating anticoagulants, malignancies, 945, 945 acquired coagulation disorders see blood coagulation acquired cobalamin deficiency, 74 acquired disorders neonatal haematology, 980–981 see also specific diseases/disorders acquired haemolytic anaemias, 158–175 immune, 158–168 classification, 159 see also alloimmune haemolytic anaemia; autoimmune haemolytic anaemia (AIHA) non-immune, 168, 168–174 chemical/physical agents, 172–174 fragmentation/mechanical, 170, 170, 170–172 infections, 168–170 see also specific infections red cell membrane, 174 acquired haemophilia, 804 acquired haemophilia A, 856, 856–858 acquired protein S deficiency, 858 acquired sideroblastic anaemia, 45 acquired thrombotic disorders, neonates, 983 acquired thrombotic thrombocytopenic purpura, 862 acquired venous thrombosis see venous thromboembolism (VTE) acquired von Willebrand disease, 811, 812, 945 acrocyanosis, cold-type AIHA, 164 acroparaesthesia, Fabry disease, 341 actin phagocyte locomotion, 300 polymerization, platelets, 786–787 red cell membrane, 227 β-actin, red cell membrane, 129 activated B cell-like diffuse large B-cell lymphomas, 623 activated clotting time, acquired coagulation disorders, 841 activated partial thromboplastin time (APTT), 748 acquired coagulation disorders, 840, 841, 841 acquired haemophilia A, 857, 857–858 antiphospholipid syndrome, 893, 894 biphasic waveform, 841, 841 DIC, 843 FVR506Q, 877 haemophilia A, 795 heritable thrombophilia, 883 in liver disease, 847–848 neonatal haemostasis, 980 990

rare bleeding disorders, 814 in trauma, 852 activated protein-1 (AP-1), Hodgkin lymphoma, 642 activated protein C (APC) assay, heritable thrombophilia, 883 DIC treatment, 845 FVR506Q, 877 resistance, 877 detection, 885 acute chest syndrome, sickle cell disease, 115, 116, 116 acute coagulopathy of trauma-shock, 852 acute graft-versus-host disease see graftversus-host disease (GVHD) acute haemolytic crises, glucose-6phosphate dehydrogenase deficiency, 151, 151 acute haemolytic transfusion reactions, 248 acute hepatitis, 848 acute hypergranular promyelocytic leukaemia, 396, 397 acute infections, anaemia, 950–951 acute infectious lymphocytosis, 364 acute intravascular haemolysis, 173–174 acute iron poisoning, 59 acute lymphoblastic leukaemia (ALL), 433–447 chemotherapy, 438–440 CNS prophylaxis, 440 consolidation therapy, 439–440 maintenance therapy, 440 relapse/resistant leukaemia, 440 remission induction therapy, 438–439, 439 classification, 433 B-lineage, 434 common ALL, 434 early T-precursor ALL, 434 mature B-cell ALL, 434 mature T-ALL, 434 pre-B ALL, 434 pro-B ALL, 434 T-lineage, 434 WHO classification, 406 clinical features, 435, 436 cytogenetic analysis, 406, 434–435 periodic acid–Schiff stain, 402, 403 phosphatase reactions, 402, 403 diagnosis, 433–435 blood film, 396, 398 bone marrow aspirates, 397 bone marrow trephine biopsy, 401 cell surface marker analysis, 433–434 flow cytometric immunophenotyping, 402 immunohistochemistry, 405

molecular genetic analysis, 434–435 morphology, 433 differential diagnosis, 437 AML vs., 433 cytochemistry, 401, 402, 403 folate deficiency, 65 genetics BCR–ABL1 chimeric gene, 487 ETV6–RUNX1, 391 fusion genes, 385 hyperdiploidy, 386 Philadelphia chromosome, 434–435 see also BCR–ABL1 chimeric gene infections, 477 initial evaluation, 437–438 laboratory evaluation, 436–437 blood counts, 436 large-volume blood transfusions, 466 management new treatment approaches, 445–446 SCT see below supportive therapy, 437–438 minimal residual disease, 435 prognostic factors, 442–445, 443 age, 442 B-precursor ALL, 443 cytogenetics, 443–444 immunophenotype, 443–444 mature B-ALL, 444 minimal residual disease, 444–445 molecular genetic analysis, 411 new integrated risk classification, 445 Philadelphia chromosome, 443–444 response to treatment, 444–445 T-lineage ALL, 443 white blood cell count, 442–443 SCT, 440–442, 442, 445 allogeneic, 441, 441 autologous, 441, 441 evidence-based recommendations, 442 indications, 442 matched unrelated donor, 441, 441 non-myeloablative, 441, 442 acute lymphoblastic leukaemia (ALL), childhood, 448–462 aetiology, 449 blood film, 450 classification, 459 clinical features, 450, 450–451 cytochemistry, 451 cytogenetics/molecular classification, 449, 452–454, 454 BCR–ABL1 rearrangements, 453, 454, 455 E2A–PBX1 (TCF3–PBX1) rearrangements, 453, 454 EBF gene, 450

Index

ETV6–RUNX (TEL–AML1) gene fusion, 448–449, 450, 452, 454, 455 HOX11 gene, 453 hyperdiploid, 452 hypodiploid, 452 IKZF1 gene, 450 IKZF3 gene, 450 LEF1 gene, 450 LMO2 (TTG-2) gene, 453 LMOI (TTG-1) gene, 453 MLL rearrangements, 453, 454, 455 novel subtypes, 454 SCL (TAL-1) gene, 453 T-cell ALL, 453–454, 454 TCF3 gene, 450 TCF3–PBX1 gene fusion, 450 differential diagnosis, 451 epidemiology, 448–449 immunophenotypic classification, 451–452 infections, 477 laboratory features, 450, 450–451 minimal residual disease, 459 morphology, 451 pathogenesis, 449–450 prognostic factors, 454–456 minimal residual disease, 455 pharmacogenetic variables, 455–456 presentation, 454–455 relapse, 459 risk classifications, 456 treatment, 456–459 allogeneic haemopoietic SCT, 458 clinical trials, 457 consolidation therapy, 457–458 immunotherapy, 459–460 intensification (consolidation), 457 remission induction, 456–457 sequelae, 458–459 tumour lysis syndrome, 479 twins, 449 acute megakaryoblastic leukaemia (AMKL) bone marrow trephine biopsy, 399, 401 transient abnormal myelopoiesis of Down syndrome, 707–708 acute microgranular/hypogranular promyelocytic leukaemia, blood film, 396, 397 acute monoblastic leukaemia blood film, 397, 399 cytochemistry, 402 acute myeloid leukaemia (AML), 415–432 age distribution, 416 childhood ALL relapse, 459 core-binding factor complex, 391–392

cytogenetics, 405, 406, 416–418, 417, 419 distribution, 418 diagnosis blood film, 398 bone marrow aspirates, 397, 398, 399, 400, 401 bone marrow trephine biopsy, 401 FISH, 409 flow cytometric immunophenotyping, 403 Diamond–Blackfan anaemia, 198, 199 differential diagnosis acquired aplastic anaemia vs., 214 adult ALL vs., 433 cytochemistry, 401, 402, 403 disease classification, 416 epidemiology, 415 essential thrombocythaemia, transformation of, 694 familial, 380 future work, 430–432 classification, 430–431 therapeutics, 431–432 genetics, 417 CEBPA gene, 380 cooperating mutations, 392 FIP1L1–PDGFRA fusion gene, 385 FLT3 gene, 392 hypomethylation, 388 KRAS gene, 391 LTA gene, 388 MYH11–CBFB, 381 NRAS gene, 391 RUNX1–ETO, 381 RUNX1 gene, 380 trisomy 8, 386 incidence, 415 minimal residual disease, 428–429 paroxysmal nocturnal haemoglobinuria, 178 pathophysiology, 415–416 post-ATG, 220 relapse management, 430 outcome, 430 relapse risk, 425–427 age, 426 CEBPA gene mutation, 427 cytogenetics, 425–426, 426 FLT3 gene mutation, 426–427, 427 induction chemotherapy, reaction to, 426 NPM1 gene mutation, 427, 427 performance score, 428 resistance proteins, 428 white cell count, 428 WT1 gene mutation, 427

severe congenital neutropenia, 314 Shwachman–Diamond syndrome, 197 treatment, 418–430 age effects, 418, 419, 421 allogeneic SCT, 423–424, 425, 521 aspirations, 418–419 autologous SCT, 424–425 chemotherapy, 419–420 consolidation treatment, 423, 425 diagnostic presentation, 420 infection prophylaxis, 421–422 large-volume blood transfusions, 466 older patients, 430 options, 423 palliative approach, 418–419 post-remission treatment, 420 prognostic factors, 429 remission induction, 420–421 RT-PCR monitoring, 420 supportive care, 421–423 WHO classification, 406 acute painful episodes, sickle cell disease, 113 acute promyelocytic leukaemia (APL), 429–430 all-trans retinoic acid, 429–430 bleeding risks, 467 DIC, 467, 945 haemostatic dysfunction, 845 phenotype–genotype correlations, 384 PML–RARA fusion gene, 384, 386 RARA–PML fusion gene, 386 relapse management, 430 thrombosis, 897 acute renal failure (ARF), fluid balance, 479 acute renal tubular necrosis (ATN) electrolyte balance, 479–480 fluid balance, 479 ADAMTS-13, 861 assays, 863–865, 864 deficiency, 780, 861, 862 diarrhoea-related haemolytic-uraemic syndrome, 869 replacement, 868 structure, 861, 861 TTP, 861, 862, 863–865, 864, 868 von Willebrand factor interaction, 755, 805, 861 ADAMTS13 gene Schulman–Upshaw syndrome, 917, 925 TTP, 862 adenopathy, AML, 415 adenosine deaminase (ADA), red cell aplasia, 973–974 adenosine, platelet inhibition, 788 991

Index

adenylic acid residue (poly-A) addition, globin gene processing, 87 adhesion molecules multiple myeloma pathogenesis, 580 phagocytes, 302 see also specific molecules adhesion, platelets, 921 adjuvant chemotherapy, localized follicular lymphoma, 663 adjuvant radiotherapy, aggressive B-cell NHL treatment, 674–675 ADP receptor defects genetics, 925 platelet responses, 922 thrombocytopathy, 922 adrenaline receptor defects, platelet responses, 922 adrenaline, von Willebrand factor, 805 α2A-adrenergic receptors, platelets, 784–785 α2-adrenergic receptors, thrombocytopathy, 922–923 adriamycin see doxorubicin adults chronic CML management, 497–498 cobalamin deficiency, 70 haematopoiesis, 3 bone marrow, 6–7 adult T-cell leukaemia lymphoma (ATLL), 567–570 aetiology, 567 clinical course, 570 clinical features, 567–568 cytogenetics, 568–569 diagnosis, 568 differential diagnosis, 569–570 epidemiology, 567 pathogenesis, 568–569 therapy, 570 treatment, 678–679 WHO lymphoma classification, 627– 628, 628 adventitia, blood vessels, 752–753 Africa, hereditary elliptocytosis, 134 African haplotypes, sickle cell disease, 112 African iron overload, 52 African trypanosomiasis, 965–966 global distribution, 965, 965 age adult ALL prognosis, 442 AML distribution, 416 AML relapse risk, 426 AML treatment, 418, 419, 421 CLL, 538 elderly see elderly patients hyposplenism, 376 multiple myeloma, 589 Sézary syndrome/mycosis fungoides, 566 992

age of onset, TTP, 864 agglutinins, infectious mononucleosis, 366 aggregation, thrombus formation, 777–778 aggressive NK-cell leukaemia, WHO classification, 627, 627 AGM see aorta–gonad–mesonephros (AGM) agranulocytosis, deferiprone, 58 AIDS-related lymphoma, 658 AIDS-related non-Hodgkin’s lymphoma, 681–682 AIHA see autoimmune haemolytic anaemia (AIHA) aircraft journeys, venous thromboembolism, 889 air embolism, blood transfusions, 288 AITL see angioimmunoblastic T-cell lymphoma (AITL) ALA (δ-aminolaevulinic acid), haem synthesis, 35 ALAS2 gene mutations, X-linked sideroblastic anaemia, 44 ALAS2 (δ-aminolaevulinic acid synthase), iron-responsive elements, 32 albendazole, filariasis treatment, 965 albumin, folate transport, 77 ALCL see anaplastic large-cell lymphoma (ALCL) alcohol, folate deficiency, 79 Alder–Reilly anomaly, neutrophils, 311, 312 alemtuzumab (Campath-1H), 666 acquired aplastic anaemia management, 220 adult T-cell leukaemia lymphoma, 570 allogeneic SCT in myelodysplastic syndrome, 521 childhood ALL treatment, 459 CLL management, 547 follicular lymphoma therapy, 667 GVHD prophylaxis, 731 Richter transformation, 549 unrelated donor allogeneic SCT conditioning regimens, 731 aleukaemic leukaemia, differential diagnosis, acquired aplastic anaemia vs., 214 AL fibrils, systemic AL amyloidosis, 601 alglucosidase alfa (Myozyme), Pompe disease, 341 algorithms, acquired aplastic anaemia management, 217 ALK1 gene, hereditary haemorrhagic telangiectasia, 854 alkaline dehydrogenase, CML, 491 alkaline phosphatase, GvHD, 735

ALK gene anaplastic large-cell lymphoma, 574 large B-cell lymphomas, WHO lymphoma classification, 625 alkylating agents B-PLL, 550 CLL management, 545 cold-type AIHA, 165 SCT conditioning regimens, 729 see also specific agents ALL see acute lymphoblastic leukaemia (ALL) allele-specific primers, molecular blood grouping techniques, 242 allelic exclusion, B cell development, 350 alloantibodies, 230 maternal, haemolytic disease of the fetus and newborn, 292–293 allogeneic bone marrow transplantation AML therapy, 431–432 paroxysmal nocturnal haemoglobinuria treatment, 181 systemic AL amyloidosis management, 609 allogeneic stem cell transplantation ALL therapy adult ALL, 441, 441 childhood ALL treatment, 458 AML therapy, 423–424, 425 antibiotic prophylaxis, 477 chronic myelomonocytic leukaemia, 527 CLL therapy, 548 CML therapy, 499 advanced CML, 498 chronic CML, 491, 496, 496–497 complications, 722, 733–741 acute GvHD, 734–735 early, 733–735 fertility, 740–741 graft failure, 733–734 graft-versus-host response, 722 growth delays, 740–741 host-versus-graft response, 722 infections, 735–737, 736, 736–737 intensive care support, 739 late complications, 739 organ toxicity, 737–739 puberty, 740–741 secondary malignancies, 740 conditioning regimens, 729 myeloablative conditioning, 730 sibling donor, 730–731 unrelated donor, 731 donor choice, 742–743 donor infection screening, 477 future developments, 744–745 mantle cell lymphoma, 673

Index

multiple myeloma management/ treatment, 593 mycosis fungoides treatment, 682 myelodysplastic syndrome therapy, 521– 522, 522, 522 NHL therapy, 669 aggressive B-cell NHL treatment, 675 aggressive NHL treatment, 681 patient factors affecting outcome, 741, 741 primary myelofibrosis treatment, 699–700 refractory anaemia with excess blasts in transformation, 521 refractory anaemia with ring sideroblasts, 521 relapsed Hodgkin lymphoma, 651 relapse management, 743–744 sibling donor conditioning regimens, 730–731 myelodysplastic syndrome, 521 T-prolymphocytic leukaemia therapy, 561 umbilical cord blood see umbilical cord stem cell transplantation unrelated donor conditioning regimens, 731 myelodysplastic syndrome, 521 alloimmune anaemia of the fetus and newborn, 293 alloimmune haemolytic anaemia, 166–168 anti-D antibodies, 167–168 drug-induced, 166–167, 167 alloimmune neonatal neutropenia, 263, 979 alloimmune thrombocytopenias, 916 allopurinol adult ALL, 437 hyperuricaemia therapy, 479 all-trans retinoic acid (ATRA), acute promyelocytic leukaemia, 429– 430, 845 α chain, T-cell receptor see T-cell receptor (TCR) α-globin chains see haemoglobin(s) α-granules, platelets, 774, 774 alternative complement pathway, 234, 235 ambulatory haemolysis, fragmentation haemolysis, 171 ambulatory peritoneal dialysis, anaemia, 947 American trypanosomiasis (Chagas disease), 966–967 blood transfusions, 274 AMG 531, congenital platelet disorder therapy, 924

δ-aminolaevulinic acid (ALA), haem synthesis, 35 δ-aminolaevulinic acid synthase (ALAS2), iron-responsive elements, 32 AMKL see acute megakaryoblastic leukaemia (AMKL) AML see acute myeloid leukaemia (AML) AML-1 gene see RUNX1 (AML-1) gene AMN, cobalamin absorption, 69 amotosalen, fresh frozen plasma preparation, 279 amphotericin, glomerular toxicity, 480 amsacrine AML treatment, 423 with fludarabine and cytarabine (FLAMSA), allogeneic SCT in myelodysplastic syndrome, 521, 525 amyloid deposits, amyloidosis, 599, 600 amyloidogenic monoclonal immunoglobulin chains, 600 amyloidosis, 599–613 AA amyloidosis, 611 breast, 610 classification, 600 coagulation disorders, 856 definition, 599 factor X deficiency, 856 future work, 612 hereditary systemic, 611–612 localised AL, 610 non-neuropathic hereditary systemic, 612 pathogenesis, 599–601 amyloid deposits, 599, 600 amyloidogenic monoclonal Ig chains, 600 glycosaminoglycans, 600–601 β2-microglobulin, 599–600, 611 β-protein, 600 serum amyloid A, 599–600 transthyretin, 600 rheumatoid arthritis, 946 systemic types, 610–612 transthyretin, 611 see also specific diseases/disorders anaemia(s) aetiology adult T-cell leukaemia lymphoma, 568 ambulatory peritoneal dialysis, 947 blood donation, 269 CLL, 534, 535 CML, 490 connective tissue disorders, 945–946 hairy cell leukaemia, 551 hepcidin, 32 hereditary spherocytosis, 130

infections see below liver disease, 948–949 malignancies, 942, 942–943 see also specific malignancies multiple myeloma, 587 myelodysplastic syndrome, 512 nephrectomy, 947 NHL, 659–660 pregnancy, 953 prematurity, 977–978 primary myelofibrosis, 698 protein deficiency, 81 renal disease, 850, 946–977, 947 rheumatoid arthritis, 945 scurvy, 81 sickle cell disease, 113 infections acute infections, 950–951 HIV infection, 954 malaria, 952, 959 tropical diseases, 968–969 viral infection, 949–950 leukaemias see leukaemias, supportive care management erythropoietin, 468 granulocyte colony-stimulating factor, 468 haematinics, 292 primary myelofibrosis treatment, 700 types acquired aplastic anaemia see acquired aplastic anaemia acquired haemolytic see acquired haemolytic anaemias acquired haemolytic anaemias see acquired haemolytic anaemias acquired sideroblastic anaemia, 45 acute, 465 AIHA see autoimmune haemolytic anaemia (AIHA) alloimmune anaemia of the fetus and newborn, 293 alloimmune haemolytic anaemia see alloimmune haemolytic anaemia aplastic anaemia see aplastic anaemia (AA) autosomal recessive megaloblastic anaemia (MGA1), 73 cardiac haemolytic anaemia, 170, 170–172 chronic haemolytic anaemia, 127 chronic non-spherocytic haemolytic anaemia, 152 cold-antibody AIHA, 942 cold-type AIHA, 164–165, 165 993

Index

congenital dyserythropoietic anaemia (CDA) see congenital dyserythropoietic anaemia (CDA) congenital non-spherocytic haemolytic anaemia see congenital non-spherocytic haemolytic anaemia (CNSHA) Diamond–Blackfan anaemia see Diamond–Blackfan anaemia drug-induced alloimmune haemolytic anaemia, 166–167, 167 drug-induced warm-type AIHA, 162–163 Fanconi anaemia see Fanconi anaemia fetal/newborn anaemia, 293 fragmentation acquired haemolytic anaemias, 170, 170, 170–172 haemolytic anaemias see haemolytic anaemias hypochromic anaemia, 37 hypochromic microcytic anaemia, 102–103 idiopathic aplastic anaemia, 187 idiopathic cold-type AIHA, 164–165 idiopathic warm-type AIHA, 162 inherited sideroblastic anaemia, 44–45 iron deficiency see iron deficiency anaemia iron deficiency anaemia see iron deficiency anaemia iron-loading see iron-loading anaemias iron-loading anaemias see ironloading anaemias iron refractory iron deficiency anaemia, 41 juvenile pernicious anaemia, 72 mechanical acquired haemolytic anaemias, 170, 170, 170–172 megaloblastic see megaloblastic anaemia megaloblastic anaemia see megaloblastic anaemia microangiopathic haemolytic anaemia see microangiopathic haemolytic anaemia (MAHA) neonatal anaemia see neonatal anaemia normochromic macrocytic anaemia, 198 normochromic normocytic anaemia see normochromic normocytic anaemia pernicious see pernicious anaemia pernicious anaemia see pernicious anaemia 994

refractory anaemia (RA) see refractory anaemia (RA) refractory anaemia with excess blasts 1 (RAEB-1), 506 refractory anaemia with excess blasts-2 (RAEB-2), 506 refractory anaemia with excess blasts in transformation see refractory anaemia with excess blasts in transformation (RAEBt) refractory anaemia with ring sideroblasts see refractory anaemia with ring sideroblasts (RARS) refractory anaemia with ring sideroblasts and thrombocytosis (RARS-T), 527–528 secondary sideroblastic anaemia, 45 sideroblastic see sideroblastic anaemia sideroblastic anaemia see sideroblastic anaemia ‘spur-cell anaemia,’ 949 thiamine-responsive megaloblastic anaemia, 81 warm-type AIHA see warm-type autoimmune haemolytic anaemia X-linked sideroblastic anaemia, 44 anaemia of chronic disease (ACD), 940–942 associated disease, 941 erythropoiesis inhibition, 941 haematological features, 941 iron metabolism, 941 pathogenesis, 940–941 treatment, 941–942 anagrelide essential thrombocythaemia treatment, 696 polycythaemia vera treatment, 690 analgesia, opiates, 481 anaplastic large-cell lymphoma (ALCL), 574–575, 677 ALK+, 633, 633, 634, 635 ALK-, 635 T-cell type, 660 treatment, 678 WHO lymphoma classification, 633, 633, 634, 635 anaplastic lymphoma kinase (ALK), T-cell lymphomas, 659 Anderson–Fabry disease see Fabry disease aneuploidy, somatic mutations, 386–387 angioimmunoblastic T-cell lymphoma (AITL), 572, 677 WHO lymphoma classification, 631, 632, 633 angiokeratoma, Fabry disease, 341

angiotensin II inhibitors, renal amyloidosis, 610 animal models, platelets, 774–775 anisocytosis, myelodysplastic syndrome, 512 ANK1 gene, hereditary spherocytosis, 131 ankylosing spondylitis, HLA-B27 association, 713 ankyrin, red cell membrane, 129, 130, 227 ankyrin–spectrin complex, hereditary spherocytosis, 131 Ann Arbor system Hodgkin lymphoma, 643, 643 NHL staging, 662, 663 anorexia, leukaemias, 480 anorexia nervosa, acquired aplastic anaemia vs., 214 antenatal screening blood transfusion recipients, 281 thalassaemias, 105 Anthony Nolan Trust, 716–717 anthracycline adult ALL chemotherapy, 438 AML treatment, 423 CLL management, 546 anti-A1 antibodies, 248 neonatal anaemia, 974 anti-A antibodies, 247, 248 anti-AB antibodies, 247 anti-ADAMTS-13 antibodies, TTP, 866 antibacterial prophylaxis, adult ALL, 437–438 anti-B antibodies, 247, 248 antibiotics diarrhoea-related haemolytic-uraemic syndrome, 869 fever of unknown origin, 476 induced neutropenia, 316 MALT lymphoma, 670 post-splenectomy, 133, 133, 935 prophylactic, splenectomy, 376 Waldenström macroglobulinaemia, 672 see also specific antibiotics antibodies, 230–232 adult ALL therapy, 445 antiphospholipid syndrome, 892, 892–893 characteristics, agglutination reactions, 238 detection, malaria diagnosis, 961 haemolytic transfusion reactions, 284 infectious mononucleosis, 365–366 Rh system, 257–258 see also immunoglobulin(s) (Igs); specific antibodies antibody conjugates, follicular lymphoma therapy, 667

Index

antibody span, agglutination reactions, 238 anti-C antibodies, 257 anti-c antibodies, 297 anti-CD20 monoclonal antibody see ofatumumab; rituximab anti-CD22 monoclonal antibody see epratuzumab anticoagulants blood storage, 276 endothelial cells, 755 venous thromboembolism therapy, 903– 906, 907–908 see also specific anticoagulants anticonvulsants, induced neutropenia, 316 anti-D antibody, 257 alloimmune haemolytic anaemia, 167–168 detection in early pregnancy, 294 fetal/newborn anaemia, 293 immunization by abortion/miscarriage, 258 injection after birth, 257–258 maternal, haemolytic disease of the fetus and newborn, 298 primary immune thrombocytopenia management, 933, 934 SHOT, 290 source, 258 transplacental haemorrhage, 258 antiembolic stockings, thrombolytic prophylaxis, 908 antiemetic drugs, 481 anti-factor VIII antibodies, 799 antifibrinolytic agents congenital platelet disorder therapy, 924 factor XI deficiency therapy, 827 see also specific agents antifolate drugs, folate deficiency, 79 antifungal prophylaxis adult ALL, 437 see also specific antifungals anti-Fya antibody, 260 anti-gastrin receptor antibodies, 72 antigen(s) definition, 228–229 detection Aspergillus infection, 471 malaria diagnosis, 960–961 Rh system, 252–253, 256 SCT, 722–723 site density, agglutination reactions, 238 antigen-presenting cells (APCs), 345 acute GvHD, 724 antigen processing/presentation CD4 T-cells, 345 HLA, 711–712 HLA class II, 712

Langerhans cells, 325 lymphocytes, 344–346 MHC class I, 344–345, 345, 346 MHC class II, 345, 345, 346 antiglobin test see Coombs (antiglobin) test anti-β2-glycoprotein antibodies, antiphospholipid syndrome, 892, 893, 894–895 anti-GPIIb/IIIa antibodies, primary immune thrombocytopenia, 928–929 anti-H antibodies, 248 anti-HCV antibodies, blood donation screening, 272, 275 anti-heparin/platelet factor 4 complex, 891 antihistamines febrile reactions, 467 mastocytosis treatment, 703 see also specific antihistamines anti-HLA-DR antibodies see apolizumab anti-HPA-Ia antibodies, neonatal alloimmune thrombocytopenia, 981 anti-HTLV antibodies, blood donation testing, 275 anti-human globulin (AHG), 239 standardization, 239 anti-I antibodies, 252 anti-i antibodies, 252 anti-idiotype antibodies, 232 anti-inflammatory agents induced neutropenia, 316 see also specific agents anti-intrinsic factor antibodies, 72 anti-Jka antibody, 260 anti-Jkb antibody, 260 anti-K antibodies, 260 fetal/newborn anaemia, 293 haemolytic disease of the fetus and newborn, 297 anti-k antibodies, 260 anti-Kell antibodies, neonatal anaemia, 974 anti-Lea antibodies, 251 anti-Leb antibodies, 251 antilymphocyte globulin (ATG), myelodysplastic syndrome, 520 anti-M antibodies, 259 antimetabolite drugs megaloblastic anaemia causing, 81 see also specific drugs antimicrobial proteins phagocytes, 306–307 see also specific proteins antimotility agents diarrhoea-related haemolytic-uraemic syndrome, 869 see also specific agents

anti-N antibodies, 259 anti-P1 antibody, 251 anti-parietal cell antibodies, 72 antiphospholipid syndrome (APS), 892–895 antibodies, 892, 892–893 diagnostic criteria, 892 laboratory diagnosis, 893–895 coagulation assays (lupus anticoagulant tests), 893–894 solid-phase assays, 894–895 management, 895 pathogenic mechanisms, 893 pregnancy, 895 α2-antiplasmin, 759, 765 haemostatic disturbance in chronic liver disease, 848 plasmin inhibition, 770 antiplatelet antibodies, primary immune thrombocytopenia, 930 antiplatelet drugs, 773 monitoring, 790 thrombolytic prophylaxis, 909 see also specific drugs anti-PP1PK antibody, 251 anti-S antibodies, 259 antithrombin (AT), 759, 763 activation, 874, 875–876 blood coagulation feedback inhibition, 749 deficiency, 872, 873, 874, 876 neonates, 983 type 1, 874 type 2, 874 disseminated vascular coagulation, 764 heparin interaction, 764 structure, 874, 875–876 antithrombin tests heritable thrombophilia, 884 neonatal thrombocytopenia, 982 antithymocyte globulin (ATG) acquired aplastic anaemia, 215, 216, 218–220 allogeneic SCT in myelodysplastic syndrome, 521 children, 220 cyclosporin with, 218–219, 219 definition, 216 development of, 218 GvHD prophylaxis, 731 late complications, 220 monitoring, 216 myelodysplastic syndrome, 520 older patients, 220 prognosis, 218, 218 repeat courses, 219 response predictors, 218–219 995

Index

side effects, 216, 218 unrelated donor allogeneic SCT conditioning regimens, 731 antithyroid drugs induced neutropenia, 316 see also specific drugs α1-antitrypsin, 765 protein C pathway inhibition, 767 antituberculous drugs, sideroblastic anaemia, 45 aorta–gonad–mesonephros (AGM) erythropoiesis, 12 haematopoiesis, 4, 5, 7 haemopoiesis, 971 aortic stenosis, acquired von Willebrand disease, 811 APC see activated protein C (APC) apixaban, venous thromboembolism therapy, 906 APL see acute promyelocytic leukaemia (APL) aplastic anaemia (AA), 206–225 acquired see acquired aplastic anaemia characterization, 206 definition, 206 differential diagnosis adult ALL vs., 437 childhood ALL vs., 451 disease severity, 207 epidemiology, 206–208 incidence, 206–207 idiopathic, 187 inherited/bone failure syndromes, 186– 205, 187 incidence, 186 ribosomal RNA processing, 197 see also specific diseases/disorders neutropenia, 316 aplastic crises, sickle cell disease, 113 apolizumab, 666 follicular lymphoma therapy, 667 apoptosis erythropoiesis, 22–23 erythropoietin receptor, 22 leukaemogenesis, 393 myelodysplastic syndrome, 508 neutrophils, 312 apoptotic cell-associated molecular patterns (ACAMPs), 304 apoptotic cells macrophage clearance, 323 phagocyte receptors, 304 apparent polycythaemias, 693 aprotinin, haemostatic disturbances in surgery, 852 APS see antiphospholipid syndrome (APS) APTT see activated partial thromboplastin time (APTT) 996

aquaporin 1 (Colton antigen), 228 biological significance, 229 aquaporin 3 (GIL antigen), 228 biological significance, 229 Arab–Indian haplotype, sickle cell disease, 112 arachidonic acid platelet signalling transduction disorders, 920 prostaglandin I2, 754 argatroban, venous thromboembolism therapy, 905 arrhythmias, American trypanosomias, 966 arsenic trioxide, acute promyelocytic leukaemia, 430 arsine, acquired haemolytic anaemias, 173 artemisin, malaria treatment, 962 arterial occlusion, polycythaemia vera, 688 arterial thrombosis, heritable thrombophilia, 882 arteriovenous malformation, 854–855 fragmentation haemolysis, 171 arthritis, haemochromatosis, type 1, 49, 49 arthropathy, haemophilia A, 794, 794 Ashkenazi Jews, factor XI deficiency, 827 asparaginase adult ALL chemotherapy, 438–439 childhood ALL treatment, 457, 458 coagulation factor inhibition, 467 venous thromboembolism, 890–891 aspartylglucosaminuria, 334 clinical features, 337 Aspergillus infections acquired aplastic anaemia, 215 allogeneic SCT infections, 737 AML treatment, 422 antigen detection, 471 ELISA, 471 leukaemias, 471 aspirations AML treatment, 418–419 bleeding risks, 467 aspirin antiphospholipid syndrome management, 895 anti-platelet effects, 773, 784 essential thrombocythaemia treatment, 696 polycythaemia vera treatment, 690 ASXL1 gene, myelodysplastic syndrome, 512 AT see antithrombin (AT) ATG see antithymocyte globulin (ATG) AT gene, knockout animal models, 763 atherosclerosis, thrombosis in inflammation, 898

ATLL see adult T-cell leukaemia lymphoma (ATLL) ATM gene deletion, mantle cell lymphoma, 386, 393 T-prolymphocytic leukaemia, 559 ATN see acute renal tubular necrosis (ATN) atovaquone, babesiosis, 962 ATP production, glycolytic pathway, 140 atransferrinaemia, iron overload, 53 ATRX gene, myelodysplastic syndrome, 510 atypical chronic myeloid leukaemia, 527 atypical haemolytic-uraemic syndrome, 869 aurora kinase inhibitors, multiple myeloma management/treatment, 596 autoantibodies definition, 230 myelodysplastic syndrome, 513 see also specific antibodies autografting, chronic CML management, 498 autohaemolysis test hereditary spherocytosis, 131 pyruvate kinase deficiency, 144 autoimmune diseases/disorders acquired aplastic anaemia, 207 antiphospholipid syndrome, 892 chronic GvHD, 739–740 CLL management, 548 drug-induced alloimmune haemolytic anaemia, 167 interferon alpha toxicity, 495–496 MHC, 347 pernicious anaemia association, 72 primary immune thrombocytopenia, 929 warm-type AIHA association, 162 see also specific diseases/disorders autoimmune haemolytic anaemia (AIHA), 158–166 antibody characteristics, 158–159 cold-acting antibodies, 159, 160 specificity, 160 warm-acting antibodies, 159, 160 blood transfusions, 282 CLL, 548 cold-type see cold-type autoimmune haemolytic anaemia complement activation, 160 Coombs test, 158 DAT, 158 destruction mechanisms, 160–161 Ig test, 159 paroxysmal cold haemoglobinuria, 165–166

Index

warm-type see warm-type autoimmune haemolytic anaemia autoimmune neutropenia, 315 neutrophil antibodies, 263–264 autoimmune thrombocytopenic purpura (ITP), CLL, 536 autoimmune thrombotic thrombocytopenic purpura, 863 autologous blood transfusions, 292 autologous stem cell transplantation complications, 741 conditioning regimens, 729 future developments, 744 myelodysplastic syndrome development, 504 peripheral blood, 608 relapse management, 744 stem cell mobilization, 727 therapy in adult ALL, 441, 441 adult T-cell leukaemia lymphoma, 678–679 aggressive B-cell NHL, 675 AML, 424–425 CLL, 548 lymphoblastic lymphoma, 679 mantle cell lymphoma, 673 multiple myeloma, 587, 591–592, 592 NHL see non-Hodgkin lymphoma (NHL) relapsed Hodgkin lymphoma, 650 systemic AL amyloidosis, 608 T-prolymphocytic leukaemia, 561 automated blood group screening, 241 automated cell counting, platelet function testing, 789 autonomous gene silencing, haemoglobin synthesis regulation, 86 autosomal dominant dyskeratosis congenita, 194 autosomal mutations, sideroblastic anaemia, 44 autosomal recessive dyskeratosis congenita, 195 autosomal recessive inheritance, rare bleeding disorders, 813, 814 autosomal recessive megaloblastic anaemia (MGA1), 73 avascular necrosis, pain, 481 5-aza-2′-deoxycytidine, myelodysplastic syndrome, 522 azacitidine, myelodysplastic syndrome, 528 5-azacytidine, myelodysplastic syndrome, 522, 523 azathioprine hepatosplenic T-cell lymphoma, 629

primary immune thrombocytopenia management, 934, 938 warm-type AIHA treatment, 163 azurocidin neutrophils, 307 phagocytes, 306 azurophil granules, neutrophils, 308–309, 310 B B1 cells, 352 B2 cells, 352 B7.1 (CD80), T cell activation, 345–346 B7.2 (CD86), T cell activation, 345–346 B7, T-cell-dependent B-cell activation, 357 babesiosis, 962 acquired haemolytic anaemias, 169 blood transfusions, 274 bacterial infections acquired haemolytic anaemias, 169 blood transfusions, 270, 271, 274 haematological disorders, 950–952 haemophagocytic lymphohistiocytosis, 326 in leukaemia, 468 see also individual species bacterial overgrowth, cobalamin malabsorption, 73 bacterial permeability-increasing protein (BPI) neutrophils, 307 phagocytes, 306 bacterial pyrogens, blood transfusions, 287–288 Bacteroides fragilis infection, polyagglutinable red cell Tk activation, 262 band 3 proteins, 228 red cell membrane, 129, 130, 227 Rh system, 255 B antigen see ABO blood group Bantu siderosis, 52 Barr bodies, neutrophils, 310–311, 311 Bartonella infections (Oroya fever), 951 acquired haemolytic anaemias, 169 basophilia, malignancies, 944 basophilic erythroblasts (Bas), 14 basophilic normoblasts, 17, 17 basophils, 321–323 CML, 490 development, 321 disorders, 321–323 FcεIR, 321 functions, 321 granules, 322 structure, 308

B cell(s), 343–344 activation selection, 357 T-cell-dependent, 356, 357 development, 349–351, 350 phenotypic changes, 349, 350 malignancies, 530–557 diagnosis, 530–531 origins, 383–384, 384 WHO classification, 530 see also specific diseases/disorders marginal zone see marginal zone B-cell(s) recovery post-SCT, 726 repertoire, 352–353, 353 B-cell acute lymphoblastic leukaemia, 434 immunophenotypic classification, 452 B-cell precursor acute lymphoblastic leukaemia, adult ALL prognosis, 443 B-cell prolymphocytic leukaemia (B-PLL), 550 CLL vs., 550 WHO lymphoma classification, 616 B-cell receptor (BCR) CLL, 533–534 generation, 347–349 genetics gene rearrangements, 347 heavy chain genes, 349 structure, 344 CDR3, 348–349 complementarity determining regions, 348 D gene segments, 348 heavy chain, 348, 348 J gene segments, 348 light chain, 348 see also immunoglobulin(s) (Igs) β chain, T-cell receptor see T-cell receptor (TCR) BCL2 antisense oligonucleotides, CLL management, 547 BCL2 gene CLL, 388, 533 NHL, 658 overexpression, follicular lymphoma, 394 BCL2–IGH fusion gene, FISH, 407 BCL-5, DLBCL not otherwise specified, 623 BCL-6 follicular lymphoma, 620 nodular lymphocyte predominant Hodgkin lymphoma, 636 BCL10 gene, extranodal marginal zone lymphoma of MALT, 619 997

Index

Bcl-XL, erythropoiesis, 16 BCNU see carmustine BCR see B-cell receptor (BCR) BCR–ABL1 chimeric gene childhood ALL, 453, 454, 455 chronic myelomonocytic leukaemia, 526 chronic neutrophilic leukaemia, 706 clonal hypereosinophilic syndromes, 705 CML see chronic myelogenous leukaemia (CML) detection FISH, 407, 408 PCR, 410 polymerase chain reaction, 410 myeloid malignancies, 382–383, 383 phosphatidylinositol 3-kinase, 489 BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine and prednisone) classical Hodgkin lymphoma, 650 infertility side effect, 652 BEAM (carmustine, etoposide, cytarabine and melphalan) fertility effects, 741 relapsed Hodgkin lymphoma, 650 Behçet disease, thrombosis in inflammation, 898 bendamustine, CLL management, 547 benign neutropenia, chronic, 315 benznidazole, American trypanosomias, 967 Bernard–Soulier syndrome (BSS), 788, 919–920 genetics, 781, 925 platelet responses, 922 thrombocytopenias, 917 von Willebrand factor, 755 β-globin chains gene deletions, β-thalassaemias, 90, 91 haemoglobin see haemoglobin(s) BFU-E cells, 2 erythroid cells, 15 bilateral temporal neuropathy, cobalamin deficiency, 66 bilateral ultrasonography, pulmonary embolism diagnosis, 903 bile pigments, haemolytic disease of the fetus and newborn, 294 bilirubin, GvHD, 735 Binet staging systems, CLL, 537, 544 bioavailability, iron absorption, 33 biopsy, Gaucher disease, 338 BIRC3–MALT1 chimeric gene, extranodal marginal zone lymphoma of MALT, 619 998

bisphosphonates mastocytosis treatment, 704 multiple myeloma management/ treatment, 584–585, 586 bivalirudin, venous thromboembolism therapy, 905 Blackwater fever acquired haemolytic anaemias, 168–169 malaria, 959 blastic transformation, CML, 490–491, 491, 499 bleeding essential thrombocythaemia, 694 mucosal, acquired aplastic anaemia, 211 obstetric haemorrhage, 851 oropharynx, haemophilia A, 795 platelet disorders, 788, 915 prevention, haemophilia A treatment, 797 therapy, leukaemias, 463 bleeding disorders inherited, 793–812 rare, 813–838 autosomal recessive inheritance, 813, 814 distribution, 814 genetics, 814 knockout mouse models, 815 laboratory diagnosis, 814 long-term prophylaxis, 833–834 molecular defects, 832 treatment, 814, 832–833, 833, 835 treatment in pregnancy, 834–835 in women, 814–815 see also specific diseases/disorders bleeding time haemostatic disturbances in renal disease, 850–851 platelet function testing, 789 bleomycin with doxorubicin, vinblastine and dacarbazine see ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine) with etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine and prednisone see BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine and prednisone) β-locus control region (β LCR), β-globin, 85 blood coagulation, 748, 748–752 amplification, 749 factors, 755–762, 758–759 structures, 757 see also specific factors

feedback inhibition, 749–750, 751, 752 antithrombin, 749 thrombomodulin, 749–750 tissue factor pathway inhibitor, 749 HIV infection, 955 initiation, 749, 749 malignancies, 944–945, 945 natural inhibitors, 762–767 classification, 762 see also specific inhibitors pregnancy, 954 thrombin, 748 tissue factor, 749 ‘waterfall’ hypothesis, 748 blood coagulation assays (lupus anticoagulant tests), antiphospholipid syndrome, 893–894 blood coagulation disorders acquired, 839–859 definition, 839 multifactorial causes, 839 point-of-care testing, 840–841 symptoms, 839 tests for, 840–841 acquired inhibitors, 856–858 connective tissue disorders, 946 platelet congenital disorders vs., 915 see also specific diseases/disorders blood coagulation factors dilution of, massive transfusions, 289 fresh frozen plasma, 279 see also specific factors blood components, 276–280 leucodepletion, 278 preparation, 276, 277, 278–279 storage, 276, 278–279 changes, 279–280 see also specific components blood counts, 395–397 adult ALL, 436 heritable thrombophilia, 883 myelodysplastic syndrome, 512 primary immune thrombocytopenia, 932–933 blood cultures, neutropenic fever, 470, 470–471 blood donation, 268–276 anaemia, 269 anti-HCV antibody testing, 275 deference, 269, 272 donation intervals, 270 haemoglobin estimation, 269–270 hazards of, 270 laboratory tests, 275, 275 minor red cell abnormalities, 268–269 protection of, 269

Index

screening, HIV, 273 volume taken, 269 blood films, 395–397 acute lymphoblastic leukaemia, 396, 398 acute microgranular/hypogranular promyelocytic leukaemia, 396, 397 acute monoblastic leukaemia, 397, 399 adult T-cell leukaemia lymphoma, 568, 568 African trypanosomiasis, 966 AML, 398 Burkitt lymphoma, 396, 396 childhood ALL, 450 CLL, 396, 397, 534, 534, 535 CML, 396, 398, 490 Diamond–Blackfan anaemia, 974 differential diagnosis, 396 FAB category, 396 filariasis diagnosis, 964, 964 follicular lymphoma, 554 glucose-6-phosphate dehydrogenase deficiency, 152 hairy cell leukaemia, 551 hairy cell leukaemia diagnosis, 551, 551 hereditary stomatocytosis, 135, 135 hyposplenism, 377 malaria diagnosis, 960, 960, 961 myelodysplastic syndrome, 399 Pearson syndrome, 974 polyclonal B-cell lymphocytosis, 544 polycythaemia vera, 689 primary myelofibrosis, 698, 698 red cell aplasia, 973 Richter syndrome, 549 Sézary syndrome, 566 splenic marginal zone lymphoma, 555 T-prolymphocytic leukaemia, 560, 560 warm-type AIHA, 161 blood flow, spleen, 369 blood group antibodies, 232–233 cold antibodies, 233 identification, 242 see also blood grouping IgG, 233 IgM, 233 naturally-occurring, 232–233 screening, 242 warm antibodies, 233 blood group antigen–antibody reactions (agglutination), 236–238 antibody characteristics, 238 antibody span, 238 antigen site density, 238 cellular contact, 237 detection, 238–242 absorption tests, 239–240

antiglobin (Coombs) test, 238–239 automated techniques, 241 direct agglutination, 238 see also direct agglutination test (DAT) ELISA, 240, 240 elution tests, 239–240 enhancement, 239 flow cytometry, 240 haemolysis, 239 indirect agglutination, 238 see also indirect agglutination test (IAT) inhibition, 239 microcolumn tests, 240, 240 microplate techniques, 240–241 reagents, 241–242 see also anti-human globulin (AHG); blood grouping electrical charge, 237–238 ionic strength, 237 pH, 237 temperature, 237 blood group antigens, 228–229, 244–267 biological significance, 229 chemistry, 226–228 null phenotypes, 229 pathogen receptors, 229 see also specific antigens blood grouping molecular techniques, 242–243 monoclonal antibodies, 233 see also blood group antigen–antibody reactions (agglutination) blood group proteins, 228 blood groups high frequency antigens, 261 low frequency antigens, 261 blood group testing, Rhesus haemolytic disease of the fetus and newborn, 295 blood group typing, blood transfusion recipient testing, 280 blood loss iron deficiency anaemia, 39–40 neonatal anaemia, 977 blood pooling, spleen, 369–370 blood products in massive blood loss, 852, 852–853 rare bleeding disorder therapy, 832–833 blood substitution, adult ALL, 437 blood tests anaemia of prematurity, 978 CML, 488 blood transfusion(s), 268–299 alternatives to, 292 associated circulatory overload, 288 autologous, 292

complications, 282–292 air embolism, 288 bacterial pyrogens, 287–288 GvHD, 287 immunological, 282–287 see also specific complications massive transfusions, 288–289 non-haemolytic febrile transfusion reactions, 286 non-immunological, 287–289 plasma protein antibodies, 287 platelet antibodies, 286–287 post-transfusion purpura, 287 red cell antigen sensitization, 282–286 see also haemolytic transfusion reactions (HTRs) reporting of, 290 septic shock, 287 thrombophlebitis, 288 transfusion-associated circulatory overload, 288 transfusion haemosiderosis, 288 transfusion-related acute lung injury see transfusion-related acute lung injury (TRALI) white cell antibodies, 286–287 cytomegalovirus-negative blood products, 465 donors see blood donation haemosiderosis, 288 haemovigilance, 289–292 infection transmission, 270–275, 271 Babesia microti, 274 bacteria, 270, 271, 274 see also specific bacteria Chagas disease, 274 Chikungunya fever virus, 274 cytomegalovirus, 273 dengue virus, 274 hepatitis viruses, 271–272, 274 HIV, 272–273 human T-cell leukaemia virus, 273 Leishmania donovani, 274 malaria, 274 parasites, 271 see also individual parasites prions, 271, 274 residual microbial risk, 275–276 SHOT, 290 syphilis, 274 viruses, 270, 271 see also specific viruses West Nile virus, 274 large volume, 466 massive transfusions see massive blood transfusions 999

Index

molecular blood grouping techniques, 242 neonatal ‘top-up’ transfusion, 282 recipient, 280–282 antenatal testing, 281 compatibility testing, 281 electronic cross-matching, 281 laboratory tests, 280–281 preoperative assessment, 280, 280 protection of, 270 repeated transfusions, 281 SHOT see SHOT storage, 276 therapy of acquired aplastic anaemia, 214–215 AIHA, 282 cold-type AIHA, 165 glucose-6-phosphate dehydrogenase deficiency, 153 myelodysplastic syndrome, 519 paroxysmal nocturnal haemoglobinuria, 180 sickle cell disease therapy, 121–122 β-thalassaemia, 92, 94 thalassaemia intermedia, 104–105 warm-type AIHA, 164 blood vessels, 752–755 endothelium, 753 platelet–vessel wall interaction, 753–754 structure, 752–753, 753 BMP4 haematopoiesis, 8 induced pluripotent embryonic stem cells, 9 BMP/SMAD signalling, hepcidin, 31 BNLI LY09 trial, classical Hodgkin lymphoma treatment, 649 Bohr effect, 84 Bombay phenotype, H antigen, 246–247 bone desferrioxamine, 57, 57 multiple myeloma pathogenesis, 582 sickle cell disease, 117–118 solid plasmacytoma, 584 bone infarction, sickle cell disease, 117 bone marrow AIHA, 161 anaplastic large-cell lymphoma, 574, 574 CLL, 536–537 fibrosis, primary myelofibrosis, 697 haematopoiesis, 3, 4 histology myelodysplastic syndrome, 513, 514 refractory anaemia with excess blasts in transformation, 514 refractory anaemia with ring sideroblasts, 514 1000

Hodgkin lymphoma, 643 malaria, 959 megaloblastic anaemia, 67, 67 multiple myeloma, 587 myelodysplastic syndrome, 513 paroxysmal nocturnal haemoglobinuria, 177 primary immune thrombocytopenia, 933, 933 thrombocytopenias, 916–917 WHO lymphoma classification, 615 bone marrow aspirates, 397–399 acquired aplastic anaemia, 213 acute lymphoblastic leukaemia, 397 adult ALL, 436 allogeneic SCT graft failure, 734 AML, 397, 398, 399, 400, 401 breast cancer, 942, 943 CLL, 534, 536, 536, 537 clonal hypereosinophilic syndromes, 705 CML, 397, 398, 490 cytopenia, 398 FISH, 398 haemophagocytic lymphohistiocytosis, 952 hairy cell leukaemia, 551 iron metabolism, 36, 38 leishmaniasis, 968, 968 mastocytosis, 702, 703 multiple myeloma, 397, 400 myelodysplastic syndrome, 397, 400 peripheral T-NHL not specified, 570 plasma cell dyscrasia, 606 polycythaemia vera, 401 primary myelofibrosis, 698–699 bone marrow transplants, 1 acquired aplastic anaemia, 209, 214–215 β-thalassaemia management, 96 bone marrow trephine biopsy, 399, 401 acquired aplastic anaemia, 213 acute lymphoblastic leukaemia, 401 acute megakaryoblastic leukaemia, 399, 401 allogeneic SCT graft failure, 734 AML, 401 amyloid, 604 CLL, 531, 534, 535, 536 CML, 490 essential thrombocythaemia, 694 hairy cell leukaemia diagnosis, 552 hepatosplenic lymphoma, 570 Hodgkin lymphoma, 399 mantle cell lymphoma, 556 mastocytosis, 702, 703 myelodysplastic syndrome, 515 NHL, 399, 531, 661 peripheral T-NHL not specified, 570

plasma cell dyscrasia assessment, 606 primary myelofibrosis, 699, 700 splenic marginal zone lymphoma, 555 T-NHL, 570 bone pain, multiple myeloma, 584 borderline diffuse large B-cell lymphomas and Burkitt lymphoma, 626 Bordetella pertussis infection, 363–364 Borrelia burgdorferi infections, 619 bortezomib Hodgkin lymphoma treatment, 652 mantle cell lymphoma, 556, 673 multiple myeloma, 593, 595, 595 systemic AL amyloidosis, 609 Waldenström macroglobulinaemia, 672 bosutinib, CML management, 494, 495 BPI see bacterial permeability-increasing protein (BPI) B-PLL see B-cell prolymphocytic leukaemia (B-PLL) BRAC2 mutations, Fanconi anaemia, 191 breast amyloidosis, 610 breast cancer bone marrow aspirates, 942, 943 risk, Hodgkin lymphoma treatment, 652 British Committee for Standards in Haematology (BCSH), polycythaemia vera diagnosis, 689, 689 British National Lymphoma Investigation (BNLI) follicular lymphoma, 663 relapsed Hodgkin lymphoma, 650 broad-spectrum antibiotics, fever of unknown origin, 474–475 bronchoalveolar lavage (BAL), 471, 472 Brucella infections, neutrophilia, 951 Brugia malayi infection see filariasis bruising, 853–854 blood donation, 270 CML, 488 haemophilia A, 795 non-accidental, 853 BSS see Bernard–Soulier syndrome (BSS) Budd–Chiari syndrome paroxysmal nocturnal haemoglobinuria, 178, 180 polycythaemia vera, 688 Burkitt lymphoma, 626, 662 blood film, 396, 396 cellular origins, 384 c-MYC gene, 384 cytogenetic analysis, 405 DLBCL vs., 412 Epstein–Barr virus infection, 656–657 FISH, 407

Index

geographic areas, 625 HIV infection, 625 immunohistochemistry, 404 phenotype–genotype correlations, 384 treatment, 679 tumour lysis syndrome, 479 WHO lymphoma classification, 625–626 burns, acquired haemolytic anaemias, 174 busulfan allogeneic SCT in myelodysplastic syndrome, 521 antiemetic effects, 481 chronic CML management, 496 cyclophosphamide with allogeneic SCT conditioning, 730 autologous SCT conditioning, 729 fludarabine with allogeneic SCT myeloablative conditioning, 730 SCT conditioning, 730 polycythaemia vera treatment, 690 C C1 complement protein, 234 C1-esterase inhibitor, 765 C1q complement protein, 234–235 Ig binding, 236 C1r complement protein, 234–235 C1s complement protein, 234–235 C3 complement protein, alternative pathway, 235 C3a complement protein, 283 C3b complement protein, 234, 284 C3 receptor mechanisms, AIHA, 160–161 C4b-binding protein (C4bBP), 766–767 C4 complement protein, 234 C5 complement protein, 235 C5a complement protein, 283, 310 C6 complement protein, 235 C7 complement protein, 235 C8 complement protein, 235 C9 complement protein, 235 CABG see coronary artery bypass graft (CABG) caeruloplasmin deficiency, iron refractory iron deficiency anaemia, 41 calcium, platelet second messenger pathways, 785 CALGB trial, 546 classical Hodgkin lymphoma treatment, 649 calpains, neutrophil apoptosis, 312 Campath-1H see alemtuzumab (Campath-1H) Campylobacter infections, 951 Campylobacter jejuni infection, extranodal marginal zone lymphoma of MALT, 619

CAMT see congenital amegakaryocytic thrombocytopenia (CAMT) cancer see malignancies Candida infections allogeneic SCT infections, 737 AML treatment, 422 leukaemias, 471 polyagglutinable red cells, Tk activation, 262 C antigen, Rh system, 253 c antigen, Rh system, 253 cap addition, globin gene processing, 87 carboxypeptidase, basophil/mast cell granules, 322 carcinoma, warm-type AIHA, 163 card agglutination test for trypanosomiasis (CATT), 966 cardiac amyloidoses, diuretics, 610 cardiac assessment, thalassaemia major, 95–96 cardiac death, Hodgkin lymphoma treatment, 652 cardiac disease autologous SCT complications, 741 systemic AL amyloidosis, 602 cardiac haemolytic anaemia, 170, 170–172 cardiac iron, iron-loading anaemias, 55, 55 cardiac surgery, massive transfusions, 289 cardiomegaly, American trypanosomias, 966 cardiopulmonary bypass (CPB) surgery, 851–852 cardiopulmonary post-perfusion syndrome, fragmentation haemolysis, 171 cardiovascular disease folate deficiency, 65 megaloblastic anaemia, 65 carmustine with etoposide, cytarabine and melphalan see BEAM (carmustine, etoposide, cytarabine and melphalan) mycosis fungoides treatment, 567, 682 Sézary syndrome, 567 carriers, β-thalassaemia management, 96 case cohort studies, CLL, 543 case–control studies, CLL, 543 Castleman disease, 680 catalase, 27 catastrophic antiphospholipid antibody syndrome, DIC vs., 844 cathepsin G neutrophils, 307 phagocytes, 306 cathepsin G-like molecule, basophil/mast cell granules, 322

causes, microangiopathic haemolysis anaemias, 171 CBL gene chronic myelomonocytic leukaemia, 526 myelodysplastic syndrome, 511–512 CC chemokines, 304, 359, 363 CCND1 gene mantle cell lymphoma, 556, 659 multiple myeloma, 578, 580 translocation, mantle cell lymphoma, 621 CCND1–IGHG1 fusion gene, FISH, 407 CCND2 gene, multiple myeloma, 580 CCND3 gene, multiple myeloma, 578, 580 Cc polymorphism, 255 CD1a, T-prolymphocytic leukaemia, 560 CD2 adult T-cell leukaemia lymphoma, 568 aggressive NK-cell leukaemia, 627 extranodal NK/T-cell lymphoma, nasal type, 628 large granular lymphocyte leukaemia, 562 peripheral T-NHL not specified, 570 Sézary syndrome, 566 T-prolymphocytic leukaemia, 560 CD3 enteropathy-associated T-cell lymphoma, 628 large granular lymphocyte leukaemia, 562 peripheral T-NHL not specified, 570 Sézary syndrome, 566 T-lineage ALL, 452 CD3ε aggressive NK-cell leukaemia, 627 extranodal NK/T-cell lymphoma, nasal type, 628 CD3 T-cells, 624 CD4 angioimmunoblastic T-cell lymphoma, 633 T-cell-dependent B-cell activation, 357 CD4 T-cells antigen recognition, 345 development, 351 functions, 353–354 HLA class II interaction, 712 maturation pathways, 358, 358 in spleen, 371 T-cell/histiocyte-rich large B-cell lymphoma, 624 CD5 adult T-cell leukaemia lymphoma, 568 B-cell prolymphocytic leukaemia, 616 CLL/small lymphocytic leukaemia, 614 1001

Index

DLBCL not otherwise specified, 622–623 mantle cell lymphoma, 555–556, 622 peripheral T-NHL not specified, 570 Sézary syndrome, 566 T-prolymphocytic leukaemia, 560 CD7 enteropathy-associated T-cell lymphoma, 628 peripheral T-NHL not specified, 570 T-lineage ALL, 452 T-prolymphocytic leukaemia, 560 CD8 T-cells acquired aplastic anaemia, 209 antigen recognition, MHC class I, 345, 345 cytokines, 353 development, 351 functions, 353–354 granzymes, 353 HLA class I interaction, 712 maturation pathways, 358 myelodysplastic syndrome, 507–508 perforins, 353 primary immune thrombocytopenia, 931, 931 CD10 angioimmunoblastic T-cell lymphoma, 633 B-cell ALL, 452 DLBCL not otherwise specified, 623, 623 follicular lymphoma, 620 plasma cell myeloma, 618 pre-B ALL, 451 CD11a/18, phagocytes, 302 CD11b see complement receptor 3 (CR3/ CD11b/CD18) CD11c/18, phagocytes, 302 CD14, 304 apoptotic cell receptor, 304 CD15 classical Hodgkin lymphoma, 641 early pre-B ALL, 451 CD18b see complement receptor 3 (CR3/ CD11b/CD18) CD19 B-cell ALL, 452 B cell development, 349 CLL, 534 early pre-B ALL, 451 mantle cell lymphoma, 555–556 multiple myeloma, 589 plasma cell myeloma, 618 pre-B ALL, 451 CD20 B-cell ALL, 452 CLL, 534, 537 FMC7 vs., 531 1002

hairy cell leukaemia diagnosis, 552, 552 mantle cell lymphoma, 555–556, 622 nodular lymphocyte-predominant Hodgkin lymphoma, 636, 642 plasma cell myeloma, 618 CD22 B-cell ALL, 452 early pre-B ALL, 451 hairy cell leukaemia diagnosis, 552 pre-B ALL, 451 CD23 B-cell prolymphocytic leukaemia, 616 CLL, 533, 537, 614 small lymphocytic leukaemia, 614 CD24, B cell development, 349 CD25 CLL, 533 regulatory T-cells, 359 CD27, CLL, 533 CD28, multiple myeloma, 589 CD30, classical Hodgkin lymphoma, 641 CD31, phagocytes, 302 CD34 acquired aplastic anaemia, 208 erythroid cell development, 18 SCT donor cells, 728–729 CD35 see complement receptor 1 (CR1/ CD35) CD36, platelets, 785 CD38, CLL, 540, 541 CD39, platelet inhibition, 787 CD40 T cell activation, 345–346 T-cell-dependent B-cell activation, 357 CD40L platelet α-granules, 774 T-cell-dependent B-cell activation, 357 CD43, mantle cell lymphoma, 622 CD44, 228 phagocytes, 302 CD45, cell reduction, autologous SCT, 744 CD45R haemopoietic stem cells, 2 nodular lymphocyte predominant Hodgkin lymphoma, 636 CD49a/29, phagocytes, 302 CD49e/29, phagocytes, 302 CD49f/29, phagocytes, 302 CD50, phagocytes, 302 CD51/61, phagocytes, 302 CD51, phagocytes, 302 CD52, plasma cell myeloma, 618 CD54, phagocytes, 302 CD55 see DAF (CD55) CD56 aggressive NK-cell leukaemia, 627

extranodal NK/T-cell lymphoma, nasal type, 573, 628 multiple myeloma, 589 plasma cell myeloma, 618 CD62E, phagocytes, 302 CD62L, phagocytes, 302 CD62P, phagocytes, 302 CD65, early pre-B ALL, 451 CD66, autologous SCT, 744 CD69, CLL, 533 CD71, CLL, 533 CD79α early pre-B ALL, 451 nodular lymphocyte predominant Hodgkin lymphoma, 636 pre-B ALL, 451 CD80 (B7.1), T cell activation, 345–346 CD86 (B7.2), T cell activation, 345–346 CD99, 228 CD102, phagocytes, 302 CD103, enteropathy-associated T-cell lymphoma, 628 CD106, phagocytes, 302 CD108, 228 CD117 (c-Kit) activating mutations, 387 deficiency, haematopoiesis, 6 erythroid cell development, 18 haemopoietic stem cells, 2 mast cell development, 321–322 mastocytosis, 701 multiple myeloma, 589 plasma cell myeloma, 618 CD147 see EMMPRIN (CD147) CD150 markers, haemopoietic stem cells, 2 CD163, 304 CD226, thrombus stabilization, 779 CD234 see Duffy blood group CD235A see glycophorin A (CD235A) CD235B see glycophorin B (CD235B) CD236 see glycophorin C (CD236); glycophorin D (CD236) CD237, regulatory T-cells, 359 CD238 see Kell blood group CD239 see Lutheran blood group CD242 see ICAM-4 (CD242) CDA see congenital dyserythropoietic anaemia (CDA) CDAN1 gene, congenital dyserythropoietic anaemia type I, 200 Cdc42, phagocyte signalling, 305 CDE, AIDS-related NHL, 681 CDH1 gene, hypermethylation, 388 CDKN2A gene adult T-cell leukaemia lymphoma, 568–569 multiple myeloma, 579–580

Index

CDKN2B gene hypermethylation, chronic myelomonocytic leukaemia, 526 multiple myeloma, 579–580 CDR3, B-cell receptor, 348–349 CDRs see complementarity determining regions (CDRs) CEBPA gene AML, 380 relapse risk, 427 myelodysplastic syndrome, 510 cell–cell adhesion receptors, phagocytes, 301 cell cycle control, leukaemogenesis, 392– 394, 393 cell differentiation antigens see under CD cell dose, HLA-matched sibling donor haematopoietic SCT, 221 cell-mediated immunity depression, CLL, 478 neutropenia, 316 red cell destruction, AIHA, 160 cell membrane B cell malignancy markers, 530–531 damage in sickle cell disease, 111–112 cell surface markers analysis, adult ALL diagnosis, 433–434 erythroid progenitor development, 14–15 erythropoiesis, 17–18 haemopoietic stem cells, 2 HSC development, 4–5 cell surface receptors erythroid cells, 22 see also specific receptors CEMPA gene, inactivating mutations, 387 central nervous system (CNS) African trypanosomiasis, 966 bleeding factor VII deficiency, 822–823 factor XIII deficiency, 829 haemophilia A, 795 lymphomas, immunosuppression, 658 prophylaxis adult ALL chemotherapy, 440 aggressive B-cell NHL treatment, 674 NHL treatment, 680–681, 681 central vein thrombosis, therapy, 880 central venous catheters (CVCs) fever of unknown origin, 475 venous thromboembolism, 890 venous thrombosis, 880 centroblasts, 356–357 centrocytes, 356–357 peptide processing, 357–358 cephalosporins, alloimmune haemolytic anaemia, 166

cerebral infarction, hereditary haemorrhagic telangiectasia, 854 cerebrospinal fluid (CSF) childhood ALL, 451 NHL, 662–663 ceroid lipofuscinosis, clinical features, 338 CFU-E cells, 2 erythroid cells, 15 CFU-G cells, 2 CFU-GEMM/Mix cells, 1–2 erythroid cells, 15 CFU-GM cells, 2 CFU-M cells, 2 CGD see chronic granulomatous disease (CGD) Chagas disease see American trypanosomiasis (Chagas disease) Charcot–Leyden crystals basophil/mast cell granules, 322 eosinophil granules, 318 Chédiak–Higashi syndrome, 923 genetics, 925 haemophagocytic lymphohistiocytosis, 326 LYST gene, 317 neutrophils, 311, 312, 317 chemical agents acquired haemolytic anaemias, 172–174 childhood ALL, 449 chemiluminescence, blood screening, HIV, 273 chemoattractant receptors eosinophils, 304 neutrophils, 304 see also specific molecules chemokine(s), 304, 359, 359, 363 see also specific chemokines chemokine receptors, neutrophils, 308 chemotaxins, phagocytes, 300 chemotherapeutic agents adult ALL see acute lymphoblastic leukaemia (ALL) advanced follicular lymphoma treatment, 664–665 AML treatment, 419–420 classical Hodgkin lymphoma treatment, 648, 650 fever, cause of, 469 Hodgkin lymphoma treatment, 645–647, 647, 652 lymphomatoid granulomatosis treatment, 680 mastocytosis treatment, 704 myelodysplastic syndrome therapy, 520–521 neutropenia, 313 NHL treatment, 664

radiotherapy combination, Hodgkin lymphoma treatment, 646 venous thromboembolism, 890–891 see also specific drugs chest radiography, venous thromboembolism diagnosis, 901 CHF see congestive heart failure (CHF) Chido–Rogers blood group, 245 Chikungunya fever virus, 274 children AML see acute lymphoblastic leukaemia (ALL), childhood ATG, 220 chronic CML management, 497 cobalamin deficiency, 71 Hodgkin lymphoma treatment, 651 myelodysplastic syndrome, 528 non-accidental bruising, 853 positive T-cell lymphoproliferative disorders, Epstein–Barr virus infection, 627, 627 positive T-cell NHL, Epstein–Barr virus infection, 573 primary immune thrombocytopenia management, 936–937 warm-type AIHA, 162 chimerism, HLA-matched sibling donor haematopoietic SCT, 222 CHL see classical Hodgkin lymphoma (CHL) Chlamydia infections extranodal marginal zone lymphoma of MALT, 619 sickle cell disease, 114, 116 chlorambucil CLL management, 545, 546 cold-type AIHA, 165 follicular lymphoma, 664 chloramphenicol, acquired aplastic anaemia, 208 chlorate, acquired haemolytic anaemias, 173 2-chlorodeoxyadenosine, follicular lymphoma treatment, 665 chloroquine, malaria treatment, 961–962 cholelithiasis see gallstones cholesterol embolism, 855 cholesterol ester storage disease, 338 cholesterol, red cell membrane, 227 chondroitin sulphate, basophil/mast cell granules, 322 CHOP (cyclophosphamide, doxorubicin, vincristine and prednisolone) adult T-cell leukaemia lymphoma, 570, 678 aggressive B-cell NHL, 674 AIDS-related NHL, 681 1003

Index

anaplastic large cell lymphoma, 678 B-PLL, 550 CLL, 546 follicular lymphoma, 665 mantle cell lymphoma, 673 multicentric Castleman disease, 680 peripheral T-cell lymphomas, 677 Sézary syndrome/mycosis fungoides, 567 Waldenström macroglobulinaemia, 672 chromogenic antithrombin heparin cofactor assay, 884, 884 chromosome(s) abnormalities AML, 415 Fanconi anaemia, 189, 189 myelodysplastic syndrome, 509 Sézary syndrome/mycosis fungoides, 567 HLA, 710 megaloblastic anaemia, 67 rearrangements leukaemogenesis, 384–385 molecular genetic analysis, 411 translocations FISH, 407 NHL, 657, 659 plasma cell myeloma, 618 somatic mutations, 385–386 chromosome 7 abnormalities, myelodysplastic syndrome, 509–510 chronic benign neutropenia, 315 chronic eosinophilic leukaemia, 500, 704 cytogenetic analysis, 406 FISH, 407 chronic granulocytic leukaemia see chronic myelogenous leukaemia (CML) chronic granulomatous disease (CGD) CYBB gene, 260 neutropenia, 317–318 chronic haemolytic anaemia, 127 chronic hypoxia, erythrocytosis, 691 chronic idiopathic neutropenia, 315 chronic idiopathic neutrophilia, 313 chronic intravascular haemolysis, Heinz bodies, 172–173 Chronic Leukaemia and Myeloma Task Force (CLMTF), multiple myeloma prognosis, 590 chronic liver disease, haemostatic disturbance, 848–849 chronic lymphocytic leukaemia (CLL), 530–557 cell-mediated immunity, 478 clinical features, 534 haematological features, 534 immune cytopenias, 535–536 1004

infections see below lymph nodes, 616 malignancies, 942, 944 monoclonal B-cell lymphocytosis, 543–544 polyclonal B-cell lymphocytosis, 544 red cell aplasia, 942 thrombocytopenia, 467 warm-type AIHA, 162 cytogenetic analysis, 405–406 diagnosis, 534, 615 blood film, 396, 397, 534, 534, 535 bone marrow aspirates, 534, 536, 536, 537 bone marrow examination, 536–537 bone marrow trephine biopsy, 531, 534, 535, 536 FISH, 407, 409 flow cytometry, 403, 532 white cell counts, 466 differential diagnosis, 542–543 B-cell prolymphocytic leukaemia vs., 550 NHL vs., 403 genetics BCL2 gene, 388 predisposition, 543 humoral immune response, 478 hypomethylation, 388 immunophenotype, 531 incidence, 531 infections, 478 laboratory features, 534 management, 544–550 alemtuzumab, 547 alkylating agents, 545 anthracycline-based therapy, 546 autoimmune complications, 548 BCL2 antisense oligonucleotides, 547 bendamustine, 547 chlorambucil, 545, 546 CHOP, 546 cladribine, 546 corticosteroids, 545–546 cyclophosphamide, 546 epratuzumab, 547 fludarabine, 546, 547 IWCLL response definition, 545 lumiliximab, 547 monoclonal antibodies, 546, 546 MRD assessment, 545 ofatumumab, 547 pentostatin, 546 purine analogues, 546 response criteria, 544–545 Richter transformation, 548–549 rituximab, 547

SCT, 547–548 secondary MDS/AML, 549–550 splenectomy, 547 spontaneous regression, 544 supportive care, 549 median age, 531 pathogenesis, 533–534 prognostic factors, 538–542 age, 538 biological, 540, 540 CD38, 541 cytogenetics, 538–540 gender differences, 542 gene expression profiling, 541 IGH chromosomal translocations, 540 IGHV mutational status, 538, 541, 541 median survival, 538 microRNAs, 541–542 therapy response, 538 ZAP-70, 541 prognostic stratification, 537–538 molecular genetic analysis, 411–412 score frequency, 533 staging systems, 537–538, 544 WHO lymphoma classification, 614–616 chronic mountain sickness, erythrocytosis, 692 chronic myelogenous leukaemia (CML), 483–502 adult ALL vs., 437 advanced phase management, 498–499 allogeneic SCT, 498 blastic transformation, 499 cytotoxic drugs, 499 aetiology, 483–484 basophils, 321 BCR–ABL1 chimeric gene, 385–386, 388, 483, 486, 486, 487 signal transduction pathways, 489 structure, 488 biphasic disease, 484 cell biology, 485–486 chronic phase management, 491–498 adults, 497–498 allogeneic SCT, 491, 496, 496–497 autografting, 498 busulfan, 496 children, 497 homoharringtonine, 496 hydroxycarbamide, 495 imatinib mesylate, 491–494 immunotherapy, 498 interferon alpha, 495–496 treatment decisions, 497–498, 498 tyrosine kinase inhibitors, second generation, 494–495

Index

classification, 484–485 clinical features, 488–489 cytogenetics, 405, 406, 486–487, 487 diagnosis blood film, 396, 398 bone marrow aspirates, 397, 398 white cell counts, 466 epidemiology, 483–484 genetics ABL1–BCR gene, 486, 486 BCR–ABL1 chimeric gene see above LTA gene, 388 Philadelphia chromosome, 483, 486 historical aspects, 483 incidence, 483 laboratory haematology, 489–491 advanced phase, 490, 490–491 biochemical changes, 491 blastic phase, 490 chronic phase, 489–490, 490 management advanced phase see above chronic phase see above SCT, 724 molecular biology, 487–488 natural history, 483–484 progenitors, 485 prognosis, 485, 485 Sokal prognostic index, 484 staging, 484, 484–485 triphasic disease, 484 variants, 499–501 Philadelphia-negative, 499 see also specific variants chronic myeloid leukaemia see chronic myelogenous leukaemia (CML) chronic myelomonocytic leukaemia (CMML), 499–500, 526–527 bone marrow histology, 514 KRAS gene, 391 NRAS gene, 391 splenomegaly, 499 chronic neutrophilic leukaemia (CNL), 500, 706–707 diagnosis, 706–707, 707 chronic non-spherocytic haemolytic anaemia, glucose-6-phosphate dehydrogenase deficiency, 152 chronic pancreatitis, cobalamin malabsorption, 73 chronic post-phlebitic symptoms, deep vein thrombosis, 888 chronic thromboembolic pulmonary hypertension, 913 Churg–Strauss syndrome, eosinophilia, 946 Chuvash polycythaemia, 691 chymase, basophil/mast cell granules, 322

ciclosporin acute GvHD therapy, 735 chronic GvHD therapy, 740 CLL, 548 with methotrexate and mycophenolate mofetil, 732 renal toxicity, 738 SCT reduced intensity conditioning regimens, 732 T-cell large granular lymphocyte leukaemia, 563 ciprofloxacin, infection prophylaxis, 473 cirrhosis see liver citrate-phosphate-dextrose-adenine (CPD-A) anticoagulant, blood donation, 269 c-Kit see CD117 (c-Kit) cladribine CLL management, 546 follicular lymphoma treatment, 665 hairy cell leukaemia treatment, 553 classical Hodgkin lymphoma (CHL), 640 histology, 640 subtypes, 640, 640 treatment, 647–648, 650 ABVD, 648 BEACOPP, 650 chemotherapy, 648, 650 randomized trials, 648 WHO lymphoma classification, 636, 636–637 classic complement pathway, 234, 234–235 class switching, Igs, 358 clear cells, peripheral T-cell lymphoma not otherwise specified, 631 CLEC-2, platelets, 785 clindamycin, malaria treatment, 962 clinically directed therapy, fever of unknown origin, 476 clinical penetrance, haemochromatosis, type 1, 50–51 clinical trials adult ALL therapy, 446 childhood ALL treatment, 457 see also specific trials CLL see chronic lymphocytic leukaemia (CLL) CLL5 trial, 546 CLN, 335 CLN 1, 335 CLN 2, 335 CLN 3, 335 CLN 4, 335 CLN 5, 335 CLN 6, 335 CLN 7, 335 CLN 9, 335

clodronate, multiple myeloma management/treatment, 585 clonal analysis, HSCs, 1–2 clonal hypereosinophilic syndromes, 704–706 clinical features, 705 investigations, 705–706 pathophysiology, 704–705 prognosis, 706 treatment, 706 clonality, leukaemogenesis, 381–382, 383 clonal selection theory, lymphocytes, 344–346 clopidogrel anti-platelet effects, 773 bleeding risks, 467 essential thrombocythaemia treatment, 696 resistance to, 790–791 Clostridium difficile infection, allogeneic SCT complications, 738 Clostridium perfringens infection, 951 acquired haemolytic anaemias, 169 polyagglutinable red cells, 262 clotting factors concentrates, haemophilia A treatment, 796–797 sickle cell disease, 119 CLOT trial, venous thromboembolism in cancer, 896 CMFB–MYH11 fusion gene, AML minimal residual disease, 428–429 CML see chronic myelogenous leukaemia (CML) CMML see chronic myelomonocytic leukaemia (CMML) CMT (combined modality treatment), classical Hodgkin lymphoma treatment, 647 c-MYC gene, Burkitt lymphoma, 384 CNL see chronic neutrophilic leukaemia (CNL) CNS see central nervous system (CNS) CNSHA see congenital non-spherocytic haemolytic anaemia (CNSHA) Coa antigen, Colton blood group, 261 coagulation see blood coagulation cobalamin, 68–76 absorption, 68–69 intrinsic factor, 68–69 tests for, 75–76 analogues, 70 dietary sources, 68 enterohepatic circulation, 69 folate relationship, 62 intracellular metabolism, 63 requirements, 68 serum measurement, 75 1005

Index

structure, 68, 68 synthesis, 68 transport, 69–70 cobalamin deficiency adults, 70 causes, 70–74, 71 dietary inadequacy, 70–71 drug-induced, 72, 74 food cobalamin absorption, 72 gastrectomy, 72 intestinal causes, 72–73 pernicious anaemia, 71–72 simple atrophic gastritis, 72 congenital, 73, 74 diagnosis, 74–75 drug-induced, 72, 74 epithelial surfaces, 64 HIV infection, 954 metabolism abnormalities, 74 neurological manifestations, 66 tests for cause, 75–76 tissue effects, 64–66 treatment, 80 see also megaloblastic anaemia cobalamin F disease, 335 clinical features, 338 Cob antigen, Colton blood group, 261 cobblestone-area forming cell (CAFC) assays, 2 CODOX-M/IVAC, AIDS-related NHL, 681 coeliac disease, acquired aplastic anaemia, 207 coenzymes, folate, 63 cold-acting antibodies AIHA, 159, 160 blood group antibodies, 233 cold-type autoimmune haemolytic anaemia, 164–165, 165 malignancies, 942 colistin with framycetin and nystatin (FRACON), 473 infection prophylaxis, 473 collagen(s) binding, von Willebrand disease diagnosis, 806 disorders, bruising, 854 platelet receptors, 781–782 thrombus formation, 777 type αI genes, β-thalassaemia modifiers, 92 vascular disorders, neutropenia, 316 collagen receptor defects genetics, 925 platelet responses, 922 colony-forming unit (CFU) assays, HSCs, 1–2 1006

colony-stimulating factor 1 (CSF-1), monocyte development, 323 Colton blood group, 245, 261 antigen see aquaporin 1 (Colton antigen) combined modality treatment (CMT), classical Hodgkin lymphoma treatment, 647 common acute lymphoblastic leukaemia (c-ALL), 434 comparative genomic hybridization (CGH), 412 myelodysplastic syndrome, 510–511 compatibility testing, blood transfusion recipients, 281 ‘compatible’ platelets, platelet transfusions, 466 complement, 233–236 activation, AIHA, 160 alternative pathway, 234, 235 blockade, paroxysmal nocturnal haemoglobinuria treatment, 181–182, 183 classic pathway, 234, 234–235 drug-induced alloimmune haemolytic anaemia, 166 extravascular haemolytic transfusion reactions, 284 fixation, Igs, 232 Ig binding, 232, 235–236 intravascular haemolysis, 161 lytic phase, 235–236 paroxysmal nocturnal haemoglobinuria, 178 receptors see complement receptor(s) see also specific proteins complementarity determining regions (CDRs) B-cell receptor, 348 CLL, 533 complement receptor(s) macrophages, 301 monocytes, 301 phagocytes, 301, 304 complement receptor 1 (CR1/CD35), 228, 303 interferon-γ effects, 303 phagocytes, 301 complement receptor 3 (CR3/CD11b/ CD18), 303 interferon-γ effects, 303 phagocytes, 301, 302 compound heterozygosity, δβ thalassaemia, 97–98 computed tomography (CT) CLL, 534 Hodgkin lymphoma, 643, 643, 645 mastocytosis, 702

NHL, 662 Richter transformation, 549 spiral, pulmonary embolism diagnosis, 902–903 spleen, 372, 373 systemic AL amyloidosis, 607 venous thromboembolism diagnosis, 902 conditional expression, haemoglobin, 85 conditioning regimens allogeneic SCT, 729, 730 autologous SCT, 729 sibling donor allogeneic SCT, 730–731 unrelated donor allogeneic SCT, 731 see also stem cell transplantation (SCT) congenital abnormalities, folate deficiency, 79 congenital amegakaryocytic thrombocytopenia (CAMT), 203, 204, 917 genetics, 925 congenital cobalamin deficiency, 74 congenital cobalamin malabsorption, 73 congenital cyanotic heart disease, erythrocytosis, 692 congenital dyserythropoietic anaemia (CDA), 187, 199–201 red cell aplasia, 973 type I, 199–200, 200, 200 type II, 200, 200, 201 type III, 200, 201, 201 congenital erythrocytosis, 691 congenital erythropoietic porphyria, 41–42 congenital intrinsic factor deficiency, 72 congenital methaemoglobulinaemia, 107 congenital methylmalonic acidaemia, 74 congenital methylmalonic aciduria, 74 congenital neonatal neutropenias, 979 congenital neutropenia, 202 congenital non-inherited thrombocytopenias, 916–917 congenital non-spherocytic haemolytic anaemia (CNSHA) glucose phosphate isomerase deficiency, 146 pyruvate kinase deficiency, 144 triose phosphate isomerase deficiency, 147 congenital thrombotic thrombocytopenic purpura, 862 congestive heart failure (CHF) folate deficiency, 79 iron deficiency anaemia, 40 Congo red staining, systemic AL amyloidosis, 604, 604 conjunctival telangiectasia, hereditary haemorrhagic telangiectasia, 854–855

Index

connective tissue disorders, 945–946, 946 anaemia, 945–946 blood coagulation disorders, 946 consolidation radiotherapy, classical Hodgkin lymphoma, 650 consolidation therapy adult ALL chemotherapy, 439–440 AML treatment, 423, 425 childhood ALL treatment, 457–458 constant region, Ig heavy chains, 231 continuous-flow autoanalysers, agglutination reactions, 241 contraceptives, oral see oral contraceptives Coombs (antiglobin) test agglutination reactions, 238–239 AIHA, 158 haemolytic disease of the fetus and newborn, 975 COPP, classical Hodgkin lymphoma, 650 copper deficiency, 81 coproporphyrinogen oxidase, haem synthesis, 35 core-binding factor complex AML, 391–392 structure, 391, 392 coronary artery bypass graft (CABG) haemostatic disturbances, 851 platelets, 851 corticosteroids acute GvHD, 735 antiphospholipid syndrome, 895 CLL, 545–546, 548 cold-type AIHA, 165 Diamond–Blackfan anaemia, 199 GvHD, infections, 477 haemophagocytic lymphohistiocytosis, 327 lymphopenia, 364 mastocytosis, 703 neutrophil development, 310 paroxysmal nocturnal haemoglobinuria, 180 primary immune thrombocytopenia, 933, 934 in pregnancy, 938 T-cell large granular lymphocyte leukaemia, 563 TTP, 867 warm-type AIHA, 163 CosmoFer (iron dextran), 41 co-trimoxazole allogeneic SCT infections, 736 infection prophylaxis, 473 Cotton glycoprotein, biological significance, 229 counselling, heritable thrombophilia, 885–886

CpG dinucleotides, myelodysplastic syndrome, 512 CR1 see complement receptor 1 (CR1/ CD35) CR3 see complement receptor 3 (CR3/ CD11b/CD18) cranial irradiation, childhood ALL treatment, 458 Cre-lox recombination, HSC development, 5 Crohn’s disease, cobalamin malabsorption, 73 Cromer blood group, 245 antigen, 227, 229 cryoglobulinaemia, essential mixed, 687 cryoprecipitate, 279 leukaemias, 467 cryoprecipitate-poor plasma (cryosupernatant), 279 cryopreservative side effects, SCT, 733 CSF see cerebrospinal fluid (CSF) CSF1R gene, myelodysplastic syndrome, 510 CT see computed tomography (CT) CTD (cyclophosphamide, thalidomide and dexamethasone), systemic AL amyloidosis management, 608–609 CTLA4, T-cell-dependent B-cell activation, 357 cubulin, cobalamin absorption, 69 cutaneous B-cell lymphoma, 683 cutaneous non-Hodgkin’s lymphoma, 682–683 CVAD, mantle cell lymphoma, 673 CVCs see central venous catheters (CVCs) CVP (cyclophosphamide, vincristine, prednisolone) follicular lymphoma, 665 Waldenström macroglobulinaemia, 672 Cw antigen, Rh system, 256 CX antigen, Rh system, 256 CXC chemokines, 304, 359, 363 CXCL13, angioimmunoblastic T-cell lymphoma, 633 CXCR4 haemopoietic stem cells, 3 overexpression, neutropenia, 316 platelets formation, 775 stem cell mobilization, 727 cyanocobalamin, cobalamin deficiency treatment, 80 CYBB gene chronic granulomatous disease, 317–318 Kell blood group, 260 cyclic neutropenia, 202, 314–315

cyclin D1 gene, mantle cell lymphoma, 393, 621 mantle cell lymphoma, 622, 622 cyclin D2, mantle cell lymphoma, 622 cyclin D3, mantle cell lymphoma, 622 cyclin-dependent cyclase inhibitors, multiple myeloma management/ treatment, 596 cyclin D genes, multiple myeloma, 580 cyclooxygenase deficiency, platelet signalling transduction disorders, 920 cyclophosphamide acquired aplastic anaemia, 220 adult ALL, 439 with bleomycin, etoposide, doxorubicin, vincristine, procarbazine and prednisone see BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine and prednisone) with busulfan see busulfan CLL, 546, 548 with dexamethasone, multiple myeloma management/treatment, 586 with doxorubicin, vincristine and prednisolone see CHOP (cyclophosphamide, doxorubicin, vincristine and prednisolone) Fanconi anaemia, 191 follicular lymphoma, 664 with mitoxantrone and fludarabine, follicular lymphoma therapy, 666 with rituximab, doxorubicin, vincristine and prednisolone see R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone) SCT conditioning, 726 allogeneic SCT, 521 with fludarabine and TBI, 731 with total body irradiation, 729, 730 Sézary syndrome/mycosis fungoides, 567 T-cell large granular lymphocyte leukaemia, 563 with thalidomide and dexamethasone, systemic AL amyloidosis management, 608–609 with total body irradiation allogeneic SCT myeloablative conditioning, 730 autologous SCT conditioning regimens, 729 with vincristine and prednisolone see CVP (cyclophosphamide, vincristine, prednisolone) warm-type AIHA, 163 1007

Index

cyclosporin, ATG with, 218–219 cystathionine β-synthase, deficiency, 879 cystinosis, clinical features, 338 cytarabine adult ALL chemotherapy, 439, 440 AML treatment, 420–421, 423 with carmustine, etoposide and melphalan see BEAM (carmustine, etoposide, cytarabine and melphalan) CML management, 499 with fludarabine and amsacrine (FLAMSA), allogeneic SCT in myelodysplastic syndrome, 521, 525 hepatotoxicity, 480 myelodysplastic syndrome, 520 cytochemistry, 401–402 acute lymphoblastic leukaemia, 401, 402, 403 acute monoblastic leukaemia, 402 AML, 401, 402, 403 childhood ALL, 451 cytochrome a, 27 cytochrome b, 27 cytochrome b1, iron absorption, 34 cytochrome c, 27 cytochrome oxidase, 27 cytogenetics, 405, 405–407 acquired aplastic anaemia, 213 acute lymphoblastic leukaemia, 406 adult ALL diagnosis, 434–435 prognosis, 443–444 adult T-cell leukaemia lymphoma, 568–569 AML see acute myeloid leukaemia (AML) Burkitt lymphoma, 405 childhood ALL see acute lymphoblastic leukaemia (ALL), childhood chronic eosinophilic leukaemia, 406 chronic myelomonocytic leukaemia, 526 CLL, 405–406, 538–540 CML, 405, 406, 486–487, 487 FISH, 405 multiple myeloma, 589 mycosis fungoides, 567 myelodysplastic 5q–syndrome, 407 myelodysplastic syndrome, 406, 508, 508–510 natural killer cell leukaemia, 406 NHL, 405, 658–659 plasma cell dyscrasia assessment, 606 Sézary syndrome, 567 T-prolymphocytic leukaemia, 560 WHO neoplasm classifications, 409 1008

cytokine(s), 359, 360–362 anaemia of chronic disease treatment, 941 CD8 T-cells, 353 Gaucher disease, 331, 332 non-haemolytic febrile transfusion reactions, 286 proinflammatory, 724 single-nucleotide polymorphisms, 720 T helper cells type 1, 358 T helper cells type 2, 358 T helper cells type 17, 358 see also specific cytokines cytokine receptors, neutrophils, 308 cytology primary immune thrombocytopenia, 933 splenic marginal zone lymphoma, 617 cytomegalovirus infection allogeneic SCT, 736–737, 738 AML, 424 infectious mononucleosis vs., 366 quantitative PCR, 471 screening allogeneic SCT, 477 blood donation, 275 transmission, blood transfusions, 273 cytopenia, bone marrow aspirates, 398 cytoreductive therapy polycythaemia vera treatment, 690 primary myelofibrosis treatment, 700 cytoskeleton phagocytes, 305 platelets, 773 cytotoxic drugs advanced CML, 499 myelodysplastic syndrome development, 504 cytotoxic T-cell precursor (CTLp) assay, HLA typing, 720 cytotoxic T-cells see CD8 T-cells D dabigatran, venous thromboembolism therapy, 905–906 dacarbazine, with doxorubicin, bleomycin and vinblastine see ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine) dactylitis, sickle cell disease, 113 DAF (CD55), 228 dalteparin, venous thromboembolism in cancer, 896 danaparoid, venous thromboembolism therapy, 904 Danon disease, 335 clinical features, 338

D antigen (Rh1) see Rhesus blood group dapsone acquired haemolytic anaemias, 173 malaria treatment, 962 dasatinib, CML management, 494, 495 DAT see direct agglutination test (DAT) daunorubicin AML treatment, 420–421 CML management, 499 myelodysplastic syndrome, 520 DC, telomerase gene complex, 211 DDAVO, acquired von Willebrand disease therapy, 858 DDAVP see desmopressin (DDAVP) decitabine, myelodysplastic syndrome, 523 deep vein thrombosis (DVT) chronic post-phlebitic symptoms, 888 diagnosis MRI, 903 ultrasound, 902 incidence, 888 morbidity, 888 prophylaxis, 908–909 recurrence risk, 888 thalidomide side effects, 596 see also pulmonary embolism (PE) defensins neutrophils, 307 phagocytes, 306–307 deferasirox, 54 acquired aplastic anaemia, 215 Diamond–Blackfan anaemia, 199 iron-loading anaemias, 59 structure, 56 β-thalassaemias, 95 deferiprone, 54 acquired aplastic anaemia, 215 iron-loading anaemias, 53, 57–58 structure, 56 β-thalassaemias, 95 degranulation, phagocytes, 305 δ-storage pool disorder, platelet responses, 922 dementia, plasma homocysteine, 66 dendritic cells, 324 antigen presentation, 345 neoplasms, treatment, 679 Dengue fever, 969 blood transfusion transmission, 274 dense granules, platelets, 774 deoxycoformycin see pentostatin (deoxycoformycin) deoxycoformycin (pentostatin), follicular lymphoma treatment, 665 deoxythymidine monophosphate (dTMP), megaloblastic anaemia, 61 deoxyuridine monophosphate (dUMP), megaloblastic anaemia, 61

Index

deoxyuridine triphosphate (dUTP), megaloblastic anaemia, 61–62 dermatan sulphate, 764 desferrioxamine (DFX), 54 acquired aplastic anaemia treatment, 215 acute iron poisoning, 59 haemochromatosis, type 1, 51 iron-loading anaemias, 53, 56–57 structure, 56 thalassaemia intermedia, 59 β-thalassaemias, 95 desirudin, venous thromboembolism therapy, 905 desmopressin (DDAVP) congenital platelet disorder therapy, 924 factor V and factor VIII combined deficiency, 821 haemophilia A treatment, 798–799 haemostatic disturbances in renal disease, 851 von Willebrand disease treatment, 809 developed countries, childhood ALL, 448 developmental growth factor signalling pathways, haematopoiesis, 8 dexamethasone childhood ALL treatment, 457, 458 with cyclophosphamide and thalidomide, systemic AL amyloidosis management, 608–609 with cyclophosphamide, multiple myeloma management/ treatment, 586 erythroid cell development, 22 multiple myeloma management/ treatment, 586, 588 systemic AL amyloidosis management, 608 with vincristine, doxorubicin see VAD (vincristine, doxorubicin, dexamethasone) DFX see desferrioxamine (DFX) D gene segments, B-cell receptor, 348 Dia antigen, Diego blood group, 261 diabetes insipidus, diabetes mellitus, optic atrophy and deafness (DIDMAOD), sideroblastic anaemia, 44 diabetes mellitus anaemia, 948 thrombosis in inflammation, 898 diacylglycerol, platelet second messenger pathways, 785–786 dialysis folate prophylaxis, 81 long-term, folate deficiency, 79

Diamond–Blackfan anaemia, 187, 197–199 cell/molecular biology, 198–199 clinical features, 197–198 family incidence, 198 genetic subtypes, 198–199, 199 myelodysplastic 5q–syndrome, 509 neonatal anaemia, 972 treatment, 199 diarrhoea allogeneic SCT complications, 738 leukaemias, 471 diarrhoea-related haemolytic-uraemic syndrome, 868–869 Dib antigen, Diego blood group, 261 DIC see disseminated intravascular coagulation (DIC) DIDMAOD (diabetes insipidus, diabetes mellitus, optic atrophy and deafness), sideroblastic anaemia, 44 Diego blood group, 245, 261 antigen, 229 diet cobalamin, 68 deficiency, 70–71 iron absorption, 34 iron deficiency anaemia, 39 diethylcarbamazine, filariasis treatment, 964–965 diffuse alveolar haemorrhage, allogeneic SCT complications, 739 diffuse large B-cell lymphomas (DLBCLs), 623 CLL/small lymphocytic leukaemia, 616 differential diagnosis Burkitt lymphoma vs., 412 primary mediastinal B-cell lymphoma vs., 412 gene expression profiling, 412 hepatitis C virus, 687 immunoblastic morphology, 624 immunohistochemistry, 404–405 intestine, 676 not otherwise specified, 622–624 with predominant extranodal location, 624–625 Richter transformation, 548 of stomach, 676 subtypes, 623 WHO lymphoma classification, 622– 624, 624–625 dilute Russell’s viper venom time (DRVVT), antiphospholipid syndrome, 893, 894 2,3-diphosphoglycerate (2,3-DPG) accumulation, pyruvate kinase deficiency, 142–143

neonatal anaemia, 972 sickle cell disease, 110 2,3-diphosphoglycerate mutase deficiency, 146 Diphyllobothrium latum infection, cobalamin malabsorption, 73 dipyridamole, essential thrombocythaemia treatment, 696 direct agglutination test (DAT), 238 AIHA, 158 babesiosis, 962 blood group screening, 238 CLL, 535–536 drug-induced alloimmune haemolytic anaemia, 167 haemolytic disease of the fetus and newborn, 295 repeated blood transfusions, 281 Rh D typing, 255 direct Coombs test, TTP, 863 direct thrombin inhibitors, venous thromboembolism therapy, 905–906 disseminated intravascular coagulation (DIC), 841–845 acute promyelocytic leukaemia, 467, 945 antithrombin, 764 associated conditions, 842 see also specific conditions clinical features, 843 diagnosis, 843–844 diarrhoea-related haemolytic-uraemic syndrome, 868 differential diagnosis, 843–844, 844 TTP vs., 865 heparin cofactor II, 764 HIV infection, 955 infections, 951 malaria, 959 malignancies, 942, 944–945 megaloblastic anaemia, 67–68 neonates, 981 pathophysiology, 841, 842, 843 purpura fulminans, 882 scoring system, 844, 844 treatment, 844–845 diuretics, induced neutropenia, 316 divalent metal transporter 1 (DMT1), 28 DKC1 gene dyskeratosis congenita, 193 reticular dysgenesis, 315 DLBCLs see diffuse large B-cell lymphomas (DLBCLs) DLI see donor lymphocyte infusion (DLI) DMT1, 29 iron absorption, 34 mutations, iron refractory iron deficiency anaemia, 41 1009

Index

DMT1hemojuvelin, 29 DNA analysis systemic AL amyloidosis, 605–606 systemic amyloidosis, 612 DNA damage Fanconi anaemia, 189 in utero, childhood ALL, 449 DNA methylation myelodysplastic syndrome, 512 somatic mutations, 387–388 DNA repair, Fanconi anaemia, 189 DNA replication, folate, 77 DNA synthesis, folate, 64 Doa antigen, 261 Dob antigen, 261 Döhle bodies, 918 Dombrock blood group, 245, 261 antigen, 227 Dombrock glycoprotein, 228 Donath–Landsteiner autoantibody, 252 donor, blood transfusions see blood transfusion(s) donor lymphocyte infusion (DLI) allogeneic SCT relapse management, 743–744 CML, 497 graft-versus-leukaemia effect, 724 donor matching, haematopoietic SCT, 223 donor registries, 742–743 dormancy, haemopoietic stem cells, 6 double strand DNA breaks, chromosome translocations, 386 Down syndrome congenital neutropenias, 979 neutrophilia, 313 transient abnormal myelopoiesis, 707–708 doxorubicin AML treatment, 421 with bleomycin, vinblastine and dacarbazine see ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine) with cyclophosphamide, vincristine and prednisolone see CHOP (cyclophosphamide, doxorubicin, vincristine and prednisolone) with rituximab, cyclophosphamide, vincristine and prednisolone see R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone) with vincristine, dexamethasone see VAD (vincristine, doxorubicin, dexamethasone) doxycycline, malaria treatment, 962 2,3-DPG see 2,3-diphosphoglycerate (2,3-DPG) 1010

D—, Rh system, 257 drug(s) acquired aplastic anaemia, 208 contraindications, glucose-6-phosphate dehydrogenase deficiency, 153 see also specific drugs drug-induced acute haemolysis, 152 drug-induced alloimmune haemolytic anaemia, 166–167, 167 drug-induced cobalamin deficiency, 72, 74 drug-induced neutropenia, 316 drug-induced thrombocytopenias, 916 drug-induced warm-type autoimmune haemolytic anaemia, 162–163 drug-resistance phenotype, multiple myeloma, 581 DRVVT (dilute Russell’s viper venom time), antiphospholipid syndrome, 893, 894 D segment, Igs, 347–348 dTMP (deoxythymidine monophosphate), megaloblastic anaemia, 61 Duffy blood group, 245, 260 antigen (CD234), 228, 229 Plasmodium vivax binding, 229 red cell membrane, 227 malaria infection, 959–960 dUMP (deoxyuridine monophosphate), megaloblastic anaemia, 61 Duncan syndrome see X-linked lymphoproliferative syndrome duodenal cytochrome b1, 29 dUTP (deoxyuridine triphosphate), megaloblastic anaemia, 61–62 DVT see deep vein thrombosis (DVT) dysfibrinogenaemia haemostatic disturbance in chronic liver disease, 848 renal disease, 947–948 dyskeratosis congenita, 187, 192–196 cell/molecular biology, 193–195 autosomal dominant, 194 autosomal recessive, 195 DKC1 gene, 193 fibroblasts, 193 genetic subtypes, 195 HUMARA gene, 193 NOP10 gene, 195 reverse transcriptase, 194 telomerase, 194 telomerase complex, 194, 194–195 TERC gene, 194 TERT gene, 194 X-chromosome inactivation patterns, 193 clinical features, 192, 192–193 premature ageing, 195

Fanconi anaemia vs., 193 mortality, 192 somatic abnormalities, 193 treatment, 195–196 dysmegakaryopoiesis, primary myelofibrosis, 698 E E2A–PBX1 (TCF3–PBX1) gene rearrangements, childhood ALL, 453, 454 E antigen, Rh system, 253 e antigen, Rh system, 253 early pre-B acute lymphoblastic leukaemia, immunophenotypic classification, 451 early T-precursor acute lymphoblastic leukaemia, 434 EATL see enteropathy-associated T-cell lymphoma (EATL) EBF gene, childhood ALL, 450 EBMT see European Group for Blood and Marrow Transplantation (EBMT) Ebola virus, 969 echinocytosis, liver disease, 948 ECG, systemic AL amyloidosis, 607 echocardiography clonal hypereosinophilic syndromes, 705–706 sickle cell disease, 116 ‘economy class syndrome,’ 889 eculizumab, paroxysmal nocturnal haemoglobinuria, 181–182 effector cells, SCT, 722–723 eflornithine, African trypanosomiasis therapy, 966 Ehlers–Danlos syndrome (EDS), 854 EKLF see erythroid Kruppel-like factor (EKLF) EKLF gene globin gene clusters, 86 Lutheran blood group, 259 ELA2 gene cyclic neutropenia, 314–315 neutropenias, 202, 314 elastase, neutrophils, 307 elderly patients AML treatment, 430 ATG, 220 multiple myeloma management/ treatment, 593–594, 594–595 NHL, 683 electrical charge, agglutination tests, 237–238 electrocardiography (ECG), systemic AL amyloidosis, 607

Index

electrolyte balance acute renal tubular necrosis, 479–480 leukaemia, 479–480 electron beam irradiation mycosis fungoides treatment, 682 Sézary syndrome/mycosis fungoides, 567 electronic cross-matching, 281 electron microscopy, systemic AL amyloidosis, 604 ELISA see enzyme-linked immunosorbent assay (ELISA) elliptocytosis, hereditary see hereditary elliptocytosis (HE) eltrombopag, congenital platelet disorder therapy, 924 elution tests, blood group screening, 239–240 EMA, nodular lymphocyte predominant Hodgkin lymphoma, 636 Embden–Meyerhof pathway see red cell metabolism embryonic haemoglobin, 83 embryonic sites, haematopoiesis, 5–6 EMMPRIN (CD147), 228 ENA078, platelet α-granules, 774 endocrine disorders, 948, 948 erythrocytosis, 693 endoglin, haemopoietic stem cells, 2 endoplasmic reticulum, HLA class I antigen processing/presentation, 711 endothelial cells activation, DIC, 843 anticoagulant activity, 755 erythropoiesis, 12 fibrinolytic factors, 755 thrombosis in inflammation, 898 tissue factor pathway inhibitor, 763 endothelial protein C receptor, 759, 766 endothelin-1 (ET-1), 754 endothelium blood vessels, 753 tissue factor, 760 vessel injury, 755 von Willebrand factor, 753 see also endothelial cells endotoxin neutrophil development, 310 thrombosis in inflammation, 897 ENG gene, 854 enhancer sequences, haemoglobin, 86 Enterobacter infection, leukaemias, 472 enterohepatic circulation cobalamin, 69 folate, 76 enteropathy-associated T-cell lymphoma (EATL), 573–574

treatment, 677–678 WHO lymphoma classification, 628, 629 environmental factors acquired aplastic anaemia, 207–208 heritable thrombophilia, 880 primary immune thrombocytopenia, 929 enzyme(s) cycling in lysosomes, 331 Gaucher disease type I, 332 pyruvate kinase deficiency, 144–145, 145 enzyme-linked immunosorbent assay (ELISA) antiphospholipid syndrome, 894–895 Aspergillus infection, 471 blood group screening, 240, 240 blood screening HIV, 273 human T-cell leukaemia virus, 273 filariasis diagnosis, 964 venous thromboembolism diagnosis, 901–902 enzyme replacement therapy (ERT) Fabry disease, 332, 341 Gaucher disease, 332, 338–340 Gaucher disease type II, 338–339 lysosomal storage disorder therapy, 336 mucopolysaccharidosis, 332 Niemann–Pick disease, 341 Pompe disease, 332 EORTC 20884 trial classical Hodgkin lymphoma, 650 classical Hodgkin lymphoma treatment, 649 EORTC H8-F, classical Hodgkin lymphoma treatment, 648 eosin-labelled maleimide binding, hereditary spherocytosis, 131–132 eosinophil(s), 318–321 activation, 319 chemoattractant receptors, 304 CML, 490 development, 318–319 disorders, 319–321 see also specific diseases/disorders eotaxin, 304 function, 318–319 granules, 318–319, 319 myeloproliferative disorders, 389 recruitment, 319 structure, 308 eosinophilia, 319–321 causes, 320, 705 Churg–Strauss syndrome, 946 definition, 319

filariasis, 964 idiopathic, 704 malignancies, 944 polyarteritis nodosa, 946 rheumatoid arthritis, 946 systemic disease, 946 systemic lupus erythematosus, 946 eosinophilia–myalgia syndrome, 319–320 eosinophilic fasciitis, acquired aplastic anaemia, 207 eosinophilic leukaemia, chronic see chronic eosinophilic leukaemia eosinophil myeloproliferative disorders, FGFR1 gene, 389, 704 eotaxin, 363 eosinophils, 304 EPCR gene, 766 ephrins, thrombus stabilization, 779 epigenetic effects, somatic mutations, 387–388 epistaxis factor V deficiency, 818 factor X deficiency, 825 epithelial surfaces cobalamin deficiency, 64 folate deficiency, 64 epitopes, 229 EPO see erythropoietin (EPO) EPOCH, AIDS-related NHL, 681 EpoR see erythropoietin receptor (EpoR) epratuzumab, 666 childhood ALL treatment, 459 CLL management, 547 follicular lymphoma therapy, 667 Epstein–Barr virus infection CD21, 364–365 extranodal NK/T-cell lymphoma, nasal type, 628 Hodgkin lymphoma, 639 large-cell lymphomas of terminally differentiated B cells, 625 NHL, 656, 656–657 positive T-cell lymphoproliferative disorders of childhood, 627, 627 positive T-cell NHL in childhood, 573 Richter transformation, 549 screening, allogeneic SCT, 477 see also infectious mononucleosis; posttransplant lymphoproliferative disease (PTLD) Epstein syndrome, 918, 918 genetics, 925 Erdheim–Chester disease, 327 ERMAP, 228 error-related incidents, SHOT, 290 ERT see enzyme replacement therapy (ERT) 1011

Index

erythroblasts iron deficiency anaemia, 39 in vitro development, 15 erythrocytes see red cell(s) erythrocyte sedimentation rate (ESR), sickle cell disease, 119 erythrocytapheresis see exchange transfusion (erythrocytapheresis) erythrocytosis, 690–693, 691 abnormal erythropoietin secretion, 693 congenital, 691 endocrine disorders, 693 idiopathic, 693 inherited/congenital, 691 primary, 691 secondary, 691–693 systemic hypoxia, 691–693 chronic hypoxia, 691 congenital cyanotic heart disease, 692 high-affinity haemoglobin, 692, 692 high altitudes, 691–692 hypopnoea, 691 methaemoglobulinaemia, 692–693 red cell metabolic defects, 692 tobacco smoking, 693 see also polycythaemias erythroid cells cell surface receptors, 22 development, erythropoietin receptor, 18 iron absorption, 34–35 erythroid dysplasia, bone marrow histology, 516 erythroid Kruppel-like factor (EKLF) erythropoiesis, 15, 16 haemoglobin synthesis regulation, 86–87 erythromycin allogeneic SCT infections, 736 post-splenectomy, 935 erythropoiesis, 12–25, 13, 14 apoptosis, 22–23 cell surface markers, 17–18 definition, 12 developmental origins, 12–13 diseases/disorders, 23–24 see also specific diseases/disorders gene expression, 18, 18–19 homeostasis, 12 inhibition, anaemia of chronic disease, 941 myelodysplastic syndrome, 513 polycythaemia vera, 686 regulation, 23 signal pathway regulation, 19–22 hypoxia sensing, 19–20, 20 see also erythropoietin (EPO); erythropoietin receptor (EpoR) 1012

sites, 369 terminal maturation, 16–17 β-thalassaemias, 91, 92 transcription factors, 12, 15–16 erythropoietic porphyrias, 42 congenital, 41–42 erythropoietin (EPO), 12, 20–22 anaemia therapy, 468 anaemia of prematurity, 978 in malignancies, 943 dyskeratosis congenita treatment, 195 erythrocytosis, 693 gene structure, 20 mechanism of action, 21 myelodysplastic syndrome therapy, 468, 519–520 production measures, 24 structure, 20 β-thalassaemias, 91, 93 erythropoietin receptor (EpoR), 20–22 apoptosis, 22 erythroid cell development, 18 expression, 19 hypoxia, 19 JAK2-STAT5 pathway, 21, 21, 22 PI3-K/Ras-MAP kinase pathway, 21, 22 structure, 21 ESAM, thrombus stabilization, 779 Escherichia coli infections, 951 leukaemias, 472 sickle cell disease, 114 splenectomy risks, 375 Escherichia coli type 0157:H7 infections, 868 ESR (erythrocyte sedimentation rate), sickle cell disease, 119 essential mixed cryoglobulinaemia, hepatitis C virus, 687 essential thrombocythaemia (ET), 693–697 clinical features, 694 diagnosis, 695, 897 epidemiology, 694 investigations, 694–696 JAK2 gene, 694 pathophysiology, 693–694 in pregnancy, 696–697 prognosis, 696 thrombosis, 896–897 treatment, 696 estrogen erythroid cell development, 22 haemostatic disturbances in renal disease, 851 estrogen receptor gene, β-thalassaemia modifiers, 92 ET see essential thrombocythaemia (ET)

etoposide AML treatment, 423 with bleomycin, cyclophosphamide, doxorubicin, vincristine, procarbazine and prednisone see BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine and prednisone) with carmustine, cytarabine and melphalan see BEAM (carmustine, etoposide, cytarabine and melphalan) Sézary syndrome/mycosis fungoides, 567 ETV6–RUNX (TEL–AML1) gene fusion acute lymphoblastic leukaemia, 391 childhood ALL, 448–449, 450, 452, 454, 455 ETV–PDGFRB gene fusion, chronic myelomonocytic leukaemia, 499 European Group for Blood and Marrow Transplantation (EBMT) multiple myeloma prognosis, 590 NHL SCT, 668–669 European Organisation for Research and Treatment of Cancer (EORTC) risk factors, Hodgkin lymphoma prognosis, 644, 644 trials see under EORTC Euro score, CML prognosis, 485, 485 EV1I gene, CML, 488 Evans syndrome TTP vs., 865 warm-type AIHA, 162 exchange transfusion (erythrocytapheresis) megaloblastic anaemia therapy, 80 Rhesus haemolytic disease of the fetus and newborn, 297 sickle cell disease, 121–122 see also plasma exchange exogenous plasminogen activator, 769 exoglycosidase, basophil/mast cell granules, 322 explant culture experiments, haematopoiesis, 7 expression profiling, myelodysplastic syndrome, 510 extramedullary disease, AML, 415 extramedullary haemopoiesis, spleen, 371–372 extramedullary plasmacytoma, 584 extranodal marginal zone lymphoma of MALT, 619 WHO lymphoma classification, 618–619 extranodal NK/T-cell lymphoma, nasal type, 572–573, 573

Index

treatment, 678 WHO lymphoma classification, 628, 628 extrapulmonary haemopoiesis, primary myelofibrosis, 697–698 extravascular haemolysis drug-induced alloimmune haemolytic anaemia, 166 eculizumab effects, 182 extravascular red cell destruction, haemolytic transfusion reactions, 284, 284–286 F F2G20210A, 878 detection, 885 heritable thrombophilia, 872, 873 F5G1691A, heritable thrombophilia, 872, 873 F5 gene, 877 F7 gene, factor VII deficiency, 822, 823, 824, 825 F13A gene factor XIII deficiency, 829–831, 830 polymorphisms, 831 F13B gene, factor XIII deficiency, 831 FAB (French–American–British) classification adult ALL, 433 AML, 416, 418 blood film, 396 CML, 491 myelodysplastic syndrome, 504, 504–505 FA-BCRA pathway, Fanconi anaemia, 190 Fabry disease, 334, 341 clinical features, 337 diagnosis, 332 enzyme replacement therapy, 332 factor I, developmental haemostasis, 980 factor II deficiency, knockout mouse model, 815 developmental haemostasis, 980 haemostatic disturbance in chronic liver disease, 848 factor V, 758, 761 deficiency, 818, 821 acquired, 858 genetics, 820 knockout mouse model, 815, 818 in pregnancy, 834 prophylaxis, 833 treatment, 833 developmental haemostasis, 980 factor VIII combined deficiency see factor V and factor VIII combined deficiency platelet α-granules, 774

factor Va generation, 877 inhibition see protein C pathway factor V and factor VIII combined deficiency, 821 genetics, 814 in pregnancy, 834 prophylaxis, 833 treatment, 833 factor V Leiden see FVR506Q (factor V Leiden) factor VII, 758, 760 blood coagulation, 749 in chronic liver disease, 848 deficiency, 821–823, 825 clinical manifestations, 822–823 genetics, 822 knockout mouse model, 815 molecular defects, 823, 825 in pregnancy, 834 prophylaxis, 834 treatment, 822–823, 833 developmental haemostasis, 980 in pregnancy, 851 factor VIII, 758, 761 antibody development, 799 assays for, 796 concentrates, 796–797 deficiency see haemophilia A developmental haemostasis, 980 factor V combined deficiency see factor V and factor VIII combined deficiency genetics, 800, 801 heritable thrombophilia, 878 porcine, 799 von Willebrand disease diagnosis, 806 von Willebrand factor complex, 805, 805–806 factor VIIIa, inhibition see protein C pathway factor IX, 758, 760 blood coagulation, 749 deficiency see haemophilia B developmental haemostasis, 980 heritable thrombophilia, 878 factor X, 758, 760 blood coagulation, 749 chronic liver disease, 848 deficiency, 825–826 amyloidosis, 856 knockout mouse model, 815 in pregnancy, 834 prophylaxis, 834 treatment, 833 developmental haemostasis, 980

factor Xa antagonists, venous thromboembolism therapy, 906 factor XI, 758, 760–761 concentrate, factor XI deficiency therapy, 827 deficiency, 748, 827 knockout mouse model, 815 in pregnancy, 835 prophylaxis, 834 treatment, 833 heritable thrombophilia, 878 factor XII deficiency, 748 developmental haemostasis, 980 factor XIII, 758, 761 deficiency, 827–831 classification, 829 clinical manifestations, 829 knockout mouse model, 815, 829 molecular defects, 829–831 neonates, 981 in pregnancy, 835 prophylaxis, 834 treatment, 829, 833 developmental haemostasis, 980 structure, 827–828 synthesis, 827–829 factor H replacement, familial haemolyticuraemic syndrome, 869 FAD (flavine adenine dinucleotide), nitric oxide synthase, 306 fainting, blood donation, 270 familial acholuric jaundice see hereditary spherocytosis (HS) familial cold urticaria and leucocytosis, 313 familial haemolytic-uraemic syndrome, 869 family history CLL, 543, 543 Diamond–Blackfan anaemia, 198 haemophilia A, 795 platelet congenital disorders, 915 systemic AL amyloidosis, 606 family testing, haemochromatosis, type 1, 51 FANC1, Fanconi anaemia, 189, 191 FANCD2, Fanconi anaemia, 189 Fanconi anaemia, 186–192, 187 cell/molecular biology, 189–191 BRAC2 mutations, 191 chromosomal abnormalities, 189, 189 complementation groups/subtypes, 190 DNA damage, 189 DNA repair, 189 FA-BCRA pathway, 190, 191 FANC1, 189, 191 1013

Index

FANCD2, 189 genes, 189 interferon-γ, 191 MAPK pathways, 191 mouse models, 191 PALB2 mutations, 191 proteins, 189, 191 see also specific proteins in vitro gene transfer, 191 clinical features, 186–187, 188, 189, 212 leukaemias, 187, 189 malignancy, 186 somatic abnormalities, 187 diagnosis, 214 dyskeratosis congenita vs., 193 incidence, 187 thrombocytopenias, 917 treatment, 191–192 haematopoietic SCT, 189, 191 Farber disease, 334 clinical features, 337 fasciitis, eosinophilic, 207 Fas-ligand, myelodysplastic syndrome, 508 Fas receptor, apoptosis in erythropoiesis, 23 fat embolism syndrome, 855 favism, glucose-6-phosphate dehydrogenase deficiency, 150, 151–152 Fc receptor(s) AIHA, 160 Ig binding, 232 natural killer, 354 phagocytes, 301 see also specific receptors FcαR, phagocytes, 303 FcεIR basophils, 321 mast cells, 321 FcγRI, phagocytes, 303, 303 FcγRIIA, platelets, 785 FcγRII, phagocytes, 303, 303 FcγRIIIA, phagocytes, 303 FcγRIIIB, phagocytes, 303 FcγRIII, phagocytes, 303, 303 Fc fragment, Ig, 231 FCM (fludarabine, cyclophosphamide, mitoxantrone), follicular lymphoma therapy, 666 febrile reactions leukaemias, 467 neutrophil antibodies, 263 Felty syndrome, neutropenia, 316, 946 femoral head, avascular necrosis, 115, 117–118 Ferrinject, iron deficiency anaemia management, 41 1014

ferritin, 26, 27 genetics, 27 heavy chain, 30 iron homeostasis, 33 light chain, 30 measurement, iron status, 37 serum haemochromatosis, type 1, 49–50 iron-loading anaemias, 54 iron metabolism measurement, 36 ferrochelatase, haem synthesis, 35 ferrokinetics, myelodysplastic syndrome, 513 ferroportin (SLC40A1), 28 ferroportin 1, 29 ferrous sulphate, iron deficiency anaemia management, 40 fertility, allogeneic SCT complications, 740–741 fetal–adult switch, haemoglobin synthesis, 84–85 fetal anaemia, anti-D antibodies, 293 fetal genotype prediction, Rh system, 258–259 fetal haemoglobin (HbF), 83, 971, 972, 972 at birth, 85 oxygen affinity, 83 Rhesus haemolytic disease of the fetus and newborn, 294, 296 β-thalassaemias, 91 β-thalassaemia, with sickle cell disease, 100 fetal liver, haematopoiesis, 5 Fetchner syndrome, 918, 918 genetics, 925 fetomaternal haemorrhage flow cytometric immunophenotyping, 404 neonatal anaemia, 977, 978 fetus, haematopoiesis, 7, 7–8 fever, infections in leukaemia, 469–470 fever of unknown origin, leukaemias see leukaemias, supportive care 18 -F-fluorodeoxyglucose positron emission tomography (FDG-PET), Hodgkin lymphoma, 643, 644 FFP see fresh frozen plasma (FFP) FGA gene, fibrinogen deficiency, 815, 816, 817 FGB gene, fibrinogen deficiency, 815, 817 FGFR1 gene chronic myelomonocytic leukaemia, 526 clonal hypereosinophilic syndromes, 705 eosinophil myeloproliferative disorders, 389 FGFR3 gene, multiple myeloma, 579

FGFR3 tyrosine kinase inhibitors, multiple myeloma management/ treatment, 596 FGFR/MMSET gene, multiple myeloma, 578 FGG gene, fibrinogen deficiency, 815, 817 fibril identification, systemic AL amyloidosis, 604–605 fibrin generation, 750 plasmin, action of, 768 fibrin D-dimers, venous thromboembolism diagnosis, 901 fibrinogen, 758, 761–762 chronic liver disease, 848 deficiency, 815–816 genetics, 817 knockout mouse model, 815 in pregnancy, 834 prophylaxis, 833 treatment, 833 developmental haemostasis, 980 heparin interaction, 764 malaria, 959 platelets, 772, 774 in pregnancy, 851 thrombus formation, 778 fibrinolysis, 751, 751, 767–770 components, 767 see also specific components factor XI deficiency, 827 heritable thrombophilia, 879 inhibitors, 769–770 plasminogen, 751 fibrinolytic factors, endothelial cells, 755 fibroblasts, dyskeratosis congenita, 193 fibronectin, thrombus formation, 778 filariasis, 962–965 biology, 962–963 clinical presentation, 963 diagnosis, 964 epidemiology, 962–963 geographical distribution, 962–963, 963 haematological abnormalities, 964 treatment implications, 964–965 fine-needle aspiration (FNA), NHL, 659 FIPIL1–PDGFRA gene fusion AML, 385 chronic eosinophilic leukaemia, 500, 704 clonal hypereosinophilic syndromes, 705 eosinophilia, 320 FISH, 407 FIPL1 gene, eosinophilia, 704 First-Line Indolent Trial (FIT), 668 FISH see fluorescence in situ hybridization (FISH) 5′ untranslated region, haemoglobin, 85

Index

FLAMSA (fludarabine, cytarabine and amsacrine), allogeneic SCT in myelodysplastic syndrome, 521, 525 flavine adenine dinucleotide (FAD), nitric oxide synthase, 306 flavine mononucleotide (FMN), nitric oxide synthase, 306 FLI1 gene, Paris–Trousseau syndrome, 919, 925 Flice inhibitory protein (FLIP), myelodysplastic syndrome, 508 FLIP (Flice inhibitory protein), myelodysplastic syndrome, 508 flow cytometry acquired aplastic anaemia, 213 adult T-cell leukaemia lymphoma, 569 agglutination reactions, 241 ALL, 402 childhood minimal residual disease, 455 AML, 403 minimal residual disease, 429 blood group screening, 240 CLL, 403, 532, 541, 542, 543 fetomaternal haemorrhage, 404 haemopoietic stem cells, 2 hairy cell leukaemia, 403 immunophenotyping, 402–404, 404 monoclonal antibody therapy, 403 monoclonal T-cell populations, 403–404 multiple myeloma, 403 myelodysplastic syndrome, 404, 508 NHL, 403 hepatosplenic T-NHL, 572 paroxysmal nocturnal haemoglobinuria, 179, 179, 180, 403–404 platelet function testing, 789 T-cell large granular lymphocyte leukaemia, 564 FLT3 gene activating mutations, 387 AML, 392 relapse risk, 426–427, 427 childhood ALL, 449 myelodysplastic syndrome, 510 fluconazole allogeneic SCT infections, 737 infection prophylaxis, 473–474 fludarabine CLL management, 546, 547 with cyclophosphamide, mitoxantrone, follicular lymphoma therapy, 666 follicular lymphoma treatment, 665 mantle cell lymphoma, 556 myelodysplastic syndrome, 520 Richter transformation, 549

SCT conditioning, 223, 726 with busulfan, 732 busulfan with, 730 with cyclophosphamide, 732 with cyclophosphamide and TBI, 731 with cytarabine and amsacrine (FLAMSA), 521, 525 with melphalan, 732 myelodysplastic syndrome, 521 total body irradiation with, 732 fluid balance acute renal failure, 479 acute renal tubular necrosis, 479 leukaemia, 478–479 polysaccharide solutions, 479 fluid replacement therapy, 292 adult ALL, 437 fluorescence in situ hybridization (FISH), 407–409 AML, 409 BCL2–IGH fusion gene, 407 BCR–ABL fusion gene, 407, 408 bone marrow aspirates, 398 Burkitt lymphoma, 407 CCND1–IGHG1 fusion gene, 407 chromosomal translocations, 407 chronic eosinophilic leukaemia, 407 CLL, 407, 409, 531, 539, 539, 540 cytogenetic analysis, 405 FIP1L1–PDGFRA fusion gene, 407 follicular lymphoma, 407 imatinib mesylate monitoring, 493 mantle cell lymphoma, 407, 556 multiple myeloma, 578–579 MYC–IGHG1 fusion gene, 407 myelodysplastic 5q–syndrome, 509 myelodysplastic syndrome, 507 spectral karyotyping, 407 fluoroquinolones, infection prophylaxis, 473 FLVCR, 29 FMC7, hairy cell leukaemia diagnosis, 552 FMN (flavine mononucleotide), nitric oxide synthase, 306 FMS-like tyrosine-3 (FLT-3), AML, 415–416 FOG-1, erythropoiesis, 15 folate, 76–79 absorption, 76 biochemical functions, 62, 63, 77 coenzymes, 63 DNA replication, 77 DNA synthesis, 64 RNA replication, 77 body stores, 76 cobalamin relationship, 62 dietary, 76 enterohepatic circulation, 76

haemolytic disease of the fetus and newborn, 975 megaloblastic anaemia, 62 prophylaxis (supplements), 80–81 anaemia of prematurity, 978 congenital dyserythropoietic anaemia treatment, 201 dialysis, 81 haemolytic anaemia, 81 hereditary spherocytosis, 132 prematurity, 81 sideroblastic anaemia treatment, 45 supplements in pregnancy, 64–65, 78, 81 requirements, 76 pregnancy, 953 serum, folate deficiency diagnosis, 79 structure, 76, 76 transport, 77 folate deficiency cardiovascular disease, 65 causes, 77, 77–79 antifolate drugs, 79 congenital abnormalities, 79 congestive heart failure, 79 excess utilization/loss, 78–79 haematological disorders, 78 homocystinuria, 79 inflammatory conditions, 78–79 liver disease, 79 long-term dialysis, 79 malabsorption, 78 nutritional, 77–78 pregnancy, 78 premature infants, 78 CLL, 535 diagnosis, 79 epithelial surfaces, 64 malignancies, 65, 943 neural tube defects, 64–65 neurological manifestations, 66 pregnancy complications, 64 scurvy, 78 sickle cell disease, 113 tissue effects, 64–66 treatment, 80 see also megaloblastic anaemia folate receptors, 77 folinic acid (5-formyl-THF), 80 follicles, lymph nodes, 355, 355 follicular lymphoma, 554, 621, 663–670 advanced disease, 663–664 chemotherapy, 664–665 interferon, 665 monoclonal antibodies, 665–667, 666 purine analogues, 665 rituximab, 665–666 1015

Index

BCL2 gene overexpression, 394 blood film, 554 chemotherapy, 664–668 antibody conjugates, 667 immunotoxins, 668 maintenance therapy, 666–667 previously untreated, 666 radioimmunoconjugates, 667, 667–668 relapsed disease, 668 relapsed low-grade, 666 therapy-naive patients, 668 cytology, 660 FISH, 407 geographical variations, 655–656 immunohistochemistry, 404 localized disease, 663 adjuvant chemotherapy, 663 radiotherapy, 663 prognostic factors, 662 transformed, treatment, 669 WHO lymphoma classification, 619–621 fondaparinux heparin-induced thrombocytopenia, 891 venous thromboembolism therapy, 904 food cobalamin absorption, 72 four T’s algorithm, heparin-induced thrombocytopenia, 891 FOXP1 gene, extranodal marginal zone lymphoma of MALT, 619 FoxP3, regulatory T-cells, 359 FRACON (framycetin, colistin and nystatin), infection prophylaxis, 473 fragmentation acquired haemolytic anaemias, 170, 170, 170–172 framycetin, with colistin and nystatin (FRACON), infection prophylaxis, 473 frataxin, 29 French–American–British classification see FAB (French–American–British) classification fresh frozen plasma (FFP), 279 DIC treatment, 845 factor V and factor VIII combined deficiency, 821 factor VII deficiency therapy, 823 factor X deficiency, 825 factor XI deficiency therapy, 827 familial haemolytic-uraemic syndrome, 869 haemostatic disturbances in pregnancy, 851 leukaemias, 467 massive blood loss, 853 1016

preparation, 276 prion disease, 276 prothrombin deficiency, 816 TTP treatment, 867 vitamin K-dependent coagulation factors deficiency, 832 Friedreich ataxia, 53 pathophysiology, 35 fructose diphosphate aldose A, 147 deficiency, 146, 147 FTL3 mutation AML classification, 430–431 AML relapse risk, 425 fucosidosis, 334 clinical features, 337 functional iron, iron metabolism measurement, 37 fungal infections allogeneic SCT infections, 737 AML treatment, 422 CLL, 478 haematological disorders, 950–952 haemophagocytic lymphohistiocytosis, 326 infections in leukaemia, 469 see also specific infections furosemide, extravascular haemolytic transfusion reactions, 284 Fusarium infection, leukaemias, 471 fusion genes, acute lymphoblastic leukaemia, 385 FUT1 gene, 250 FUT2 gene, 250 FUT3 gene, 250 FUT genes, 246 FVR506Q (factor V Leiden), 877 activated partial thromboplastin time, 877 activated protein C, 876 detection, 885 FX gene, 825, 826 FXI gene, 827, 828 Fya antigen, 260 Fyb antigen, 260 G G6PD see glucose-6-phosphate dehydrogenase (G6PD) galactosialidosis, 335 clinical features, 338 gallstones Gaucher disease, 338 hereditary spherocytosis, 132 paroxysmal nocturnal haemoglobinuria, 178 pyruvate kinase deficiency, 143, 144 gametocytes, malaria life cycle, 958

G antigen, Rh system, 256 Gardos pathway, sickle cell disease, 110 gastrectomy, cobalamin deficiency, 72 gastric biopsy, pernicious anaemia diagnosis, 72 gastrin, pernicious anaemia, 71 gastrin receptor antibodies, antibodies to, 72 gastrointestinal system allogeneic SCT complications, 737–738 autologous SCT complications, 741 deferiprone side effects, 58 GvHD, 735 mastocytosis, 702 GAT (granulocyte agglutination test), 264 GATA-1 apoptosis, erythropoiesis, 23 congenital erythropoietic porphyria, 41 erythropoiesis, 15–16 globin gene clusters, 86 primary myelofibrosis, 697 GATA1 gene grey platelet syndrome, 918 myelodysplastic syndrome, 510 GATA-2 erythropoiesis, 15, 16 haematopoiesis, 7–8 Gaucher disease, 334, 336–371 bone marrow, 340 clinical course, 340 clinical features, 336, 337 gallstones, 338 skeletal changes, 340, 340–341 diagnosis, 332 serum Igs, 338 tissue biopsy, 338 GBA gene, 336 β-glucocerebrosidase, 336 laboratory features, 336, 338 pathophysiology, cytokines, 331 prevalence, 331 thrombocytopenias, 917 treatment, 338–341 enzyme replacement therapy, 332, 338–340 haematopoietic SCT, 332 supportive therapy, 340–341 Gaucher disease type I, 336 clinical features, 338 cytokines, 332 enzymes, 332 Gaucher disease type II, 336 clinical features, 338 enzyme replacement therapy, 338–339 Gaucher disease type III, clinical features, 338 GBA gene, 336

Index

GCNT2 gene, 252 G-CSF see granulocyte colony-stimulating factor (G-CSF) GCSFR gene, 510 GDF-15, hepcidin regulation, 32 GELA H89 trial, classical Hodgkin lymphoma, 649, 650 gelatinase, neutrophils, 310 gemcitabine angioimmunoblastic T-NHL, 572 Hodgkin lymphoma treatment, 652 gemtuzumab ozogamicin (Mylotarg) acute promyelocytic leukaemia, 430 AML therapy, 423, 431 childhood ALL treatment, 459 gender differences, CLL, 542 gene(s) competition, haemoglobin synthesis regulation, 86 defects, hereditary spherocytosis, 131–132 deletions δβ thalassaemia, 98, 98 β-thalassaemias, 89 α thalassaemias, 100, 102 environment interactions heritable thrombophilia, 873–874 leukaemogenesis, 380–381 expression, erythropoiesis, 18, 18–19 mutations glucose-6-phosphate dehydrogenase deficiency, 149–150 molecular genetic analysis, 411 rearrangements, T-cell receptor, 347, 351 see also specific genes gene expression profiling (GEP), 412 acquired aplastic anaemia, 209 CLL, 541 DLBCL, 412 DLBCL not otherwise specified, 623 peripheral T-NHL not specified, 570 primary mediastinal B-cell lymphoma, 412 gene therapy dyskeratosis congenita treatment, 196 Fanconi anaemia treatment, 191 haemophilia A, 803 haemophilia B, 803 platelet congenital disorders, 925–926 sickle cell disease therapy, 123–124 genetic counselling, sickle cell disease, 120 genetic markers, heritable thrombophilia, 878 genetic predisposition acquired aplastic anaemia, 211 leukaemogenesis, 380–381

genotype–phenotype relationship rare bleeding disorders, 832 α thalassaemias, 102–103 gentamicin, vancomycin and nystatin (GVN), infection prophylaxis, 473 GEP see gene expression profiling (GEP) Gerbich blood group, 245, 261 antigen, 227, 229 German High-Grade NHL Study Group, aggressive treatment, 674 germinal centres B cell-like DLBCL, 623 lymph nodes, 355–356 germline mutations, chromosome translocations, 386 Gf-1, 16 Gf-1b, 16 GFI1 gene, 202 GGCX gene combined deficiency of vitamin K proteins, 814 vitamin K-dependent coagulation factors deficiency, 832 GHSG HD7 trial, 648 GHSG HD9 trial, 649 GIEMMA trial, 594 GIL antigen see aquaporin 3 (GIL antigen) Gilbert syndrome, 106 haemolysis with, 127 Gill blood group, 245 Glanzmann thrombasthenia, 788, 921, 922 genetics, 925 platelet responses, 922 Gleevec see imatinib mesylate Glivec see imatinib mesylate globins expression, 18–19 gene promoter mutations, hereditary persistence of fetal haemoglobin, 100 synthesis disorders, 83–108 see also specific diseases/disorders haem synthesis coordination, 19 thalassaemias, 88 globoid cell leucodystrophy, clinical features, 337 Globoside blood group, 245 GLRX5 gene mutations, 44 Glucksberg staging, acute GvHD, 735, 735 β-glucocerebrosidase, Gaucher disease, 336 glucocorticoids, childhood ALL treatment, 457 glucose-6-phosphate dehydrogenase (G6PD), 149–153 genetics, 149

pentose phosphate pathway, 140 reactions, 149 structure, 149, 149 glucose-6-phosphate dehydrogenase deficiency, 149–153 acute haemolytic crises, 151, 151 chronic granulomatous disease, 317–318 chronic non-spherocytic haemolytic anaemia, 152 clinical features, 150 drug contraindications, 153 drug-induced acute haemolysis, 152 epidemiology, 149–150, 151 favism, 150, 151–152 genetics, 150 gene mutations, 149–150 laboratory diagnosis, 152–153 management, 153 neonatal anaemia, 976 neonatal jaundice syndrome, 150–151, 151 phototherapy, 151 Plasmodium falciparum malaria resistance, 150 WHO classification, 150 glucose phosphate isomerase, 146–147 deficiency, 146–147 glucose phosphate isomerase deficiency, 146 GLUT-1, 228 γ-glutamyl carboxylase (GGCX), 831 γ-glutamylcysteine synthetase, glutathione synthesis, 153, 154 glutaredoxin-5 mutations, sideroblastic anaemia, 44 glutathione cycle, red cell metabolism see red cell metabolism glutathione deficiency, 154 glutathione peroxidase, 154 glutathione reductase, 154 glutathione S-transferase, 455 glutathione synthetase, 153, 154 glutatredoxin-5, haem synthesis, 35 gluten-induced enteropathy folate deficiency, 78 iron deficiency anaemia, 40 glyceraldehyde-3-phosphate dehydrogenase deficiency, 146 glycogen storage diseases, 335 clinical manifestations, 337 type II see Pompe disease (glycogen storage disease type II) type 7, 147 glycolytic pathway see red cell metabolism glycophorin(s) red cell membrane, 227 see also specific types 1017

Index

glycophorin A (CD235A), 228 erythroid cell development, 18 red cell membrane, 129, 130 glycophorin B (CD235B), 228 red cell membrane, 129, 130 glycophorin C (CD236), 228 red cell membrane, 129, 130 glycophorin D (CD236), 228 glycoproteinoses, 330 see also specific diseases/disorders glycoproteins ABH antigens, 248, 249 red cell membrane, 227 glycosaminoglycans, amyloidosis, 600–601 glycosylation, ferritin, 27 glycosylphosphatidylinositol (GPI) anchor acquired aplastic anaemia, 209 CML, 485–486 paroxysmal nocturnal haemoglobinuria, 176 red cell membrane, 129 GM1 gangliosidosis, 334 clinical features, 337 GM2 gangliosidosis, 334 clinical features, 337 GM3 gangliosidosis, 334 GM-CSF see granulocyte-macrophage colony-stimulating factor (GM-CSF) gout, polycythaemia vera, 689 GP1BA gene Mediterranean macrothrombocytopenia, 925 pseudo von Willebrand disease, 925 GP1BB gene, 918 GPIa/IIa antigen, primary immune thrombocytopenia, 930 GPIbβ gene, Bernard–Soulier syndrome, 920, 925 GPIb/IX antigen, primary immune thrombocytopenia, 930 GPIb–IX–V receptor complex Bernard–Soulier syndrome, 920, 925 platelet receptors, 780–781 GPIIb/IIIa antigen, primary immune thrombocytopenia, 930 GPIV, primary immune thrombocytopenia, 930 GPIX gene, Bernard–Soulier syndrome, 920, 925 G protein-coupled receptors, platelet receptors, 783–784 GPVI platelet receptors, 779, 780, 781, 781–782 primary immune thrombocytopenia, 930 1018

graft failure, allogeneic SCT complications, 733–734 graft rejection HLA-matched sibling donor haematopoietic SCT, 221, 222 HLA typing in SCT, 723 treatment, 222 graft-versus-host disease (GvHD), 734–735 acute, 724 allogeneic SCT, 734–735 immunological basis, 724, 725 allogeneic SCT acute, 734–735 AML, 424 myelodysplastic syndrome, 521 blood transfusions, 287 chronic, 739–741, 740 classification, 740 SCT, 724, 725 corticosteroid therapy, infections, 477 HLA-matched sibling donor haematopoietic SCT, 222 HLA matching, 716, 723 multiple myeloma allogeneic SCT, 593 prophylaxis, SCT conditioning regimens, 731 SCT, 467–468 allogeneic see above umbilical cord, 728 skin, 734, 734–735 graft-versus-host (GVH) response, allogeneic SCT, 722 graft-versus-leukaemia (GVL) effect, 722, 724 allogeneic SCT ALL, 441 AML, 424 CML, 497 myelodysplastic syndrome, 521, 522 granules eosinophils, 318–319, 319 mast cells, 322 neutrophils, 308–309, 310 thrombus formation, 777 granulocyte(s) concentrates, 278–279 cross-matching, 278–279 direct donations, 278 function tests, myelodysplastic syndrome, 513 infusions, leukaemias, 477–478 spleen, 370 transfusions, acquired aplastic anaemia, 215 granulocyte agglutination test (GAT), 264 granulocyte colony-stimulating factor (G-CSF) adult ALL, 438

AML treatment, 422 anaemias, 468 ATG, 218 dyskeratosis congenita treatment, 195 haematopoiesis, 3 haematopoietic SCT donor dosing, 221 imatinib mesylate co-administration, 492 leukaemia therapy, 477–478 multiple myeloma, 587 myelodysplastic syndrome therapy, 520 neutrophil apoptosis, 312 neutrophil development, 308 SCT, 743 stem cell mobilization, 727 granulocyte immunofluorescence test (GIFT), 264 granulocyte-macrophage colonystimulating factor (GM-CSF), 361 AML treatment, 422 dyskeratosis congenita treatment, 195 macrophage activation, 323 neutrophil development, 308 granulocytopenia malignancies, 944 multiple myeloma, 587, 588 granulocytosis, malignancies, 943–944 granulopoiesis, 308 myelodysplastic syndrome, 513 granzymes, CD8 T-cells, 353 grey platelet syndrome (GPS), 918 Griscelli syndrome, haemophagocytic lymphohistiocytosis, 326 GRO-α, 363 GRO-β, 363 GRO-γ, 363 growth/development delay as allogeneic SCT complications, 740–741 sickle cell disease, 113 growth differentiation factor 15 (GDF-15), 28 growth factors anaemia therapy, 468 haemopoietic see haemopoietic growth factors leukaemias, 477–478 myelodysplastic syndrome therapy, 519–520 neutrophilia, 313 polycythaemia vera, 686 growth monitoring, thalassaemia major, 95 growth programme, Epstein–Barr virus infection, 656 GSTM1 gene, 455 GSTT1 gene, 455

Index

gut investigations, infections in leukaemia, 471 GVN (gentamicin, vancomycin and nystatin), infection prophylaxis, 473 gynaecological problems, factor X deficiency, 825 GYPA gene, MNS blood group, 259 H haemangioblast, 4, 4 erythropoiesis, 12 haemarthroses Bernard–Soulier syndrome, 920 factor V deficiency, 818 factor XIII deficiency, 829 haematinics, anaemia management, 292 haematocrit, TTP, 863 haematological neoplasms laboratory investigation, 395–414 WHO classification, 413–414 see also specific tests/investigations see also specific neoplasms haematology folate deficiency, 78 splenectomy, 377–378 haematomas, factor V deficiency, 818 haematopoiesis, 1–11 acquired aplastic anaemia, 208–209 adult sites, 3 aorta–gonad–mesonephros, 7 embryonic sites, 5–6 regeneration/replacement therapy, 8–9 supportive environments, 6–8 adult bone marrow, 6–7 in fetus, 7, 7–8 transcription factors, 2 haematopoietic growth factors (HGFs), acquired aplastic anaemia, 209 haematopoietic stem cell transplantation (HSCT) see stem cell transplantation (SCT) haematoxylin and eosin (H&E) staining, systemic AL amyloidosis, 604 haematuria haemophilia A, 794 sickle cell disease, 117 haemochromatosis, 47–53 classification, 47, 48 HFE gene, 47 HLA-A*03 association, 713 neonatal, 52 type 1 see below type 2, 47, 48, 52 type 3, 47, 48, 52 type 4, 47, 48, 52

haemochromatosis protein, 29 haemochromatosis, type 1, 47–51, 48 associated conditions, 51 causes, 48 clinical diagnosis, 49 clinical penetrance, 50–51 family testing, 51 hepcidin synthesis, 48 HFE mutations, 47, 48–49 frequency, 48–49 iron status, 49 morbidity, 49 iron status, 49–50 population screening, 51 treatment, 51 haemodialysis, familial haemolytic-uraemic syndrome, 869 haemogenic endothelial cells, SCT, 8–9 haemoglobin(s), 12–13, 27–32, 83–108 α-globin chains, 85 multispecies consensus regions, 85–86 analysis, β-thalassaemias, 93 Bart’s, α thalassaemias, 100–102 β-globin chains, 85 β-locus control region, 85 Bethesda, erythrocytosis, 692 breakdown of, 36 degradation, 127 electrophoresis myelodysplastic syndrome, 513 sickle cell disease, 119, 120 embryonic, 83 fetal see fetal haemoglobin (HbF) genetics conditional expression, 85 enhancer sequences, 86 5’ untranslated region, 85 poly-A addition sites, 85 promoter region, 85 splicing acceptor sites, 85 splicing donor sites, 85 Gower 1, 83, 972 Gower 2, 83, 972 Gun Hill, 106 HbA, 972 sickle cell disease, 110 HbA2, 972 HbC, β-thalassaemia, with, 96–97 HbE, β-thalassaemia, with, 97 HbF see fetal haemoglobin (HbF) HbH, α thalassaemias, 100–102 HbS blood donation, 268–269 malaria infection, 960 β-thalassaemia, with, 96 Helsinki, erythrocytosis, 692

iron metabolism measurement, 37 Kansas, 107 measurement, blood transfusion donor, 269–270 neonatal haematology, 972 Portland, 83, 972 San Diego, erythrocytosis, 692, 692 structure, 27, 83 synthesis, 84–87 autonomous gene silencing, 86 fetal–adult switch, 84–85 gene competition, 86 genetic control, 84, 84–87 mRNA transcription/processing, 86, 87 regulation, 84–87 translation, 87 variants β-thalassaemia associations, 96–97 see also specific variants haemoglobin Bart’s hydrops syndrome, 103 haemoglobin disorders, 88 classification, 87–88 congenital methaemoglobulinaemia, 107 high-oxygen affinity variants, 107 low-oxygen affinity variants, 107 splenomegaly, 372 structural variants, 106, 106–107 see also specific diseases/disorders haemoglobinopathies, neonatal anaemia, 976–977 haemoglobinuria, immediate haemolytic transfusion reactions, 284 haemoglobulinaemia, 284 haemolysis, 126–127 blood group screening, 239 cold-type AIHA, 164 drug-induced, 152 extravascular see extravascular haemolysis intravascular see intravascular haemolysis paroxysmal nocturnal haemoglobinuria, 177 sickle cell disease, 111 haemolytic anaemias acquired see acquired haemolytic anaemias chronic, 127 clinical features, 127 CLL, 535 congenital non-spherocytic (CNSHA) see congenital non-spherocytic haemolytic anaemia (CNSHA) folate prophylaxis, 81 TTP, 863 1019

Index

haemolytic disease of the fetus and newborn (HDFN), 232, 292–298, 974–975 ABO incompatibilities, 293, 297 anti-c antibodies, 297 anti-K antibodies, 297 blood film, 975 clinical features, 293 maternal alloantibodies, 292–293 maternal O blood group, 247 prevention, 297–298 maternal anti-D antibody, 298 Rh incompatibilities, 293–297 blood group testing, 295 disease severity assessment, 294 exchange transfusion, 297 fetal haemoglobin concentrations, 294, 296 maternal blood assessment, 294 premature delivery, 295 severity assessment in newborn, 295–297 haemolytic transfusion reactions (HTRs), 282–286 antibodies, 284 delayed, 286 extravascular red cell destruction, 284, 284–286 immediate, 283–286 investigations, 285 intravascular red cell destruction, 283–284 repeated blood transfusions, 281 haemolytic–uraemic syndrome (HUS), 868–869 historical aspects, 860 HIV infection, 955 variants, 868 haemopexin, haemolysis, 127 haemophagocytic lymphohistiocytosis, 326–327, 952–953 associated conditions, 952 bone marrow aspirate, 952 Chédiak–Higashi syndrome, 326 diagnosis, 327 juvenile rheumatoid arthritis, 952 haemophagocytic syndrome, acquired haemolytic anaemias, 169–170 haemophilia A, 748, 793–801 acquired, 856, 856–858 associated disease, 857 care organization, 803–804 clinical features, 794–795 clinical severity, 794 in females, 801 gene therapy, 803 inhibitor development, 799–800 immune tolerance, 800 1020

investigation, 795–796 laboratory diagnosis, 796 molecular genetics, 800, 801 mutations, 802, 802 neonates, 980 pathophysiology, 793 presentation, 795 prophylaxis, 798 treatment, 796–799 clotting factor concentrates, 796–797 complications, 799–800 desmopressin, 798–799 guidelines for, 797 haemorrhagic episode prevention, 797 haemophilia B, 748, 802–803 diagnosis, 802 gene therapy, 803 inheritance, 802 inhibitor development, 803 neonates, 980 treatment, 802–803 guidelines for, 797 Haemophilus influenzae type B infection allogeneic SCT infections, 736 multiple myeloma, 588 post-splenectomy, 935 sickle cell disease, 114 spleen, 371 splenectomy risks, 133, 375, 376 Haemophilus pneumoniae infections, 114 haemopoiesis developmental, 971 extrapulmonary, primary myelofibrosis, 697–698 megaloblastic anaemia, 67–68 suppression, HIV infection, 954 haemopoietic growth factors adult ALL, 438 T-cell large granular lymphocyte leukaemia, 563, 565 haemopoietic stem cells (HSCs), 1–11 cell surface markers, 2 definition, 1 development, 3–6, 5, 14 aorta–gonad–mesonephros, 4, 5 cell surface markers, 4–5 haemangioblast, 4, 4 lineage tracer experiments, 5 mesodermal precursor cells, 4, 5 transdifferentiation, 4, 5 see also haematopoiesis dormancy, 6 erythroid progenitor development, 13–15, 14 cell surface markers, 14–15 microarray analyses, 13–14 models, 15

flow cytometry, 2 hierarchy, 1–2, 3 immunophenotype, 2 see also specific cells haemorrhages see bleeding haemorrhagic disease of the newborn see vitamin K deficiency bleeding (VKDB) haemorrhagic fever, acquired haemolytic anaemias, 169 haemosiderin, 26, 27–28 haemostasis, 746–771, 747 definition, 746 developmental, 980 disturbances, renal disease, 947–948 fibrin generation, 750 neonatal haematology, 979–983 in pregnancy, 767 see also blood coagulation haemovigilance, blood transfusions, 289–292 haem oxidase, haemoglobin breakdown, 36 haem proteins, 26, 27–32 structure, 35 haem-regulated inhibitor (HRI), 19 haem synthesis, 19, 35–36 δ-aminolaevulinic acid, 35 iron absorption, 35, 35 pathological alterations, 41–45 see also porphyria(s) hair dye, NHL, 658 hairy cell leukaemia, 550–553 acquired aplastic anaemia vs., 214 blood film, 551 clinical findings, 551 diagnosis, 551–553 blood film, 551, 551 bone marrow aspirates, 551 bone marrow trephine, 552 CD20 staining, 552, 552 CD22, 552 FMC7, 552 reticulin assays, 551, 552 spleen histology, 551–552 tartrate-resistant acid phosphatase, 551–552 flow cytometric immunophenotyping, 403 immunohistochemistry, 404 laboratory findings, 551 treatment, 553 Hallervorden–Spatz syndrome, 53 halofantrine, malaria treatment, 962 hand–foot syndrome, sickle cell disease, 113 Hand–Schüller–Christian disease, 325, 326 H antigen see ABO blood group

Index

haptocorrin, in disease, 70 haptoglobin haemolysis, 127 neutrophils, 309 Hasenclever score, Hodgkin lymphoma prognosis, 644, 644 Hasford score, CML prognosis, 485, 485 HAX1 gene Kostmann syndrome, 314 neutropenias, 202 Hb Bart’s hydrops fetalis syndrome, 103 HbD disease, with sickle cell disease, 119 HbE disease, with sickle cell disease, 119 HbH disease, 103 Hb Lepore disease, with sickle cell disease, 119 H blood group, 245 HbM disease, 692–693 HbO Arab disease, with sickle cell disease, 119 HBsAg, blood donation screening, 272 HbSC disease, 118 hCOT-1 (human organic cation transporter), imatinib mesylate response prediction, 493 HDFN see haemolytic disease of the fetus and newborn (HDFN) HE see hereditary elliptocytosis (HE) heavy chain(s) B-cell receptor, 348, 348, 349 ferritin, 30 genes, 349 Igs see immunoglobulin(s) (Igs) Hedgehog signalling, haematopoiesis, 8 Heinz bodies, haemoglobin structural variants, 106 Helicobacter pylori infection extranodal marginal zone lymphoma of MALT, 619 iron deficiency anaemia, 40 MALT lymphoma, 656, 670 pernicious anaemia, 72 primary immune thrombocytopenia, 930–931 HELLP syndrome pregnancy, 953–954 TTP vs., 865 hemojuvelin mutations, haemochromatosis type 2, 52 HEMPAS, congenital dyserythropoietic anaemia type II, 200 heparan sulphate, 764 heparin, 764 basophil/mast cell granules, 322 DIC treatment, 845 venous thromboembolism therapy, 903–904

venous thrombosis in heritable thrombophilias, 879 heparin cofactor II, 759, 763–764 disseminated vascular coagulation, 764 heritable thrombophilia, 878 heparin-induced thrombocytopenia (HIT), 891, 903–904 anti-heparin/platelet factor 4 antibodies, 904 heparin sulphate, mucopolysaccharidosis type III, 331 hepatic vein thrombosis, paroxysmal nocturnal haemoglobinuria, 180 hepatitis virus transmission, blood transfusions, 271–272, 274 hepatitis A virus (HAV), blood transfusions, 272, 274 hepatitis B virus (HBV) testing, blood donation, 275 transmission blood transfusions, 271, 272 factor VIII concentrates, 796 hepatitis C virus (HCV) DLBCL, 687 haemostatic disturbance in chronic liver disease, 848 hepatocellular carcinoma, 480 NHL, 657 splenic marginal zone lymphoma, 555, 617 testing, blood donation, 275 transmission blood transfusions, 272 factor VIII concentrates, 796 hepatitis E virus, blood transfusions, 272, 274 hepatitis G virus, blood transfusions, 272 hepatobiliary complications, sickle cell disease, 116 hepatocellular carcinoma (HCC), hepatitis C, 480 hepatocytes, hepcidin release, 941 hepatoma, polycythaemia, 943 hepatomegaly hairy cell leukaemia, 551 large granular lymphocyte leukaemia, 562 hepatosplenic T-cell non-Hodgkin lymphoma, 571 treatment, 678 WHO lymphoma classification, 629, 629 hepatosplenomegaly, mastocytosis, 702 hepatotoxicity cytarabine, 480 imatinib mesylate, 492 hepcidin, 19, 28, 29, 31–32 anaemia of chronic disease, 940 anaemia, response to, 32

BMP/SMAD signalling, 31 expression regulation, 28, 31–32 haemochromatosis, type 1, 48 hepatocyte release, 941 hypoxia, response to, 32 measurement, 32 regulation, 31 hephaestin, 29 iron absorption, 34 hereditary acanthocytosis (abetalipoproteinaemia), acquired haemolytic anaemias, 174 hereditary elliptocytosis (HE), 133–135 in Africa, 134 laboratory investigations, 135 mild common, 134, 134 neonatal anaemia, 975 neonates, 134 Plasmodium falciparum resistance, 134 silent carriers, 134 spherocytic, 134 treatment, 135 hereditary haemorrhagic telangiectasia (HHT), 854–855 hereditary hyperferritinaemia–cataract syndrome, 53 hereditary neutrophilia, 312–313 hereditary persistence of fetal haemoglobin (HPFH), 88, 97–100 deletions, 98, 98 globin gene promoter mutations, 100 heterocellular, 100 point mutations, 98, 99 with sickle cell disease, 118–119 hereditary pyropoikilocytosis (HPP), 133– 135, 135 laboratory investigation, 135 neonates, 134, 975–976, 976 silent carriers, 134 treatment, 135 hereditary spherocytosis (HS), 130–133 clinical course, 132 clinical features, 130–131 complications, 132 gene defects, 131–132 laboratory diagnosis, 131 management, 132–133 molecular pathology, 131 neonatal anaemia, 975 protein abnormalities, 131–132 hereditary stomatocytosis, 135, 135–136, 136 hereditary systemic amyloidosis, 611–612 heritable thrombophilias see thrombophilias, heritable 1021

Index

Hermansky–Pudlak syndrome, 923 genetics, 925 herpes simplex virus infection, allogeneic SCT, 737 screening, 477 Herpesvirus infections, allogeneic SCT infections, 736–737 HES see hypereosinophilic syndrome (HES) heterocellular hereditary persistence of fetal haemoglobin, 100 heterogenous nucleation, sickle cell disease, 110 heterotrimeric proteins, platelet receptors, 780 hexokinase, 145–146 deficiency, 145–146, 146 hexose phosphate shunt, red cell metabolism see red cell metabolism HFE gene haemochromatosis, 47 mutations, haemochromatosis, type 1 see haemochromatosis, type 1 porphyria cutanea tarda, 42 β-thalassaemia modifiers, 92–93 HFE, hepcidin expression, 31 HHT (hereditary haemorrhagic telangiectasia), 854–855 HHV-8 see human herpesvirus 8 (HHV-8) HICI gene, hypermethylation, 388 HIF see hypoxia-inducible factor (HIF) high-affinity haemoglobin, erythrocytosis, 692, 692 high altitudes, erythrocytosis, 691–692 high-density microarrays, AML classification, 430–431, 431 high endothelial venules, lymph nodes, 355, 355 highly active antiretroviral therapy (HAART), AIDS-related lymphoma, 658 high-molecular weight kininogen (HMWK) deficiency, 748 high-oxygen affinity variants, haemoglobin disorders, 107 high-performance liquid chromatography (HPLC), 119 high-throughput screening, haematopoiesis, 8 hirudin, venous thromboembolism therapy, 905 histamine basophil/mast cell granules, 322 von Willebrand factor, 805 histamine H1 receptor blockers, 703 histamine H2 receptor blockers, 703 histidine-rich protein 2 (HRP2), 960 histiocytic cell neoplasms, treatment, 679 1022

histiocytic disorders, 324–327, 325 see also specific diseases/disorders histiocytosis, Langerhans cell see Langerhans cell histiocytosis histiocytosis X see Langerhans cell histiocytosis histocompatibility, 710–721 see also HLA histology, 402 adult T-cell leukaemia lymphoma, 568 classical Hodgkin lymphoma, 640 NHL, 659 nodular lymphocyte-predominant Hodgkin lymphoma, 641 systemic AL amyloidosis, 602, 604 histone deacetylase inhibitors, peripheral T-cell lymphomas, 677 histone modification, myelodysplastic syndrome, 512 HIT see heparin-induced thrombocytopenia (HIT) HIV infection, 954, 954–955 anaemia, 954 antibody development, 272–273 blood coagulation, 955 blood donation screening, 273, 275 cobalamin malabsorption, 73 DIC, 955 haemopoiesis suppression, 954 HLA-B27, 713 HLA-B35, 713 HUS, 955 leucopenia, 954–955 lymphomas, 955 neutropenia, 954–955 platelets, 955 protein S deficiency, 955 thrombocytopenia, 955 transmission blood transfusions, 272–273 factor VIII concentrates, 796 TTP, 955 vitamin B12 deficiency, 954 HLA, 263 allogeneic SCT, 723 antigen processing/presentation, 711–712 chromosomal location, 710 disease associations, 713 functions, 712 matched sibling donor haematopoietic SCT see stem cell transplantation (SCT) nomenclature, 713–714, 714–715 non-haemolytic febrile transfusion reactions, 286 polymorphism, 712–713, 713

protein structure, 711, 711 self/non-self discrimination, 710 see also major histocompatibility complex(es) (MHC); specific haplotypes HLA-A, 710, 711 matching in SCT, 717, 723 HLA-A2, 717–718 HLA-A*03, 713 HLA-B, 710, 711 matching in SCT, 717, 723 HLA-B27 ankylosing spondylitis association, 713 HIV infection, 713 HLA-B35, 713 HLA-B*53:01, 713 HLA-B57, 713 HLA-C, 710, 711 matching in SCT, 717, 723 HLA class I, 710 antigen processing/presentation, 711–712 natural killer cells, 354 platelets, 264 HLA class II, 710 antigen processing/presentation, 712 DNA typing, 719 tissue distribution, 713 HLA-DM, 710 HLA-DO, 710 HLA-DP, 710 HLA-DPA, 711 HLA-DQ, 710 HLA-DQA, 711 HLA-DQB, 711 HLA-DQB1, 717, 723 HLA-DR, 710 HLA-DR4, 713 HLA-DR15, 507 HLA-DRA, 711 HLA-DRB, 711 HLA-DRB1, 717, 723 HLA-E, 710 HLA-F, 710 HLA-G, 710 HLA typing, 714, 716–717, 717–720 cellular assays, 719–720 DNA-based methods, 717–719 direct sequencing, 718–719 PCR, 717 PCR sequence-specific oligonucleotides, 718 PCR sequence-specific primers, 718, 718 platelet transfusions, 466 serology, 717 HMGA2 gene, 176

Index

Hodgkin lymphoma, 639–654 aetiology, 639–640 clinical presentation, 642–643 diagnosis, 643–644 bone marrow trephine biopsy, 399 epidemiology, 639 histology, 640 immunohistochemistry, 404 immunophenotype, 640 infectious mononucleosis, 366, 367 malignancies, 641–642, 942 CLL/small lymphocytic leukaemia, 616 Richter transformation, 549 pathogenesis, 640–642 malignant cell characteristics, 640–641 primary mediastinal B-cell lymphoma vs., 412 prognostic factors, 644 EORTC risk factors, 644, 644 Hasenclever score, 644, 644 response assessment, 644–645 staging, 643–644 surrounding tissue reaction, 642 treatment, 645–652 ABVD, 646 chemotherapy/radiotherapy combination, 646 childhood, 651 cytotoxic chemotherapy, 645–647, 647, 652 immunotherapy, 652 late effects, 651–652 MOPP, 645–646 new therapy, 652 in pregnancy, 651 progressive disease, 650–651 radiotherapy, 645 relapsed disease, 650–651 warm-type AIHA, 162 WHO lymphoma classification see World Health Organization (WHO), lymphoma classification see also classical Hodgkin lymphoma (CHL); nodular lymphocytepredominant Hodgkin lymphoma (NLPHL) Hollenhorst plaques, cholesterol embolism, 855 holotranscobalamin (holoTCII, holoTC), cobalamin deficiency, 75 homocysteine heritable thrombophilia, 879 neurological manifestations, 66 serum, cobalamin deficiency, 75 homocystinuria folate deficiency, 79 megaloblastic anaemia, 65

homogenous nucleation, sickle cell disease, 110 homoharringtonine, chronic CML management, 496 hookworm infection, iron deficiency anaemia, 40 hormonal deficiencies, erythropoiesis disorders, 24 hormone replacement therapy (HRT) allogeneic SCT, 740–741 thrombosis prevention, 881 venous thromboembolism, 890 hospital-acquired venous thromboembolism, 909 host-versus-graft (HVG) response, 722 HOVON trial, multiple myeloma management/treatment, 594 Howell–Jolly bodies, post-splenectomy, 377 HOX11 (TLX1) gene childhood ALL, 453 congenital amegakaryocytic thrombocytopenia, 925 T-cell acute lymphoblastic leukaemia, 385 T-cell lymphomas, 659 thrombocytopenia with absent radii, 917 Hoyeraal–Hreidarsson (HH) syndrome, 193 HPA see human platelet antigens (HPA) HPFH see hereditary persistence of fetal haemoglobin (HPFH) HPP see hereditary pyropoikilocytosis (HPP) HPS1 gene, 923, 925 HPS2 gene, 923, 925 HPS3 gene, 925 HPS4 gene, 923, 925 HPS5 gene, 925 HPS6 gene, 923, 925 HPS7 gene, 923, 925 HPS8 gene, 923, 925 HRAS gene, 387 HRI (haem-regulated inhibitor), 19 HRT see hormone replacement therapy (HRT) HS see hereditary spherocytosis (HS) HSCs see haemopoietic stem cells (HSCs) 5-HT2A receptors, platelets, 784 HTRs see haemolytic transfusion reactions (HTRs) Hu1D10 see apolizumab human herpesvirus 8 (HHV-8) large-cell lymphomas of terminally differentiated B cells, 625 NHL, 657 human leucocyte antigen see HLA

human organic cation transporter (hOCT1), imatinib mesylate response prediction, 493 human platelet antigens (HPA), 264–266, 265 molecular typing, 265 typing, 264–265 human recombinant factor VIIa, 799–800 human T-cell leukaemia virus 1 (HTLV-1) adult T-cell leukaemia lymphoma, 567, 569 blood transfusions, 273 geographical variations, 656 NHL, 657 Sézary syndrome/mycosis fungoides, 565 human T-cell leukaemia virus 2 (HTLV-2), 565 HUMARA gene, 193 humoral immune responses depression, CLL, 478 neutropenia, 316 Hunter syndrome see mucopolysaccharidosis type II (Hunter syndrome) Hurler Scheie disease see mucopolysaccharidosis type I (Hurler Scheie disease) HUS see haemolytic–uraemic syndrome (HUS) hydroa vacciniforme-like lymphoma, 627 hydrogen peroxide, phagocytes, 305 hydrolases, lysosomes, 330 hydrops fetalis, 293 hydroxocobalamin, 80 hydroxycarbamide (hydroxyurea) antiemetic effects, 481 chronic CML management, 495 clonal hypereosinophilic syndromes treatment, 706 essential thrombocythaemia treatment, 696 HbF effects, 122 polycythaemia vera treatment, 690 sickle cell disease therapy, 122–123 thalassaemia intermedia, 105 hydroxyl radical, phagocytes, 305 hydroxyurea see hydroxycarbamide (hydroxyurea) hyperbilirubinaemia extravascular haemolytic transfusion reactions, 284 leukaemias, 480 hypercalcaemia adult T-cell leukaemia lymphoma, 569 CML, 491 multiple myeloma, 582, 584 Richter transformation, 549 1023

Index

hypercoagulability arterial thrombosis, 882 liver disease, 850 hyperdiploidy ALL, 386 childhood ALL, 452 early pre-B ALL, 451 hypereosinophilia, childhood ALL, 451 hypereosinophilic syndrome (HES), 319, 704 lymphoproliferative variant, 320–321 treatment, 321 hyperfibrinolysis, DIC vs., 844 hyperhomocysteinaemia, heritable thrombophilia, 878 hyper-IgM syndrome, neutropenia, 315 hyperkalaemia CML, 491 leukaemias, 479 massive transfusions, 289 hypermethylation, CDH1 gene, 388 hyperphosphataemia, leukaemias, 479 hyperploidy, Sézary syndrome/mycosis fungoides, 567 hypersegmented neutrophils, cobalamin deficiency, 75 hypersplenism, 374 CLL, 535 malignancies, 944 tropical diseases, 968 hypertension polycythaemia vera, 689 renal amyloidosis, 610 hyperthyroidism acquired von Willebrand disease, 858 pernicious anaemia, 948 hyperunstable β-chain variants, β-thalassaemias, 97 hyperuricaemia leukaemia, 479 urinary alkalinization, 479 hypervariable regions, Igs, 231 hyperviscosity, neonatal polycythaemia, 978–979 hypoadrenalism, pernicious anaemia, 948 hypocalcaemia leukaemias, 479 massive transfusions, 289 hypochromic anaemia, differential diagnosis, 37 hypochromic microcytic anaemia, α thalassaemias, 102–103 hypochromic red cells, iron metabolism measurement, 38 hypocomplementaemia, AIHA, 161 hypodiploid childhood ALL, 452 1024

hypogammaglobulinaemia CLL, 535 pernicious anaemia, 72 hypoglycaemic agents induced neutropenia, 316 see also specific drugs hypomethylating drugs myelodysplastic syndrome therapy, 522– 523, 523 see also specific drugs hypomethylation, 388 hypoparathyroidism, pernicious anaemia, 948 hypoplastic myodysplasia syndrome, 516 hypopnoea, erythrocytosis, 691 hyposplenism, 376–378 age-dependence, 376 blood film, 377 causes, 377 essential thrombocythaemia, 694 haematological effects, 377–378 management, 376 systemic AL amyloidosis, 602 hypothrombinaemia, prothrombin deficiency, 816 hypothyroidism acquired von Willebrand disease, 811, 858 Hodgkin lymphoma treatment, 652 hypoxia chronic, erythrocytosis, 691 erythropoietin receptor, 19 hepcidin response, 32 hypoxia-inducible factor (HIF), 19–20 hepcidin regulation, 32 hypoxia sensing, erythropoiesis, 19–20, 20 I I antigen, 252 i antigen, 252 iatrogenic venous thromboembolism, 890–891 I blood group, 245, 252 ibritumomab, 666 90 Y-ibritumomab tiuxetan, 667–668 Waldenström macroglobulinaemia, 672 ICAM-1 (intracellular adhesion molecule 1), 301 ICAM-1 (intercellular adhesion molecule 1), multiple myeloma pathogenesis, 580 ICAM-2 (intracellular adhesion molecule 2), 301 ICAM-4 (CD242), 228 I cell syndrome see mucolipidosis type II (I cell syndrome) ICOS, angioimmunoblastic T-cell lymphoma, 633

icterus gravis neonatorum, 293 idarubicin, AML treatment, 421 idiopathic aplastic anaemia, 187 idiopathic cold-type autoimmune haemolytic anaemia, 164–165 idiopathic erythrocytosis, 693 idiopathic neutropenia, chronic, 315 idiopathic neutrophilia, chronic, 313 idiopathic thrombocytopenic purpura (ITP) see primary immune thrombocytopenia idiopathic warm-type autoimmune haemolytic anaemia, 162 idiotopes, 231–232 IF see intrinsic factor (IF) IFM 01 trial, 594 IFM 99 trial, 594 IFN-α see interferon-α (IFN-α) IFN-γ see interferon-γ (IFN-γ) IGH gene mantle cell lymphoma, 384 translocations CLL, 540 multiple myeloma, 578, 578–579, 589 IGHV genes B-cell prolymphocytic leukaemia, 550, 616 classic Hodgkin lymphoma, 637 CLL, 533, 533, 538, 541, 541 IKZF1 gene childhood ALL, 450 CML, 488 IKZF3 gene, 450 IL-6R gene, haemopoietic stem cells, 2 IL15 gene, childhood ALL prognosis, 455 ileal dysfunction, cobalamin malabsorption, 74 imaging, venous thromboembolism diagnosis, 902–903 imatinib mesylate adult ALL prognostic factors, 444 clinical trials, 492–493 clonal hypereosinophilic syndromes treatment, 706 CML management, 499 chronic, 491–494 G-CSF co-administration, 492 IRIS trial, 492–493 mastocytosis treatment, 704 mechanism of action, 493 monitoring, 493 resistance, 494 response definition, 493–494, 494 response failure, 493–494 response prediction, 493 side effects, 492 Imerslünd–Grasbeck syndrome, 73 Imerslünd syndrome, 73

Index

immobility, venous thromboembolism, 889, 900 immune complexes drug-induced alloimmune haemolytic anaemia, 166, 167 primary immune thrombocytopenia, 930–931 immune cytopenias, CLL, 535–536 immune factors, pernicious anaemia, 72 immune haemolysis, neonatal anaemia, 974–975 immune recognition, SCT, 724, 726 immune response, 355–359 acquired aplastic anaemia, 209 anatomy, 343–344 primary immune thrombocytopenia, 929 immune thrombocytopenia, malignancies, 944 immune thrombocytopenic purpura, acquired aplastic anaemia vs., 213 immune tolerance, haemophilia A inhibitor therapy, 800 immunizations, splenectomy, 375 immunochromatography, filariasis diagnosis, 964 immunocompromised patient, NHL, 681–682 immunodeficiency, chronic GvHD, 739–740 immunoglobulin(s) (Igs), 230–232 AIHA testing, 159 biochemistry, 230–232 class switching, 358 complement binding, 232, 235–236 C1q complement protein, 236 definition, 230 diversity, 232 effector functions, 230 Fc fragment, 231 Fc receptor binding, 232 genetics, 230–232 CLL/small lymphocytic leukaemia, 616 gene rearrangement, 381 heavy chain genes, 349 NHL mutations, 658 splenic marginal zone lymphoma, 617 variable region, 232 V–D–J gene joining, 232 V–J gene joining, 232 heavy chains, 230 constant region, 231 D segment, 347–348 J segment, 347–348 NHL, 658–659 V segment, 347–348

intravenous see intravenous immunoglobulin (IVIg) light chain, 230 kappa (κ), 230–231 lambda (λ), 230–231 physical properties, 232 specificity, 344 structure, 230, 231 heavy chains see above hypervariable regions, 231 light chains see above transplacental passage, 232 see also antibodies; B-cell receptor (BCR); specific types immunoglobulin A (IgA), 230 paraprotein, acquired von Willebrand disease, 811 secretory, effector functions, 230 structure, 231 immunoglobulin A1 (IgA1), 230 immunoglobulin A2 (IgA2), 230 immunoglobulin D (IgD), 230 immunoglobulin E (IgE), 230 basophil/mast cell binding, 321 effector functions, 230 immunoglobulin G (IgG), 230 blood group antibodies, 233 paraprotein, acquired von Willebrand disease, 811 structure, 231 immunoglobulin G1 (IgG1), 230 immunoglobulin G2 (IgG2), 230 immunoglobulin G3 (IgG3), 230 immunoglobulin G4 (IgG4), 230 immunoglobulin M (IgM), 230 blood group antibodies, 233 classical complement activation, 234 effector functions, 230 multiple myeloma, 584 structure, 231 immunohistochemistry, 404–405 acute lymphoblastic leukaemia, 405 Burkitt lymphoma, 404 DLBCL, 404–405 extranodal T/NK lymphoma, nasal type, 573 follicular lymphoma, 404 hairy cell leukaemia, 404 Hodgkin lymphoma, 404, 643 mantle cell lymphoma, 404 NHL, 404 systemic AL amyloidosis, 605 immunological effects myelodysplastic syndrome, 507–508 spleen, 371 splenectomy, 378 immunological therapy see immunotherapy

immunophenotype adult ALL prognosis, 443–444 adult T-cell leukaemia lymphoma, 568 CML, 491 haemopoietic stem cells, 2 Hodgkin lymphoma, 640 large granular lymphocyte leukaemia, 562 plasma cell dyscrasia assessment, 606 SCT donor cells, 728–729 Sézary syndrome, 566–567 T-prolymphocytic leukaemia, 560 immunoproliferative small intestinal disease (IPSID), 676 immunoreceptor tyrosine-based activation motifs (ITAMs), 304 immunoreceptor tyrosine-based inhibition motifs (ITIMs) platelet inhibition, 787 platelet signalling pathways, 779 immunostaining, CLL, 537 immunosuppression acquired aplastic anaemia see acquired aplastic anaemia acquired haemophilia A therapy, 858 ATG with, 218 chronic GvHD, 740 haemophagocytic lymphohistiocytosis, 952 Hodgkin lymphoma, 639–640 myelodysplastic syndrome therapy, 520 NHL, 656, 658 post-transplant lymphoproliferative disease, 682 Richter transformation, 549 T-cell large granular lymphocyte leukaemia, 563 warm-type AIHA treatment, 163 immunotherapy aggressive B-cell NHL treatment, 674 childhood ALL treatment, 459–460 chronic CML management, 498 Hodgkin lymphoma treatment, 652 lymphomatoid granulomatosis treatment, 680 mantle cell lymphoma, 673 TTP treatment, 867, 867–868 see also specific treatments immunotoxins, follicular lymphoma therapy, 668 IMWG see International Myeloma Working Group (IMWG) incentive spirometry, sickle cell disease therapy, 122 incorrect blood component, SHOT, 291 increased iron uptake, iron overload, 52 1025

Index

indanediones, venous thromboembolism therapy, 904 Indian blood group, 245 antigen (CD44), 229 indirect agglutination test (IAT), 238–239 blood group screening, 238 Lewis blood group, 251 induced pluripotent embryonic stem cells, 9, 9 induction therapy AML relapse risk, 426 multiple myeloma, 581–582 multiple myeloma management/ treatment, 591, 592 infantile pyknocytosis, neonatal anaemia, 976 infants see children infection(s) acquired aplastic anaemia, 213, 215 acquired haemolytic anaemias, 168–170 adult ALL therapy, 437–438 allogeneic SCT complications, 735–737, 736 AML, 424 anaemia of chronic disease, 940 blood transfusion transmission see blood transfusion(s) childhood ALL, 477 CLL, 549 cold-type AIHA, 165 DIC, 951 eculizumab risks, 182 favism, 152 haematological diseases, 949–952 leukaemia care see leukaemias, supportive care microangiopathic haemolysis anaemias, 172 multiple myeloma, 588 neutropenias, 202, 313 neutrophilia, 313, 951 prophylaxis, AML treatment, 421–422 sickle cell disease, 113–114 sickle cell disease therapy, 121 thrombocytopenias, 917 see also specific infections infectious mononucleosis, 364–367 clinical features, 365 differential diagnosis, 366 childhood ALL vs., 451 secondary associations, 366–367 serological changes, 365–366 infectious organism clearance, neutrophils, 309 inferior vena cava filters, venous thromboembolism therapy, 907 1026

infertility BEACOPP side effect, 652 Hodgkin lymphoma treatment, 651, 652 post-haematopoietic SCT, 222 inflammation African trypanosomiasis, 965 anaemia of chronic disease, 940 DIC, 843 folate deficiency, 78–79 venous thromboembolism, 897, 897–898 inflammatory bowel disease (IBD) thrombosis in inflammation, 898 infliximab, hepatosplenic T-cell lymphoma, 629 inherited aplastic anaemia, acquired vs., 207 inherited disorders neonates, 980–981 see also specific diseases/disorders inherited erythrocytosis, 691 inherited neonatal neutropenias, 979 inherited sideroblastic anaemia, 44–45 inherited thrombotic disorders, neonates, 982–983 inhibitors, haemophilia A, 796 innate immune response, spleen, 371 in situ follicular lymphoma, WHO lymphoma classification, 620 insulin, erythroid cell development, 22 insulin-like growth factor 1 (IGF-1), 22 integrin(s) phagocytes, 301, 302 platelet signalling pathways, 781, 781 thrombus formation, 776, 777 see also specific types integrin α2β1, platelet receptors, 781–782 integrin αIIbβ3 Glanzmann thrombasthenia, 922 platelet α-granules, 774 platelet receptors, 782–783 thrombocytopathy, 922 thrombus formation, 778 integrin αvβ3, apoptotic cell receptor, 304 integrin αvβ5, apoptotic cell receptor, 304 integrin β1, multiple myeloma pathogenesis, 580 intensive care support, allogeneic SCT complications, 739 intercellular adhesion molecule 1 (ICAM1), multiple myeloma pathogenesis, 580 interferon-α (IFN-α), 362 adult T-cell leukaemia lymphoma treatment, 678 chronic CML management, 495–496 clonal hypereosinophilic syndromes treatment, 706

essential thrombocythaemia treatment, 696 hairy cell leukaemia treatment, 553 polycythaemia vera treatment, 690 toxicity, 495–496 interferon(s), advanced follicular lymphoma treatment, 665 interferon-γ (IFN-γ), 362 complement receptor 1, 303 complement receptor 3, 303 Fanconi anaemia, 191 macrophage activation, 323 secreting cells, allogeneic SCT, 744–745 T helper cells type 1, 358 Intergruppo Italiano Linfomi trial, 649 interleukin-1 (IL-1) anaemia of chronic disease, 940 Gaucher disease, 331 interleukin-1α (IL-1α), 360 anaemia of chronic disease, 940 interleukin-1β (IL-1β), 360 anaemia of chronic disease, 940 hepcidin expression, 32 interleukin-2 (IL-2), 360 adult T-cell leukaemia lymphoma, 569 macrophage activation, 323 receptor, adult T-cell leukaemia lymphoma, 569 T helper cells type 1, 358 interleukin-3 (IL-3), 360 Hodgkin lymphoma, 642 neutrophil development, 308 interleukin-4 (IL-4), 360 macrophage activation, 323 nitric oxide synthase, 306 T helper cells type 2, 358 interleukin-5 (IL-5), 360 interleukin-6 (IL-6), 360 anaemia of chronic disease, 940 Gaucher disease, 331 hepcidin expression, 32 Kupffer cell secretion, 941 T helper cells type 2, 358 thrombosis in inflammation, 897–898 interleukin-7 (IL-7), 360 interleukin-8 (IL-8), 361, 363 nitric oxide synthase, 306 interleukin-9 (IL-9), 361 interleukin-10 (IL-10), 361 Gaucher disease, 331 nitric oxide synthase, 306 T helper cells type 2, 358 interleukin-11 (IL-11), 361 interleukin-12 (IL-12), 361 interleukin-13 (IL-13), 361 interleukin-14 (IL-14), 361 interleukin-15 (IL-15), 361

Index

intermediate diffuse large B-cell lymphoma and classical Hodgkin lymphoma, WHO lymphoma classification, 626–627 internal tandem duplications (ITDs), AML, 416 International Haemovigilance Network, 289–290 International Myeloma Working Group (IMWG) multiple myeloma classification, 582 multiple myeloma prognosis, 590, 590–591 International Prognostic Index (IPI), NHL, 661 International Prognostic Scoring System (IPSS) see myelodysplastic syndrome (MDS) International Prognostic Staging System for Waldenström Macroglobulinaemia (IPSSWM), 671 International Rare Bleeding Disorders Database (RBDD), 813 International Society for Thrombosis and Haemostasis (ISTH), 790 International Society of Blood Transfusion, blood group antigens, 244 International Staging System (ISS), multiple myeloma, 589–590 International Workshop on CLL (IWCLL), 534 CLL management, 545 intestine, DLBCL, 676 intima, blood vessels, 752–753 intra-abdominal vein thrombosis, therapy, 880 intracellular adhesion molecule 1 (ICAM1), 301 intracellular adhesion molecule 2 (ICAM2), 301 intracellular cobalamin metabolism, 63 intracellular iron homeostasis, 32–33, 33 intracellular transit iron, 36 intracorpuscular causes, haemolysis, 127 intrafollicular neoplasia, WHO lymphoma classification, 620 intraperitoneal transfusion, haemolytic disease of the fetus and newborn, 294–295 intravascular haemolysis eculizumab effects, 181 paroxysmal nocturnal haemoglobinuria, 178 intravascular large B-cell lymphoma treatment, 676 WHO lymphoma classification, 624, 625

intravascular red cell destruction, haemolytic transfusion reactions, 283–284 intravenous immunoglobulin (IVIg) haemolytic disease of the fetus and newborn, 975 primary immune thrombocytopenia in pregnancy, 938 primary immune thrombocytopenia management, 933, 934, 937 warm-type AIHA treatment, 163 intrinsic factor (IF) antibodies to, 72 cobalamin absorption, 68–69 congenital deficiency, 72 pernicious anaemia, 71 structure, 69 intron removal, globin gene processing, 87 invariant chain, HLA class II, 712 invasive pulmonary aspergillosis (IPA), allogeneic SCT infections, 737 in vitro gene transfer, Fanconi anaemia, 191 ionic strength, agglutination tests, 237 ionizing radiation, childhood ALL, 449 IP-10, 363 IPA (invasive pulmonary aspergillosis), allogeneic SCT infections, 737 IPI (International Prognostic Index), NHL, 661 IPSID (immunoproliferative small intestinal disease), 676 IPSS (International Prognostic Scoring System) see myelodysplastic syndrome (MDS) IPSSWM (International Prognostic Staging System for Waldenström Macroglobulinaemia), 671 IREs see iron-responsive elements (IREs) IRF4, primary cutaneous DLBCL, leg type, 624 IRIDA, hypochromic anaemia vs., 37 IRIS trial, 492–493 iron distribution in body, 26 serum, iron metabolism measurement, 36–37 iron absorption, 33–34 erythroid cells, 34–35 molecular factors, 34 iron chelation therapy acquired aplastic anaemia management, 215 Diamond–Blackfan anaemia treatment, 199 haemochromatosis, type 1, 51 iron-loading anaemias see iron-loading anaemias

myelodysplastic syndrome therapy, 519 β-thalassaemia management, 94 iron deficiency anaemia, 38–41 causes, 39, 39–40 clinical features, 39 CLL, 535 diagnosis, 36 hypochromic anaemia vs., 37 iron-deficient erythropoiesis, 38–39 iron refractory type, 41 management, 40–41 sickle cell disease, 113 store depletion, 38 tissue effects, 39 iron-deficient erythropoiesis, 38–39 iron dextran (CosmoFer), 41 iron homeostasis, 33, 33 intracellular, 32–33, 33 iron-loading anaemias, 53–57 iron chelation therapy, 56–59 combination therapy, 58, 58 deferasirox, 59 deferiprone, 53, 57–58 desferrioxamine, 53, 56–57 organ function tests, 56 tests for, 54, 54–56 cardiac iron, 55, 55 liver iron, 54–55 non-transferrin-bound iron, 56 serum ferritin, 54 urine iron excretion, 55 iron metabolism, 26–46 anaemia of chronic disease, 941 diagnostic methods, 36–38 bone marrow aspiration, 36, 38 confounding factors, 37 functional iron, 37 haemoglobin, 37 hypochromic red cells, 38 iron-binding capacity, 36–37 red cell mean corpuscular volume, 37 red cell protoporphyrin, 38 reticulocyte haemoglobin content, 38 serum ferritin, 36 serum iron, 36–37 serum transferrin receptors, 37, 37–38 storage iron, 36, 37 supply to tissues, 36–38, 37 intracellular transit iron, 36 plasma non-transferrin-bound iron, 36 see also haem proteins iron overload, 47–60 acute iron poisoning, 59 atransferrinaemia, 53 increased iron uptake, 52 neurodegeneration, 53 see also haemochromatosis 1027

Index

iron refractory iron deficiency anaemia, 41 iron regulatory protein(s) (IRPs), 33 iron-regulatory protein 1 (IRP1), 19, 30, 32 iron-regulatory protein 2 (IRP2), 19, 30, 32 iron-responsive elements (IREs), 32 δ-aminolaevulinic acid synthase, 32 erythropoiesis, 19 iron homeostasis, 33 iron–sucrose (Venofer), 41 iron supplements, anaemia of prematurity, 978 iron transport proteins, 29–30 IRP1 (iron-regulatory protein 1), 19, 30, 32 IRP2 (iron-regulatory protein 2), 19, 30, 32 IRPs (iron regulatory proteins), 33 irradiation CML risk, 484 transfusions, SCT, 467–468 irradiation-based conditioning, haematopoietic SCT, 223 isouramil, favism, 152 ISS (International Staging System), multiple myeloma, 589–590 ISTH (International Society for Thrombosis and Haemostasis), 790 ITAMs (immunoreceptor tyrosine-based activation motifs), 304 ITDs (internal tandem duplications), AML, 416 ITIMs see immunoreceptor tyrosine-based inhibition motifs (ITIMs) ITP see primary immune thrombocytopenia itraconazole, infection prophylaxis, 474 IVC filters, thrombolytic prophylaxis, 908 ivermectin, filariasis treatment, 965 IWCLL see International Workshop on CLL (IWCLL) J Jacobsen syndrome, 919 genetics, 925 JAK2 gene, 686 chronic myelomonocytic leukaemia, 526 essential thrombocythaemia, 693, 694 myelodysplastic syndrome, 510 polycythaemia vera, 686–687 primary myelofibrosis, 697 refractory anaemia with ring sideroblasts and thrombocytosis, 527–528 JAK2 inhibitors, polycythaemia vera treatment, 690 1028

JAK/STAT signalling pathway erythropoietin receptor, 21, 21, 22 Hodgkin lymphoma, 642 JAM-A, thrombus stabilization, 779 JAM-C, thrombus stabilization, 779 jaundice blood donation, 272 haemolysis, 127 haemophagocytic syndrome, 169–170 hereditary spherocytosis, 130 leukaemias, 480 neonatal anaemia, 974, 975 pyruvate kinase deficiency, 143 warm-type AIHA, 161 J gene segments, B-cell receptor, 348 Jk gene, Kidd blood group, 260 JMML see juvenile myelomonocytic leukaemia (JMML) John Milton Hagen blood group, 245 joint pain, deferiprone, 58 J segment, Igs, 347–348 J-stain immunostaining, nodular lymphocyte-predominant Hodgkin lymphoma, 642 juvenile myelomonocytic leukaemia (JMML), 500–501, 527 clinical features, 501 NF1 gene, 391, 501 PTPN11 gene, 501 RAS family signalling, 389, 390, 391 juvenile pernicious anaemia, 72 juvenile rheumatoid arthritis, haemophagocytic lymphohistiocytosis, 952 K kala-azar, 967 kaolin cephalin clotting time (KCCT) see prothrombin time (PT) kaolin clotting time (KCT), antiphospholipid syndrome, 893, 894 Kaposiform haemangioendothelioma, 855 kappa (κ) light chain B cell development, 351 Igs, 230–231 karyotypes multiple myeloma, 578 myelodysplastic syndrome, 516 Kasabach–Merritt syndrome, 855 KCT (kaolin clotting time), antiphospholipid syndrome, 893, 894 Kell blood group, 245, 259–260 antigen, 228 biological significance, 229 McLeod phenotype, 136–137

red cell membrane, 227 structure, 260 Kidd blood group, 245, 260 antigen, 229 kidney see entries under renal killer inhibitory receptors (KIRs), natural killer, 354 kindlin-3, platelet activation, 786 KIT gene, myelodysplastic syndrome, 510 kit-ligand deficiency, haematopoiesis, 6 Klebsiella infections leukaemias, 472 sickle cell disease, 114 Kleihauer acid elution test, 298 knockout mouse models factor V deficiency, 815, 818 factor VII deficiency, 815 factor X deficiency, 815 factor XI deficiency, 815 factor XIII deficiency, 815, 829 fibrinogen deficiency, 815 rare bleeding disorders, 815 vitamin K-dependent coagulation factors deficiency, 815 Knops blood group, 245 antigen, 229 Kostmann syndrome, 202, 314 Krabbe disease (globoid cell dystrophy), 334 haematopoietic SCT, 332 prevalence, 331 KRAS gene activating mutations, 387 AML, 391 chronic myelomonocytic leukaemia, 391 juvenile myelomonocytic leukaemia, 527 multiple myeloma, 579 myelodysplastic syndrome, 391 K typing, blood transfusion recipient testing, 281 Kupffer cells, 323 interleukin-6 secretion, 941 kwashiorkor, folate deficiency, 78 Kx blood group, 245 L LA (lupus anticoagulant), antiphospholipid syndrome, 892–893 lactate dehydrogenase (LDH) adult ALL, 437 CML, 491 leukaemias, 480 NHL, 660–661 TTP, 863 warm-type AIHA, 162 lactoferrin, 28 neutrophils, 307

Index

lactoperoxidase, 27 LAD (leucocyte adhesion deficiency), 312 lambda (λ) light chain B cell development, 351 Igs, 230–231 LAMPs (lysosome-associated membrane proteins), 330 Landsteiner–Wiener blood group, 245 Langerhans cell histiocytosis, 324–326 aetiology, 325 diagnosis, 325 lesions, 325 prognosis, 326 pulmonary, 325–326 spontaneous resolution, 326 treatment, 326 Langerhans cells, 324 antigen recognition/presentation, 325 large B-cell lymphoma, 660 large-cell lymphomas of terminally differentiated B cells, WHO lymphoma classification, 625 large granular lymphocytic (LGL) leukaemia, red cell aplasia, 942, 943 Lassa fever, 969 latent membrane protein-1 (LMP-1), Hodgkin lymphoma, 642 LDH see lactate dehydrogenase (LDH) Lea antigen, 250 lead poisoning acquired haemolytic anaemias, 174 iron deficiency, 42–43 Leb antigen, 250 lectins, 233 LEF1 gene, childhood ALL, 450 Legionella infections antigen detection, 471 sickle cell disease, 116 leg ulcers, sickle cell disease, 115, 118 leishmaniasis, 967–968 global distribution, 967, 967 transmission, blood transfusions, 274 lenalidomide mantle cell lymphoma, 556, 673 multiple myeloma, 591, 592–593, 595, 595 myelodysplastic syndrome, 523–524, 528 systemic AL amyloidosis, 609 Waldenström macroglobulinaemia, 672 lepirudin, venous thromboembolism therapy, 905 lesions, Langerhans cell histiocytosis, 325 leucocyte(s) count, CML, 489–490 post-splenectomy, 377

recruitment, platelets, 772–773 see also lymphocyte(s) leucocyte adhesion deficiency (LAD), 312 leucocytosis, infectious mononucleosis, 365 leucodepletion blood components, 278 non-haemolytic febrile transfusion reactions, 286 sickle cell disease, 121 leucodystrophy, metachromatic see metachromatic leucodystrophy leukaemias development see leukaemogenesis electrolyte balance, 479–480 essential thrombocythaemia, transformation of, 694 Fanconi anaemia, 187, 189 methicillin-resistant Staphylococcus aureus, 472 molecular basis, 380–394 oesophagitis, 471 platelet transfusions, 466 primary myelofibrosis, development from, 698 Staphylococcus aureus infection, 472 Staphylococcus epidermis infection, 472 supportive care see below types acute hypergranular promyelocytic leukaemia, 396, 397 acute lymphoblastic leukaemia (ALL) see acute lymphoblastic leukaemia (ALL) acute megakaryoblastic leukaemia (AMKL) see acute megakaryoblastic leukaemia (AMKL) acute microgranular/hypogranular promyelocytic leukaemia, 396, 397 acute monoblastic leukaemia see acute monoblastic leukaemia acute promyelocytic leukaemia see acute promyelocytic leukaemia (APL) adult T-cell leukaemia lymphoma see adult T-cell leukaemia lymphoma (ATLL) aggressive NK-cell leukaemia, 627, 627 aleukaemic leukaemia, 214 AML see acute myeloid leukaemia (AML) B-cell acute lymphoblastic leukaemia see B-cell acute lymphoblastic leukaemia B-cell prolymphocytic leukaemia, 616

B-cell prolymphocytic leukaemia (B-PLL) see B-cell prolymphocytic leukaemia (B-PLL) B-precursor acute lymphoblastic leukaemia, 443 chronic eosinophilic leukaemia see chronic eosinophilic leukaemia chronic myelomonocytic leukaemia see chronic myelomonocytic leukaemia (CMML) chronic neutrophilic leukaemia (CNL) see chronic neutrophilic leukaemia (CNL) CLL see chronic lymphocytic leukaemia (CLL) CML see chronic myelogenous leukaemia (CML) common acute lymphoblastic leukaemia, 434 early pre-B acute lymphoblastic leukaemia, 451 early T-precursor acute lymphoblastic leukaemia, 434 hairy cell leukaemia see hairy cell leukaemia juvenile myelomonocytic leukaemia see juvenile myelomonocytic leukaemia (JMML) large granular lymphocytic leukaemia, 942, 943 mast cell leukaemia, 322–323 mature B-cell acute lymphoblastic leukaemia see mature B-cell acute lymphoblastic leukaemia mature T-cell acute lymphoblastic leukaemia, 434 natural killer cell leukaemia, 406 neutrophilic chronic myeloid leukaemia, 707 plasma cell leukaemia, 583 pre-B-cell acute lymphoblastic leukaemia see pre-B-cell acute lymphoblastic leukaemia pro-B-cell acute lymphoblastic leukaemia, 434 small lymphocytic leukaemia, 614–616 T-cell acute lymphoblastic leukaemia (T-ALL) see T-cell acute lymphoblastic leukaemia (T-ALL) T-cell large granular lymphocyte leukaemia (T-cell LGL) see T-cell large granular lymphocyte leukaemia (T-cell LGL) thymic (cortical) T-cell acute lymphoblastic leukaemia, 434 1029

Index

T-lineage adult acute lymphoblastic leukaemia see T-lineage adult acute lymphoblastic leukaemia T-prolymphocytic leukaemia see T-prolymphocytic leukaemia (TPLL) see also specific diseases/disorders leukaemias, supportive care, 463–482 anaemia, 463, 464–468 growth factor support, 468 see also blood transfusion(s) bleeding, 463 febrile reactions, 467 fever of unknown origin, 474–477 broad-spectrum antibiotics, 474–475 central venous catheters, 475 clinically directed therapy, 476 microbiological therapy, 476 subsequent therapy rules, 476, 476–477 granulocyte infusions, 477–478 growth factors, 477–478 infections, 463, 468–478 bacterial pathogens, 468 fever, 469–470 fungal infections, 469 gut investigations, 471 neutropenia, 469 non-cultural screening, 471–472 oral/rectal swabs, 471 prophylaxis, 473–474 pulmonary infection specimens, 471 sites of, 469–470 skin lesions, 471 sources, 470 surveillance cultures, 472 urinary tract, 471 metabolic complications, 464, 478–480 electrolyte balance, 479–480 fluid balance, 478–479 hyperuricaemia, 479 liver function abnormalities, 480 renal function abnormalities, 479–480 tumour lysis syndrome, 479 nausea and vomiting, 464, 481 neutropenic fever, 470–472, 472–473 antibiotic cessation, 477 blood cultures, 470, 470–471 mucosal barrier injury, 470, 470 nutritional complications, 464, 480 pain, 464, 481 psychological complications, 463, 464 reproductive complications, 463, 464 specific bleeding problems, 467–468 thrombocytopenia, 464–468 see also blood transfusion(s) white cell counts, 465–466 1030

leukaemia stem cells, 382–384, 383 leukaemic phase, NHL, 544–556 leukaemic transformation myelodysplastic syndrome, 518 paroxysmal nocturnal haemoglobinuria, 178 polycythaemia vera, 689 leukaemogenesis, 380–385 apoptosis/cell cycle control, 393 chromosome rearrangements, 384–385 clonal evolution, 381–382, 383 clonality, 381 definition, 380 gene–environment interactions, 380–381 genetic predisposition, 380–381 Ig gene rearrangement, 381 leukaemia stem cells, 382–384, 383 lymphoid malignancies, 383–384 molecular basis, 388–394 cell cycle control, 392–394 RAS family signalling, 389, 391 tyrosine kinase, 388–389, 389, 390 myeloid malignancies, 382–383 phenotype–genotype correlations, 384–385 risk factors, 381 T-cell receptor gene rearrangement, 381 see also somatic mutations leukaemoid reaction, 951 leukopenia, HIV infection, 954–955 leukotriene antagonists, mastocytosis treatment, 704 levodopa, warm-type AIHA, 162 levofloxacin, infection prophylaxis, 473 Lewis blood group, 245, 250–251 antibodies, 251 antigen, 250 lifespan, neutrophils, 309 light chain, 230 B-cell receptor, 348 ferritin, 30 light transmission aggregometry (LTA), platelet function testing, 789 Lille scoring system, primary myelofibrosis prognosis, 701 lineage tracer experiments, HSC development, 5 lipid scavenger receptors, 304 lipids, red cell membrane see red cell membrane lipid storage diseases, 335 clinical manifestations, 338 lipoprotein A, plasmin inhibition, 770 β-lipoproteins, heparin interaction, 764 Listeria infection CLL, 478 cold-type AIHA, 165

livedo reticularis, 897 antiphospholipid syndrome, 892 liver AIHA, 160 allogeneic SCT complications, 738, 738 autologous SCT complications, 741 biopsy, haemochromatosis, type 1, 50, 50 cirrhosis folate deficiency, 66 leukaemias, 480 function abnormalities, leukaemia, 480 GvHD, 735 hereditary haemorrhagic telangiectasia, 854 iron-loading anaemias, 54–55 thrombopoietin production, 931–932 liver disease, 948–949, 949 acquired haemolytic anaemias, 174 anaemia, 948–949 childhood ALL, 451 chronic, 848–849 DIC, 843 differential diagnosis, DIC vs., 844 echinocytosis, 948 folate deficiency, 79 haemostatic disturbance, 847, 847–850 causes, 847 hypercoagulability, 850 macrocytosis, 948 liver function tests, acquired aplastic anaemia, 213 liver transplantation, 949 acquired aplastic anaemia, 207 haemostatic disturbance, 849–850 as immunoprivileged site, 716 LMAN1 gene, 814, 821 LMO1 (TTG-1) gene, 453 LMO2 (TTG-2) gene childhood ALL, 453 erythropoiesis, 15 T-cell lymphomas, 659 LMP-1 (latent membrane protein-1), Hodgkin lymphoma, 642 LMPP (lymphoid primed multipotent progenitor), 2 LMWH see low-molecular weight heparin (LMWH) Loa loa infection, 963 localised AL amyloidosis, 610 localized Castleman disease, 680 local radiotherapy aggressive B-cell NHL treatment, 674 lymphoblastic lymphoma treatment, 679 multiple myeloma management/ treatment, 588 locomotion, phagocytes, 300–301

Index

long-term bone marrow culture (LTBMC), acquired aplastic anaemia, 208 long-term culture-initiating cell (LTC-IC) assays, 2 long-term dialysis, folate deficiency, 79 low-molecular weight heparin (LMWH), 764, 904 activated prothrombin time monitoring, 907 antiphospholipid syndrome management, 895 thalidomide side effects, 596 thrombolytic prophylaxis, 909, 911 in cancer, 912 in pregnancy, 912–913 venous thromboembolism, 911, 912 venous thromboembolism therapy, 906, 907 low-oxygen affinity variants, haemoglobin disorders, 107 LRF CLL4 trial, 536 LTA gene AML, 388 CML, 388 LTA (light transmission aggregometry), platelet function testing, 789 LTBMC (long-term bone marrow culture), acquired aplastic anaemia, 208 LTC-IC (long-term culture-initiating cell) assays, 2 LUC7L gene, α thalassaemias, 100 lumbar punctures, adult ALL, 436–437 lumiliximab, CLL management, 547 lung cancer, Hodgkin lymphoma treatment, 651–652 lung disease, autologous SCT complications, 741 lung(s), hereditary haemorrhagic telangiectasia, 854 lung resistance protein (LRP), AML relapse risk, 428 lupus anticoagulant (LA), antiphospholipid syndrome, 892–893 Lutheran blood group, 245, 259 antigen (CD239), 228, 229 LW glycoprotein, biological significance, 229 LY03 trial, MALT lymphoma, 670 lymphadenopathy African trypanosomiasis, 965–966 NHL, 659 lymphatic organization, spleen, 370 lymph, flow of, 355 lymph node(s), 355 biopsy classical Hodgkin lymphoma, 640 multicentric Castleman disease, 680

nodular lymphocyte-predominant Hodgkin lymphoma, 641 enlargement, CLL, 534 follicles, 355, 355 germinal centres, 355–356 high endothelial venules, 355, 355 histology, peripheral T-NHL not specified, 570 medullary cords, 355, 355 medullary cortex, 355, 355 section, Sézary syndrome, 566 structure, 355 T zones, 355, 355 WHO lymphoma classification, 615 lymphoblastic lymphoma, treatment, 679 lymphocyte(s), 343–367 antigen recognition, 344–346 clonal selection theory, 344–346 CML, 490 counts, 359, 363–364, 364 active immune response, 363 T-prolymphocytic leukaemia, 559 malignancies, 944 myelodysplastic syndrome, 513 see also leucocyte(s); specific cell types lymphocytosis non-malignant causes, 364 polyclonal B-cell see polyclonal B-cell lymphocytosis lymphohistiocytosis, haemophagocytic see haemophagocytic lymphohistiocytosis lymphoid organs, secondary, 344 lymphoid primed multipotent progenitor (LMPP), 2 lymphoma(s) B-cell activated B cell-like DLBCLs, 623 borderline DLBCLs and Burkitt lymphoma, 626 WHO lymphoma classification, 626 cutaneous B-cell lymphoma, 683 DLBCLs see diffuse large B-cell lymphomas (DLBCLs) germinal-centre B cell-like DLBCLs, 623 intermediate DLBCL and classical Hodgkin lymphoma, 626–627 intravascular large B-cell lymphoma see intravascular large B-cell lymphoma large B-cell lymphoma, 660 large-cell lymphomas of terminally differentiated B cells, 625 marginal zone B-cell lymphomas, 661, 670–671

primary cutaneous DLBCLs, leg type, 624–625 primary cutaneous large B-cell lymphoma, leg type, 683 primary mediastinal B-cell lymphoma see primary mediastinal B-cell lymphoma T-cell/histiocyte-rich large B-cell lymphoma, 624 Burkitt see Burkitt lymphoma development see leukaemogenesis differential diagnosis, acquired aplastic anaemia vs., 214 HIV infection, 955 malignancies, 944 molecular basis, 380–394 p15INK4b gene deletion, 394 p16INK4a gene deletion, 394 T-cell adult T-cell leukaemia lymphoma (ATLL) see adult T-cell leukaemia lymphoma (ATLL) angioimmunoblastic T-cell lymphoma (AITL) see angioimmunoblastic T-cell lymphoma (AITL) enteropathy-associated T-cell lymphoma (EATL) see enteropathy-associated T-cell lymphoma (EATL) enteropathy-associated T-cell NHL, 573–574 extranodal NK/T-cell lymphoma, nasal type see extranodal NK/Tcell lymphoma, nasal type hepatosplenic T-cell lymphoma see hepatosplenic T-cell nonHodgkin lymphoma hepatosplenic T-cell NHL, 571 peripheral T-cell lymphomas (PTCLs) see peripheral T-cell lymphomas (PTCLs) primary cutaneous γδ T-cell lymphomas, 630–631 primary cutaneous T-cell lymphomas, 682–683 subcutaneous panniculitis-like T-cell lymphoma see subcutaneous panniculitis-like T-cell lymphoma T-cell/histiocyte-rich large B-cell lymphoma, 624 T-cell NHL see T-cell non-Hodgkin lymphoma (T-NHLs) WHO classification see World Health Organization (WHO), lymphoma classification lymphomatoid granulomatosis, treatment, 680 1031

Index

lymphomatoid papulosis mycosis fungoides, 565 WHO lymphoma classification, 630 lymphopenia, 364 causes, 365 corticosteroids, 364 lymphoplasmacytic lymphoma, WHO lymphoma classification, 617 lymphoproliferative disease cold-type AIHA, 165 warm-type AIHA, 162 LYN gene, CML, 488 lysosomal storage disorders, 330–342 biochemistry, 339 clinical manifestations, 336–341, 337–338 diagnosis, 331–332 pathophysiology, 330–331 prevalence, 331 prognosis, 336 therapy, 332, 336 see also specific diseases/disorders lysosome-associated membrane proteins (LAMPs), 330 lysosomes, 330 definition, 330 enzyme cycling, 331 functions, 330 platelets, 774 lysozyme, neutrophils, 307 LYST gene, 317, 925 lytic phase, complement, 235–236 M macrocytosis cobalamin deficiency, 75 liver disease, 948 α2-macroglobulin, 765 macroglobulinaemia, Waldenström see Waldenström macroglobulinaemia macroglossia, systemic AL amyloidosis, 602, 602 macrophage(s), 323–327 activation, 323–324 apoptotic cell clearance, 323 complement receptors, 301 disorders, 324–327 see also specific diseases/disorders spleen, 371 structure, 308 macrophage colony-stimulating factor (M-CSF) macrophage activation, 323 neutrophil development, 308 macrophage mannose receptor (MMR), 303–304 1032

MAFB gene, 578 MAF gene, 578, 579 Maf recognition elements (MAREs), erythropoiesis transcription factors, 16 MAG91 study, multiple myeloma autologous SCT, 591 magnetic resonance imaging (MRI) clonal hypereosinophilic syndromes, 706, 706 deep vein thrombosis diagnosis, 903 haemochromatosis, type 1, 50 Hodgkin lymphoma, 644 iron overload, 54, 55, 55 multiple myeloma, 588 NHL, 662 pulmonary embolism diagnosis, 903 spleen, 372, 373 systemic AL amyloidosis, 607 β-thalassaemia management, 95 MAHA see microangiopathic haemolytic anaemia (MAHA) MAIA, malignancy treatment, 945 MAIGA assay (monoclonal antibody immobilization of granulocyte antigens), 264 maintenance therapy adult ALL chemotherapy, 440 follicular lymphoma therapy, 666–667 multiple myeloma management/ treatment, 592–593 MAIPA assay see monoclonal antibodyspecific immobilization of platelet antigens (MAIPA) assay major basic protein (MBP), basophil/mast cell granules, 322 major histocompatibility complex(es) (MHC) autoimmunity, 347 polymorphisms, 346–347, 347 primary immune thrombocytopenia, 929 T cell antigen recognition, 344–345 see also HLA major histocompatibility complex class I (MHC class I) antigen presentation, 344–345, 345, 346 CD8 T-cell antigen recognition, 345, 345 see also HLA class I major histocompatibility complex class II (MHC class II) antigen presentation, 345, 345, 346 B cell development, 349 T-cell-dependent B-cell activation, 357 see also HLA class II malabsorption folate deficiency, 78 iron deficiency anaemia, 40

malaria, 952, 956–962 anaemias, 952, 959 acquired haemolytic anaemias, 168 biology, 956–958 clinical features, 958 diagnosis, 960–961 epidemiology, 956–958 genetic protection mechanisms, 959–960 haematological abnormalities, 959 HLA-B*53:01 association, 713 life cycle, 957, 957–958 Plasmodium falciparum, 958, 958 Plasmodium malariae, 958 Plasmodium ovale, 958–959 Plasmodium vivax, 958–959 resistance to glucose-6-phosphate dehydrogenase deficiency, 150 hereditary elliptocytosis, 134 sickle cell disease, 109 splenectomy, 376 splenomegaly, 372 transmission, 957 blood transfusions, 274 treatment, 961–962 malignancies, 942–945 acquired circulating anticoagulants, 945, 945 anaemia, 940, 942, 942–943 coagulation changes, 944–945, 945 DIC, 944–945 Fanconi anaemia, 186 folate deficiency, 65 granulocytopenia, 944 granulocytosis, 943–944 microangiopathic haemolysis anaemias, 171–172 platelets, 944 polycythaemia, 943 post-haematopoietic SCT, 222–223 primary fibrinolysis, 945 splenomegaly, 372 thrombocytopenia, 944, 944 thrombocytosis, 944 treatment-induced bleeding disorders, 945 venous thromboembolism see venous thromboembolism (VTE) white cell changes, 944, 944–945 malnutrition, hereditary spherocytosis, 132 maloprim, acquired haemolytic anaemias, 173 MALT1 gene, 619 MALT lymphoma, 670–671 α-mannosidosis, 334 clinical features, 337

Index

β-mannosidosis, 334 clinical features, 337 mannosyl/fucosyl receptors, phagocytes, 303–304 Mansonella ozzardi infection, 964 Mansonella perstans infection, 963 M antigen, 228 mantle cell lymphoma, 555–556, 622 ATM gene deletion, 386, 393 B-cell prolymphocytic leukaemia vs., 550 bone marrow trephine biopsy, 556 cellular origins, 384 cyclin D1 gene, 393 cytology, 661 FISH, 407 genetics, 659 IGH gene, 384 immunohistochemistry, 404 management, 672–673 p16INK4A gene deletion, 393 prognostic factors, 662 WHO lymphoma classification, 621–622 MAPK pathways, Fanconi anaemia, 191 Marburg virus, 969 March haemoglobinuria, 172 MAREs (Maf recognition elements), erythropoiesis transcription factors, 16 marginal zone B-cell(s), 352–353, 353 lymphomas, 661, 670–671 spleen, 371 marginating pool, monocytes, 323 Maroteaux–Lamy syndrome see mucopolysaccharidosis type VI (Maroteaux–Lamy syndrome) massive blood loss, coagulopathy, 853 massive blood transfusions, 282 complications, 288–289 DIC vs., 844 mass spectrometry, systemic AL amyloidosis, 606 mast cell(s), 321–323 development, 321 disorders, 321–323 FcεIR, 321 functions, 321 granules, 322 leukaemia, 322–323 proliferation, 322 mastocytosis, 701–704 classification, 701 clinical features, 702 urticaria pigmentosa, 701, 702, 702 investigations, 702 pathophysiology, 701

prognosis, 704 treatment, 702–704 matched unrelated donor stem cell transplantation, adult ALL, 441, 441 maternal alloantibodies, haemolytic disease of the fetus and newborn, 292–293 maternal anti-D antibody, haemolytic disease of the fetus and newborn, 298 maternal blood assessment, Rhesus haemolytic disease of the fetus and newborn, 294 matriptase-2 (TMPRSS6), 30, 32 iron refractory iron deficiency anaemia, 41 mature B-cell acute lymphoblastic leukaemia, 434 adult ALL prognosis, 444 mature B-cell neoplasms see World Health Organization (WHO), lymphoma classification mature NK-cell/T-cell neoplasms see World Health Organization (WHO), lymphoma classification mature T-cell acute lymphoblastic leukaemia, 434 maximum surgical blood ordering schedule (MSBOS), 281 May–Hegglin anomaly, 918, 918 genetics, 925 neutrophils, 311, 312 m-BACOD, AIDS-related NHL, 681 MBI (mucosal barrier injury), neutropenic fever, 470, 470 MBL see monoclonal B-cell lymphocytosis (MBL) MBP (major basic protein), basophil/mast cell granules, 322 MCFD2 gene, 814, 821, 823 MCH see mean corpuscular haemoglobin (MCH) MCL1 gene, CLL, 533 MCL2 study, mantle cell lymphoma, 673 McLeod phenotype, 136–137, 137 McLeod syndrome, CYBB gene, 260 MCP-1, 363 M-CSF see macrophage colony-stimulating factor (M-CSF) MCV see mean corpuscular volume (MCV) MDS see myelodysplastic syndrome (MDS) mean corpuscular haemoglobin (MCH) hereditary spherocytosis, 131 pyruvate kinase deficiency, 144, 144 β-thalassaemias, 93

mean corpuscular volume (MCV) HbF effects, 122 hereditary spherocytosis, 131 iron deficiency anaemia, 39 iron metabolism measurement, 37 megaloblastic anaemia, 62–63, 66 neonatal anaemia, 975 β-thalassaemias, 93 mean platelet volume (MPV), platelet function testing, 789 mechanical acquired haemolytic anaemias, 170, 170, 170–172 median age blood vessels, 752–753 CLL, 531 median survival, CLL, 538 mediastinal mass, Hodgkin lymphoma, 642 Medical Research Council (MRC), multiple myeloma prognosis, 590 Mediterranean macrothrombocytopenia, 918 genetics, 925 medullary cords, lymph nodes, 355, 355 medullary cortex, lymph nodes, 355, 355 mefenamic acid drug-induced alloimmune haemolytic anaemia, 167 warm-type AIHA, 162 mefloquine, malaria treatment, 962 MEGA (Multiple Environmental and Genetic Assessment), 879 megakaryocytes, 515 myelodysplastic syndrome, 513 platelets formation, 775 primary immune thrombocytopenia, 933 megaloblastic anaemia, 61–82 biochemistry, 61–62 causes, 81 clinical features, 62–66 cardiovascular disease, 65 mean corpuscular volume, 62–63 folate, 62 haematological findings, 66–68 bone marrow, 67, 67 chromosomes, 67 haemopoiesis, 67–68 peripheral blood, 66–67, 67 treatment, 79–80 see also cobalamin deficiency; folate deficiency megathrombocytopenia, 918–919 melarsoprol, African trypanosomiasis therapy, 966 1033

Index

melphalan with carmustine, etoposide and cytarabine see BEAM (carmustine, etoposide, cytarabine and melphalan) with prednisone, multiple myeloma management/treatment, 591 SCT conditioning allogeneic SCT, 521 autologous SCT, 729 with fludarabine, 732 systemic AL amyloidosis management, 608, 609 memory T cells, allogeneic SCT, 745 menadiol, acquired haemolytic anaemias, 173 menorrhagia factor V deficiency, 818 factor VII deficiency, 823 factor X deficiency, 825 menstruation, iron homeostasis, 33 mental development, iron deficiency anaemia, 39 mental retardation syndrome, with α thalassaemias, 103–104 mercaptopurine adult ALL chemotherapy, 440 childhood ALL, 455 childhood ALL treatment, 458 mesenchymal stem/progenitor cells, haematopoiesis, 7 mesodermal precursor cells, HSC development, 4, 5 metabolic diseases leukaemias see leukaemias, supportive care neutropenia, 316 metachromatic leucodystrophy, 334 clinical features, 337 haematopoietic SCT, 332 prevalence, 331 metaclopramide, antiemetic effects, 481 methaemoglobulinaemia acquired haemolytic anaemias, 173–174 congenital, 107 erythrocytosis, 692–693 methicillin-resistant Staphylococcus aureus (MRSA), 472 methionine synthase reductase, childhood ALL prognosis, 455 methotrexate adult ALL chemotherapy, 439, 440 childhood ALL prognosis, 455 childhood ALL treatment, 457, 458 with ciclosporin and mycophenolate mofetil, SCT reduced intensity conditioning regimens, 732 1034

primary CNS lymphoma, 676–677 Sézary syndrome/mycosis fungoides, 567 T-cell large granular lymphocyte leukaemia, 563 methylene blue, fresh frozen plasma preparation, 279 5,10-methylene tetrahydrofolate polyglutamate, megaloblastic anaemia, 61, 62 5,10-methylene tetrahydrofolate reductase (MTHFR) childhood ALL prognosis, 455 deficiency, 879 neural tube defects, 65 methylmalonate, cobalamin deficiency, 75 methylmalonic acidaemia, congenital, 74 methylmalonic aciduria, congenital, 74 methylprednisolone acute GvHD therapy, 735 primary immune thrombocytopenia in pregnancy, 938 MGUS see monoclonal gammopathy of undetermined significance (MGUS) mHA see minor histocompatibility antigens (mHA) MHC see major histocompatibility complex(es) (MHC) microangiopathic haemolytic anaemia (MAHA), 171–172 causes, 171 DIC vs., 844 malignancies, 942 microarrays Burkitt lymphoma, 626 erythroid progenitor development, 13–14 erythropoiesis, 18 gene expression, 412–413 see also gene expression profiling (GEP) high-density, AML classification, 430– 431, 431 molecular blood grouping techniques, 242 multiple myeloma, 579 NHL, 659 microbicidal proteins, neutrophils, 307 microcolumns, agglutination reactions, 241 microcolumn tests, blood group screening, 240, 240 microcytosis, myelodysplastic syndrome, 512 β2-microglobulin amyloidosis, 599–600, 611 CLL, 540 multiple myeloma, 589

microplates agglutination reactions, 241 blood group screening, 240–241 microRNAs, CLL, 541–542 microscopy malaria diagnosis, 960 sideroblastic anaemia, 43 microthrombotic disease, 855 mild common hereditary elliptocytosis, 134, 134 minimal residual disease (MRD) adult ALL, 435 flow cytometric immunophenotyping, 402 prognosis, 444–445 AML, 428–429 childhood ALL, 459 prognosis, 455 CLL management, 545 molecular genetic analysis, 411 quantitative PCR, 410 minor histocompatibility antigens (mHA), 720 allogeneic SCT, 723 minor red cell abnormalities, blood transfusion donor, 268–269 MIP-1α, 363 eosinophils, 304 MIR15 gene, 542 MIR16 gene, 542 MIR34α gene, 542 miscarriage see spontaneous abortion missense mutations factor V deficiency, 818 RHD gene, 256 mitochondria haem synthesis, 35 myopathy, sideroblastic anaemia, 44 sideroblastic anaemia, 44–45 mitoferrin, 29 mitoxantrone AML treatment, 421 with cyclophosphamide and fludarabine, follicular lymphoma therapy, 666 mixed cryoglobulinaemia, essential, hepatitis C virus, 687 mixed lymphocyte culture (MLC) assay, HLA typing, 719–720 MLC (mixed lymphocyte culture) assay, HLA typing, 719–720 MLL, erythropoiesis, 15 MLL gene early pre-B ALL, 451 myelodysplastic syndrome, 510 rearrangements, childhood ALL, 453, 454, 455 MMR (macrophage mannose receptor), 303–304

Index

MMSET gene, 579 MNS blood group, 245, 259 molecular genetics, 410–413 acute lymphoblastic leukaemia, 411 adult ALL diagnosis, 434–435 childhood ALL see acute lymphoblastic leukaemia (ALL), childhood chromosomal rearrangement, 411 CLL, 411–412 gene mutations, 411 MRD, 411 prognostic stratification, 411–412 TCR chain rearrangement, 411 molecular mimicry, primary immune thrombocytopenia, 930 Monge disease, erythrocytosis, 692 monoclonal antibodies, 233 advanced follicular lymphoma treatment, 665–667, 666 AML minimal residual disease, 429 blood grouping, 233 CLL management, 546, 546 see also specific antibodies monoclonal antibody immobilization of granulocyte antigens (MAIGA) assay, 264 monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay, 264–265 primary immune thrombocytopenia, 930 monoclonal antibody therapy cold-type AIHA, 165 flow cytometric immunophenotyping, 403 Waldenström macroglobulinaemia, 672 warm-type AIHA treatment, 163–164 see also specific antibodies monoclonal B-cell lymphocytosis (MBL) CLL, 543–544 WHO lymphoma classification, 615 monoclonal gammopathy of undetermined significance (MGUS), 582, 601 multiple myeloma, 579 WHO lymphoma classification, 618 monoclonal immunoglobulin, multiple myeloma, 577–578 monoclonal light chains, systemic AL amyloidosis, 601 monocytes, 323–327 circulating, 323 CML, 490 complement receptors, 301 disorders, 324–327 see also specific diseases/disorders malignancies, 944

marginating pool, 323 structure, 308 tissue factor, 760 monocytopenia, 324 monocytosis, 324, 324 myelodysplastic syndrome, 513 monosomy 13, multiple myeloma, 579 monospot test, infectious mononucleosis, 366 Montreal platelet syndrome, 918–919 MOPP (mustine, vincristine, procarbazine and prednisolone) classical Hodgkin lymphoma treatment, 647 Hodgkin lymphoma treatment, 645–646 Morquio syndrome see mucopolysaccharidosis type IV (Morquio syndrome) mouse models acquired aplastic anaemia, 209, 211 Fanconi anaemia, 191 moyamoya transformation, sickle cell disease, 115 MPL gene congenital amegakaryocytic thrombocytopenia, 204 essential thrombocythaemia, 693 primary myelofibrosis, 697 M-protein, multiple myeloma pathogenesis, 582 MPV (mean platelet volume), platelet function testing, 789 MRC PT-1 trial, essential thrombocythaemia treatment, 696 MRD see minimal residual disease (MRD) MRI see magnetic resonance imaging (MRI) mRNA transcription/processing, haemoglobin synthesis, 86, 87 MRSA (methicillin-resistant Staphylococcus aureus), 472 MSBOS (maximum surgical blood ordering schedule), 281 MTHFR see 5,10-methylene tetrahydrofolate reductase (MTHFR) MTHFR gene, 455 MTTRR gene, 455 mucolipidoses, 333 clinical manifestations, 337 prevalence, 331 see also specific types mucolipidosis type I (sialidosis I), 333 clinical features, 337 mucolipidosis type II (I cell syndrome), 333 clinical features, 337

mucolipidosis type III (pseudo-Hurler syndrome), 333 clinical features, 337 mucolipidosis type IV, 333 clinical features, 337 mucopolysaccharidoses, 330, 333 clinical manifestations, 337 see also specific diseases/disorders mucopolysaccharidosis type I (Hurler Scheie disease), 333 clinical features, 337 therapy, 332 mucopolysaccharidosis type II (Hunter syndrome), 333 clinical features, 337 enzyme replacement therapy, 332 mucopolysaccharidosis type III (Sanfilippo syndrome), 333 clinical features, 337 enzyme replacement therapy, 332 pathophysiology, 330–331 mucopolysaccharidosis type IV (Morquio syndrome), 333 clinical features, 337 enzyme replacement therapy, 332 mucopolysaccharidosis type VI (Maroteaux–Lamy syndrome), 333 clinical features, 337 mucopolysaccharidosis type VII (Sly syndrome), 333 clinical features, 337 mucopolysaccharidosis type X, 333 mucosal-associated lymphoid tissue lymphoma see MALT lymphoma mucosal barrier injury (MBI), neutropenic fever, 470, 470 mucosal factors, iron absorption, 34 mucosal tract bleeding acquired aplastic anaemia, 211 factor XIII deficiency, 829 mucositis, allogeneic SCT complications, 737 multicentric Castleman disease, 680 HIV infection, 657 multidrug resistance protein 1, AML relapse risk, 428 multimer analysis, von Willebrand disease, 807 Multiple Environmental and Genetic Assessment (MEGA), 879 multiple myeloma, 577–598 aetiology, 577 bone marrow aspirates, 397, 400 cellular origins, 384 clinical manifestations, 585 coagulation disorders, 856 1035

Index

definition, 577 differential diagnosis, 582–583 disease complications, 584–588 anaemia, 587 bone marrow failure, 587 infections, 588 nervous system, 588 renal failure, 586–587 skeletal involvement see below epidemiology, 577 flow cytometric immunophenotyping, 403 genetic abnormalities, 578–580 1q gains, 579 17p deletion, 579 aneuploidy, 579 CCND1, 578, 580 CCND2, 580 CCND3, 578, 580 cyclin D genes, 580 FGFR3, 579 FGFR/MMSET, 578 FISH, 578–579 IGH translocations, 578, 578–579 karyotypic instability, 578 late events, 579–580 MAF, 578, 579 MAFB, 578 MGUS display, 579 microarray analysis, 579 MMSET, 579 molecular classification, 580 monosomy 13, 579 IgM-type, 584 laboratory work-up, 583 management/treatment, 584–588, 591–597 allogeneic SCT, 593 aurora kinase inhibitors, 596 autologous SCT, 587, 591–592, 592 bisphosphonates, 584–585, 586 bortezomib, 593, 595, 595 clodronate, 585 cyclin-dependent cyclase inhibitors, 596 cyclophosphamide/dexamethasone, 586 dexamethasone, 586, 588 elderly patients, 593–594, 594–595 FGFR3 tyrosine kinase inhibitors, 596 induction therapy, 591, 592 lenalidomide, 591, 592–593, 595, 595 local radiation therapy, 588 maintenance therapy, 592–593 melphalan–prednisone, 591 newly diagnosed transplant candidates, 591–593 1036

non-transplant candidates, 593–594 pamidronate, 585 plasma cell receptor blockers, 596 proteasome inhibitors, 596 at relapse, 594–596, 595 signalling pathway inhibitors, 596 thalidomide, 591, 592, 593, 595, 595 VAD, 586, 591 zoledronic acid, 584–585, 585–586 molecular classification, 580 non-secretory, 584 pathogenesis, 577–582 drug-resistance phenotype, 581 induction, 581–582 monoclonal Ig, 577–578 myeloma cell origin, 578 plasma cell–microenvironment interaction, 580–581, 581 signalling pathways, 581, 581 prognostic factors, 588–591, 589 disease complications, 589–590 host factors, 588 malignant clone factors, 589 therapy response, 590–591 tumour burden, 589–590 skeletal involvement, 584–586 bone pain, 584 hypercalcaemia, 584 osteonecrosis, 586 smouldering, 582–583 symptomatic, 583, 583 multiple sclerosis, infectious mononucleosis, 367 multiple sulphate deficiency, 335 clinical features, 338 multispecies consensus regions, α-globin chains haemoglobin, 85–86 MUM1, primary cutaneous DLBCL, leg type, 624 µ heavy chain, B cell development, 349–350 Munc13–4, haemophagocytic lymphohistiocytosis, 326 Munc-18, platelet activation, 786 mustine, with vincristine, procarbazine and prednisolone see MOPP (mustine, vincristine, procarbazine and prednisolone) mutations factor VII deficiency, 825 factor X deficiency, 825–826, 826 leukaemogenesis, 381 MYC gene Burkitt lymphoma, 626, 657 CML, 488 DLBCL not otherwise specified, 623 multiple myeloma, 579

nodular lymphocyte predominant Hodgkin lymphoma, 636 translocation, NHL, 658 MYC–IGHG1 fusion gene, FISH, 407 mycophenolate mofetil allogeneic SCT, 744 ATG with, 218 with ciclosporin and methotrexate, 732 CLL, 548 with methotrexate and ciclosporin, 732 primary immune thrombocytopenia management, 934 SCT reduced intensity conditioning regimens, 732 warm-type AIHA treatment, 163 Mycoplasma infections, 951 sickle cell disease, 116 Mycoplasma pneumoniae infections cold-type AIHA, 165 sickle cell disease, 114 mycosis fungoides, 565–567, 682 aetiology, 565–566 clinical course, 567 clinical features, 566 cytogenetics, 567 diagnosis, 566–567 differential diagnosis, 567 prognosis, 567 staging, 682 treatment, 567, 682 WHO lymphoma classification, 630, 630 see also Sézary syndrome myelodysplasia with isolated del(5q), WHO classification, 506, 506 malignancy therapy, 943 with α thalassaemias, 104 myelodysplastic 5q–syndrome, 386–387 cytogenetics, 407, 509 myelodysplastic/myeloproliferative disease, 526–528 see also specific diseases/disorders myelodysplastic syndrome (MDS), 503–529 acquired aplastic anaemia vs., 214 aetiology, 504 Diamond–Blackfan anaemia, 198 epigenetic abnormalities, 512 erythropoietin, 468 essential thrombocythaemia, transformation of, 694 malignancies, 944 post-ATG, 220 severe congenital neutropenia, 314 apoptosis, 508 childhood, 528

Index

classification, 504–507 FAB classification, 504, 504–505 WHO classification, 505, 505–507 clinical features, 512 cytogenetic analysis, 406 diagnosis, 512–513 blood film, 399 bone marrow aspirates, 397, 400 bone marrow histology, 513, 514 bone marrow morphology, 513 bone marrow trephine biopsy, 515 FISH, 507 flow cytometric immunophenotyping, 404 future work, 528 genetic abnormalities, 510–512, 511 AML1–EVI1 gene fusion, 510 AML1 gene mutations, 510 ASXL1 gene, 512 ATRX gene, 510 CBL gene, 511–512 CEBPA gene, 510 comparative genome hybridization, 510–511 CSF1R gene, 510 expression profiling, 510 FLT3 gene, 510 GATA1 gene, 510 GCSFR gene, 510 JAK2 gene, 510 KIT gene, 510 KRAS gene, 391 MLLgene, 510 NRAS gene, 391, 510 PDGFRB gene, 510 PU.1 gene, 510 RAS gene mutations, 510 single-nucleotide polymorphisms, 510 TERC mutations, 195 TET2 gene, 511 TP53 gene, 510 uniparental disomy, 510 historical aspects, 503–504 immunological abnormalities, 507–508 incidence, 504 International Prognostic Scoring System, 516, 516–517 management indicators, 525–526 median survival, 517 outcomes, 517 WHO classification-based prognostic scoring system vs., 517–518 management/treatment, 519–524 allogeneic SCT, 521–522, 522, 522 chelation therapy, 519 chemotherapy, 520–521 goals, 524–525

growth factors, 519–520 hypomethylating drugs, 522–523, 523 immunosuppression, 520 IPSS intermediate-1, 525 IPSS intermediate-2/high, 525–526 IPSS low, 525 lenalidomide, 523–524 response assessment, 525 SCT, 519, 520–521 strategy, 524, 524–526 supportive care, 519 molecular basis, 508–512 cytogenetics, 508, 508–510 natural history, 513, 516 pathogenesis, 507–508 subtype frequencies, 505 WHO classification-based prognostic scoring system, 505, 505–507, 517, 518, 519 cumulative survival, 518 IPSS vs., 517–518 leukaemic transformation, 518 median survival, 518 overall survival, 518 see also specific diseases/disorders myelofibrosis essential thrombocythaemia, transformation of, 694 haemophagocytic lymphohistiocytosis, 952 polycythaemia vera, 689, 689 primary see primary myelofibrosis myelofibrotic myodysplasia syndrome, 516 myeloid malignancies BCR–ABL fusion gene, 382–383, 383 leukaemogenesis, 382–383 myeloma cell origin, multiple myeloma, 578 myeloma kidney, 586 myeloma, multiple see multiple myeloma myeloperoxidase deficiency, neutropenia, 318 myeloproliferative neoplasms, 686–709 acquired von Willebrand disease, 811 definition, 686 see also polycythaemias myeloproliferative syndromes, WHO classification, 484 MYH9 gene Epstein syndrome, 925 Fetchner syndrome, 925 May–Hegglin anomaly, 925 Sebastian syndrome, 925 MYH11–CBFB, AML, 381 Mylotarg see gemtuzumab ozogamicin (Mylotarg) myoglobin, structure, 27

Myozyme (alglucosidase alfa), Pompe disease, 341 N NADPH oxidase, phagocytes, 305 NADPH: quinone oxidoreductase polymorphisms, 449 NAITP (neonatal alloimmune thrombocytopenia), 298, 981–982 naive B cells, 352 N antigen, 228 narrowband UVA (NB-UVA), mycosis fungoides treatment, 682 NAT (nucleic acid test), HIV blood screening, 273 National Amyloidosis Centre, systemic amyloidosis, 610–611 natural killer (NK) cell(s), 344, 354 disorders, neutropenia, 316 leukaemia, cytogenetic analysis, 406 lytic mechanisms, 354, 354 SCT rejection, 723 tumour response, 712 viral infection response, 712 natural killer T cells, 354–355 nausea and vomiting, leukaemia, 464, 481 NB-UVA (narrowband UVA), mycosis fungoides treatment, 682 NCIC/ECOG study, classical Hodgkin lymphoma treatment, 648 NCRI AML 16 trial, 525–526 Ncx1 gene, haematopoiesis, 5 negative selection, T cell development, 351 Neisseria meningitidis infection spleen, 371 splenectomy risks, 133, 375, 376 nelarabine, lymphoblastic lymphoma treatment, 679 neonates anaemia see neonatal anaemia autoimmune thrombocytopenia, 982 congenital neutropenias, 979 exchange transfusion, haemolytic disease of the fetus and newborn, 975 haematology see neonatal haematology haemochromatosis, 52 hereditary elliptocytosis, 134 hereditary pyropoikilocytosis, 134 polycythaemia, 978–979 causes, 978 rare bleeding disorders, 814–815 screening, sickle cell disease, 119–120 stroke, 882–883 thalassaemia screening, 106 ‘top-up’ blood transfusions, 282 1037

Index

neonatal alloimmune thrombocytopenia (NAITP), 298, 981–982 neonatal anaemia, 971–979 anti-D antibodies, 293 causes, 972, 972–977 blood loss, 977 haemoglobinopathies, 976–977 immune haemolysis, 974–975 parvovirus B19 infection, 973 red cell aplasia, 973–974 red cell destruction, 974–977 red cell enzymopathies, 976 red cell membrane disorders, 975–976 reduced red cell production, 972 definition, 971–972 diagnosis, 978 algorithm, 973 pathophysiology, 971–972 red cell transfusion, 978 neonatal haematology, 971–984, 979 acquired disorders, 980–981 acquired thrombotic disorders, 983 anaemia see neonatal anaemia haemoglobins, 972 haemostasis, 979–983 inherited disorders, 980–981 inherited thrombotic disorders, 982–983 thrombosis, 979–983 white cell disorders, 979 neonatal jaundice syndrome, glucose-6phosphate dehydrogenase deficiency, 150–151, 151 nephrectomy, anaemia, 947 nerve root compression, multiple myeloma, 588 nervous system, multiple myeloma, 588 neural tube defects (NTDs), 64–65 neurodegeneration, iron overload, 53 neuroferritinopathy, 53 neurological features cobalamin deficiency, 66 folate deficiency, 66 hereditary haemorrhagic telangiectasia, 854 polycythaemia vera, 688 sickle cell disease, 114, 116 neutropenias, 313–316 adult ALL, 436, 438 AML treatment, 422, 422 aplastic anaemia, 316 autoimmune, 315 autologous SCT complications, 741 causes, 314 cellular immune responses, 316 Chédiak–Higashi syndrome, 317 childhood ALL, 450 chronic benign, 315 1038

chronic granulomatous disease, 317–318 chronic idiopathic, 315 CLL, 549 collagen vascular disorders, 316 congenital, 202 congenital neonatal, 979 CXCR4 overexpression, 316 cyclic, 314–315 deferiprone, 58 definition, 313 drug-induced, 316 ELA2 gene, 202 Felty syndrome, 316, 946 function disorders, 316–318, 317 genetic subtypes, 202 hairy cell leukaemia, 551 HIV infection, 954–955 humoral immune responses, 316 hyper-IgM syndrome, 315 imatinib mesylate, 492 infections in leukaemia, 469 infectious mononucleosis, 365 malignancies, 944 metabolic diseases, 316 myelodysplastic syndrome, 512 myeloperoxidase deficiency, 318 natural killer cell disorders, 316 neonates, 979 nutritional deficiencies, 316 Papillon–Lefevre syndrome, 318 peripheral destruction, 313 pure white cell aplasia, 315 reticular dysgenesis, 315 rheumatoid arthritis, 316 specific granule deficiency, 318 splenomegaly, 370 systemic lupus erythematosus, 316 thalidomide side effects, 596 tropical diseases, 968 viral infections, 316 WHIM syndrome, 316 X-linked agammaglobulinaemia, 315 neutropenic fever see leukaemias, supportive care neutrophil(s), 307–318 Alder–Reilly anomaly, 311, 312 antigens, 263, 263–264 apoptosis, 312 azurocidin, 307 azurophil granules, 308–309, 310 bacterial permeability-increasing protein, 307 Barr bodies, 310–311, 311 cathepsin G, 307 Chédiak–Higashi syndrome, 311, 312 chemoattractant receptors, 304 counts, ethnic variations, 956, 957

development, 307–312, 308 disorders, 312–318 see also specific diseases/disorders function, 307–312 granules, 308–309, 310 hypersegmented, cobalamin deficiency, 75 infectious organism clearance, 309 lifespan, 309 malaria, 959 May–Hegglin anomaly, 311, 312 microbicidal proteins, 307 Pelger–Huët anomaly, 310, 311, 312 peroxisome proliferator-activated receptor-γ, 309 priming, 309–310 structure, 308 neutrophil elastase, 202 neutrophilia, 312, 312–313 Brucella infections, 951 infections, 951 malignancies, 944 Rickettsia infections, 951 Salmonella infections, 951 neutrophilic chronic myeloid leukaemia, 707 new integrated risk classification, adult ALL prognosis, 445 newly diagnosed transplant candidates, multiple myeloma management/ treatment, 591–593 New York study, classical Hodgkin lymphoma treatment, 648 NF1 gene, 391, 501, 527 NF-E2, globin gene clusters, 86 NFκB pathway DLBCL not otherwise specified, 623 Hodgkin lymphoma, 641–642 NHL see non-Hodgkin lymphoma (NHL) nicotinic acid deficiency, 81 Niemann–Pick disease(s), 341 see also specific types Niemann–Pick disease A, 334 clinical features, 337 Niemann–Pick disease B, 334 Niemann–Pick disease C, 335 nifurtimox, American trypanosomias, 967 Nijmegen breakage syndrome (NBS), 191 nilotinib, CML management, 494, 495 nitrates, acquired haemolytic anaemias, 173 nitric oxide (NO) endothelium interactions, 753, 754 paroxysmal nocturnal haemoglobinuria, 177, 179 phagocytes, 305–306 platelet inhibition, 772, 787–788

Index

sickle cell disease, 111, 112 vasodilation, 111, 112 nitric oxide synthase (NOS), 305–306 nitrites, acquired haemolytic anaemias, 173 nitrogen mustard, mycosis fungoides treatment, 682 nitrous oxide inhalation, cobalamin deficiency, 74 NK cells see natural killer (NK) cell(s) NKG2D, natural killer cells, 354 NLPHL see nodular lymphocytepredominant Hodgkin lymphoma (NLPHL) NMSG, multiple myeloma management/ treatment, 594 NO see nitric oxide (NO) nodal marginal zone lymphoma, 619 nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL), 640 clinical presentation, 642–643 prognosis, 644 treatment, 647 WHO lymphoma classification, 635, 636 non-cultural screening, infections in leukaemia, 471–472 non-gastric MALT lymphomas, 671 non-haemolytic febrile transfusion reactions, blood transfusions, 286 non-Hodgkin lymphoma (NHL), 655–685 aetiology, 656–658 AIDS-related, 681–682 chromosomal translocations, 657 Epstein–Barr virus, 656, 656–657 genetics, 658 hepatitis C virus, 657 human herpesvirus 8, 657 human T-cell leukaemia/lymphoma virus type 1, 657 immunosuppressed individuals, 656 immunosuppression, 658 occupational factors, 658 virus infections, 656–657 aggressive B-cell, treatment, 673–676 adjuvant radiotherapy, 674–675 allogeneic SCT, 675 autologous SCT, 675 CNS prophylaxis, 674 localized disease, 674 stage II-IV, 674 autologous SCT, 668–669 non-conjugated monoclonal antibodies, 669 radioimmunoconjugates with, 669 clinical features, 659

cutaneous lymphomas, 682–683 cytogenetics, 658–659 diagnosis, 431 bone marrow trephine biopsy, 399, 531 cytogenetic analysis, 405 differential diagnosis, 542–543 adult ALL vs., 437 CLL vs., 403 flow cytometric immunophenotyping, 403 epidemiology, 655–656 elderly patients, 683 geographical variations, 655–656 frequency, 655 immunocompromised patient, 681–682 immunohistochemistry, 404 International Prognostic Index, 661 laboratory investigations, 659–662 anaemia, 659–660 fine-needle aspiration, 659 histology, 659 lactate dehydrogenase, 660–661 paraproteins, 660–661 leukaemic phase, 544–556 malignancies, 942 oncogenes, 658–659 red cell aplasia, 942 splenic marginal zone, 657 staging, 662–663 treatment, 663 aggressive, 680–681 allogeneic SCT, 669 autologous SCT see above chemotherapy, 664 CNS prophylaxis, 680–681, 681 high-grade lymphoma, 679 SCT, 668–669 therapy algorithms, 670 warm-type AIHA, 162 see also specific diseases/disorders non-myeloablative stem cell transplantation, adult ALL, 441, 442 non-neuropathic hereditary systemic amyloidosis, 612 non-secretory multiple myeloma, 584 nonsense-mediated mRNA decay (NMD), globin gene translation, 87 non-spherocytic haemolytic anaemia, chronic, glucose-6-phosphate dehydrogenase deficiency, 152 non-steroidal anti-inflammatory drugs (NSAIDs), sickle cell disease therapy, 122 non-transferrin-bound iron (NTBI) iron-loading anaemias, 56 β-thalassaemias, 94

non-transplant candidates, multiple myeloma management/ treatment, 593–594 Noonan syndrome PTPN11 gene, 391 thrombocytopenias, 917 NOP10 gene, 195 Nordic Myeloma Study Group, 587 norfloxacin, infection prophylaxis, 473 normochromic macrocytic anaemia, Diamond–Blackfan anaemia, 198 normochromic normocytic anaemia leishmaniasis, 967 malaria, 959 NOTCH1 gene mutations, 449–450 NOTCH signalling pathway haematopoiesis, 8 Hodgkin lymphoma, 642 NPM1 gene AML relapse risk, 427, 427 anaplastic large-cell lymphoma, 574 function alteration, 387 NPM–ALK gene translocations, 633 NRAS gene activating mutations, 387 AML, 391 chronic myelomonocytic leukaemia, 391 multiple myeloma, 579 myelodysplastic syndrome, 391, 510 NSAIDs (non-steroidal anti-inflammatory drugs), sickle cell disease therapy, 122 NT5C3 gene, pyrimidine 5’-nucleotidase, 154 NTBI see non-transferrin-bound iron (NTBI) N-terminal pro-brain natriuretic peptide (NT-pro-BNP), 607 NT-pro-BNP (N-terminal pro-brain natriuretic peptide), 607 nucleic acid test (NAT), HIV blood screening, 273 nucleotide metabolism, red cell metabolism, 154–155 null phenotypes, blood group antigens, 229 nutritional deficiencies CLL vs., 534–535 erythropoiesis, 24 folate deficiency, 77–78 leukaemia, 464, 480 neutropenia, 316 NYH9 gene, thrombocytopenias, 918 nystatin with framycetin and colistin (FRACON), 473 with gentamicin and vancomycin (GVN), 473 1039

Index

O obstetric haemorrhage, 851 occupational factors, NHL, 658 ocular complications, sickle cell disease, 117 oedema, pulmonary, 738–739 oesophagitis, leukaemias, 471 ofatumumab CLL management, 547 follicular lymphoma therapy, 667 ofloxacin, infection prophylaxis, 473 Ok blood group, 245 older patients see elderly patients oligosaccharidoses, 334 clinical manifestations, 337 prevalence, 331 oncogenes, NHL, 658–659 opiates, analgesia, 481 opportunistic infections adult T-cell leukaemia lymphoma, 568, 678 see also specific infections opportunistic/overwhelming postsplenectomy infection (OPSI), 133, 375–376, 935 opsonic receptors, phagocytes, 303 opsonization, 234–235 oral contraceptives counselling in heritable thrombophilia, 885–886 thrombosis prevention, 881 venous thromboembolism, 890 oral swabs, infections in leukaemia, 471 organ involvement allogeneic SCT complications, 737–739 systemic AL amyloidosis, 606, 606–607 organomegaly, AML, 415 oropharyngeal bleeding, haemophilia A, 795 orotic aciduria, megaloblastic anaemia causing, 81 Oroya fever see Bartonella infections (Oroya fever) orthochromatic erythroblasts (Ort), 14 osmotic fragility test, hereditary spherocytosis, 131, 132 osteomyelitis, sickle cell disease, 114 osteonecrosis, multiple myeloma, 586 osteoporosis, mastocytosis, 702 osteosclerotic myeloma (POEMS syndrome), 584 oxidative damage defence, glutathione cycle, 148 oxidative haemolysis, acquired haemolytic anaemias, 172, 173, 173 oxidative stress defences, red cell metabolism, 147–148 1040

oxidoreductases, 29–30 oxygen affinity, fetal haemoglobin, 83 oxygen dissociation curve, 83–84 oxymetholone, Fanconi anaemia treatment, 191 P P1 antigen, 238, 251, 252 P2 antigen, 251 P2X1 ATP receptor, 785 P2Y1 ADP receptor, 783 signalling transduction disorders, 920 thrombocytopathy, 922 P2Y12 ADP receptor, 783 thrombocytopathy, 922 p15INK4b gene deletion, 394 p16INK4a gene deletion lymphomas, 394 mantle cell lymphoma, 393 hypermethylation, 388 p53 gene, inactivating mutations, 387 paediatric follicular lymphoma, 620 paediatric protocol indications, adult ALL therapy, 445 PAI-1 haemostatic disturbance in chronic liver disease, 849 protein C pathway inhibition, 767 pain leukaemia, 464, 481 sickle cell disease therapy, 122 total body radiation, 481 vinca alkaloids, 481 PALB2 gene mutations, 191 palliative care, 464, 481 AML treatment, 418–419 pamidronate, multiple myeloma management/treatment, 585 pancreatic enzymes, Shwachman–Diamond syndrome, 197 pancreatitis, chronic, 73 pancytopenia, acquired aplastic anaemia, 213 P antigen, 238 pantothenic acid deficiency, 81 Papillon–Lefevre syndrome, 318 Pappenheimer bodies, myelodysplastic syndrome, 512 PAR-1 thrombin receptor, 784, 897 PAR-4 thrombin receptor, 784 paracellular diapedesis, phagocytes, 301 paracetamol, non-haemolytic febrile transfusion reactions, 286 paraproteinaemia, coagulation disorders, 856 paraproteins, NHL, 660–661

parasite lactate dehydrogenase (pLDH), malaria diagnosis, 960–961 parasitic infections blood transfusion transmission, 271 haematological disorders, 950–952 spleen, 371 see also specific infections paratopes, 229 parenteral fluid administration, adult ALL, 437 parenteral iron therapy, iron deficiency anaemia management, 40–41 parietal cells, antibodies to, 72 Paris–Trousseau syndrome, 919 genetics, 925 Parkinson’s disease, plasma homocysteine, 66 paroxysmal cold haemoglobulinuria (PCH), 159, 165–166 viral infections, 950 paroxysmal nocturnal haemoglobinuria (PNH), 176–185 clinical features, 177–179 bone marrow failure, 177 cholelithiasis, 178 haemolysis, 177 leukaemic transformation, 178 nitric oxide consumption, 179 renal disease, 178 spontaneous remission, 178–179 thrombosis, 177–178 venous thromboembolism, 898 epidemiology, 177 flow cytometric immunophenotyping, 403–404 future work, 184 glycosylphosphatidylinositol defects, 176 HMGA2 gene, 176 investigations, 179 pathophysiology, 176–177 PIGA gene, 176 post-ATG, 220 pregnancy, 182 prognosis, 184 treatment, 179–182 allogeneic bone marrow transplantation, 181 complement blockade, 181–182, 183 supportive care, 179–180 thrombosis, 180 partial D, Rh system, 255 partial remission (PR), CLL, 537 partial thromboplastin time with kaolin (PTTK) see prothrombin time (PT) parvovirus 9 infection hereditary spherocytosis, 132 red cell aplasia, 950

Index

parvovirus B19 infection, 973 pathogen inactivation techniques, blood donation, 275–276 pathogen receptors, blood group antigens, 229 patient-controlled analgesia (PCA), sickle cell disease therapy, 122 PAX5 gene, nodular lymphocyte predominant Hodgkin lymphoma, 636 P blood group, 245, 251–252 PCA (patient-controlled analgesia), sickle cell disease therapy, 122 PCC see prothrombin complex concentrate (PCC) PCH see paroxysmal cold haemoglobulinuria (PCH) PCI, protein C pathway inhibition, 767 PCR see polymerase chain reaction (PCR) PD1, angioimmunoblastic T-cell lymphoma, 633 PDGF see platelet-derived growth factor (PDGF) PDGFRA gene chronic myelomonocytic leukaemia, 526 eosinophilia, 704 eosinophil myeloproliferative disorders, 389 PDGFRB gene chronic myelomonocytic leukaemia, 526 clonal hypereosinophilic syndromes, 705 eosinophil myeloproliferative disorders, 389 myelodysplastic syndrome, 510 PE see pulmonary embolism (PE) Pearson syndrome, 196 red cell aplasia, 973 PECAM-1 phagocytes, 301 thrombus stabilization, 779 pedigrees, heritable thrombophilia, 878 pefloxacin, infection prophylaxis, 473 Pel–Ebstein fever, Hodgkin lymphoma, 642 Pelger–Huët anomaly myelodysplastic syndrome, 512–513 neutrophils, 310, 311, 312 penicillin allogeneic SCT infections, 736 drug-induced alloimmune haemolytic anaemia, 166 pentamidine African trypanosomiasis therapy, 966 allogeneic SCT infections, 736 pentose phosphate pathway see red cell metabolism

pentostatin (deoxycoformycin) CLL management, 546 follicular lymphoma treatment, 665 hairy cell leukaemia treatment, 553 T-prolymphocytic leukaemia therapy, 561 perforins CD8 T-cells, 353 haemophagocytic lymphohistiocytosis, 326 performance score, AML relapse risk, 428 periodic acid–Schiff stain, ALL, 402, 403 peripheral blood megaloblastic anaemia, 66–67, 67 morphology, myelodysplastic syndrome, 512 sickle cell disease, 119 peripheral destruction, neutropenia, 313 peripheral neuropathy, systemic AL amyloidosis, 607 peripheral T-cell lymphomas (PTCLs), 662, 677–678 not otherwise specified, 570, 631, 631 periprosthetic leaks, fragmentation haemolysis, 171 perivalvular leaks, fragmentation haemolysis, 171 pernicious anaemia autoimmune disease association, 72 cobalamin deficiency, 71–72 Helicobacter pylori infection, 72 hyperthyroidism, 948 hypoadrenalism, 948 hypogammaglobulinaemia, 72 hypoparathyroidism, 948 immune factors, 72 intrinsic factor, 71 juvenile, 72 steroid therapy reaction, 72 peroxisome proliferator-activated receptor-γ (PPAR-γ), neutrophils, 309 peroxisomes, platelets, 774 pesticides, acquired aplastic anaemia, 208 PET see positron emission tomography (PET) petechiae, 915 PETHEMA-94 trial, 591 PF4, platelet α-granules, 774 PGE2 see prostaglandin E2 (PGE2) P-glycoprotein AML, 430 relapse risk, 428 imatinib mesylate resistance, 494 pH agglutination tests, 237 blood component changes, 279 complement, lytic phase, 235

phagocytes, 300–329 adhesion molecules, 302 cell–cell adhesion receptors, 301 intracellular adhesion molecule 1, 301 intracellular adhesion molecule 2, 301 vascular cell adhesion molecule-1, 301 antimicrobial proteins, 306–307 azurocidin, 306 bacterial permeability-increasing protein, 306 cathepsin G, 306 cytoskeletal proteins, 305 degranulation, 305 dysfunction, 307–327 integrins, 301, 302 locomotion, 300–301 mechanisms of function, 300–307 nitric oxide, 305–306 opsonic receptors, 303 paracellular diapedesis, 301 PECAM-1, 301 production, 307–327 receptors, 301, 303–304 apoptotic cells, 304 complement receptors, 301, 304 Fc receptors, 301 mannosyl/fucosyl receptors, 303–304 respiratory burst, 305 secretion, 305 selectins, 301, 302 signalling, 304–305 structure, 307–327, 308 see also specific cells phagocytosis, red cells in spleen, 371 pharmacogenetic variables, childhood ALL prognosis, 455–456 phenazopyridine, acquired haemolytic anaemias, 173 phenotype–genotype correlations, leukaemogenesis, 384–385 phenoxymethylpenicillin, postsplenectomy, 935 phenytoin, folate deficiency, 79 Philadelphia (Ph) chromosome adult ALL, 434–435 prognosis, 443–444 CML, 483, 486 negative chronic myelogenous leukaemia, 499 phlebotomy, haemochromatosis, type 1, 50, 51 phosphatase reactions, ALL, 402, 403 phosphatidylcholine, red cell membrane, 128 phosphatidylethanolamine, red cell membrane, 128 1041

Index

phosphatidylinositol 3-kinase (PI3-K) BCR–ABL1 chimeric gene, 489 platelet second messenger pathways, 786 phosphatidylinositol glycan A, red cell membrane, 129 phosphatidylserine, red cell membrane, 128 phosphofructokinase, 147 deficiency, 146, 147 6-phosphogluconate dehydrogenase, 140 phosphoglycerate kinase, 147 deficiency, 146, 147 phototherapy acute GvHD therapy, 735 glucose-6-phosphate dehydrogenase deficiency, 151 phox (phagocyte oxidase) proteins, phagocytes, 305 physical examination, primary immune thrombocytopenia, 932 physiological reactions, SHOT, 290 PI3-K see phosphatidylinositol 3-kinase (PI3-K) PI3-K/Ras-MAP kinase pathway, erythropoietin receptor, 21, 22 PIFT (platelet suspension immunofluorescence test), 264 PIGA gene acquired aplastic anaemia, 209 acquired haemolytic anaemias, 174 myelodysplastic syndrome, 507 paroxysmal nocturnal haemoglobinuria, 176 PIM1 gene, 636 PK antigen, 238 PKG-1, platelet inhibition, 787 placenta, haematopoiesis, 5–6, 7 plasmablastic lymphoma, WHO lymphoma classification, 625 plasma cell(s), 343–344 development, 355 dyscrasia, systemic AL amyloidosis, 601, 606 leukaemia, 583 microenvironment interaction, multiple myeloma, 580–581, 581 neoplasms, WHO lymphoma classification, 618 proliferative activity, multiple myeloma, 589 receptor blockers, multiple myeloma management/treatment, 596 plasma cell myeloma, WHO lymphoma classification, 618 plasmacytoma, 618 bone, 584 extramedullary, 584 WHO lymphoma classification, 618 1042

plasma exchange cold-type AIHA, 165 diarrhoea-related haemolytic-uraemic syndrome, 869 TTP treatment, 866–867 see also exchange transfusion (erythrocytapheresis) plasma expanders, anticoagulant effects, 852 plasma iron, erythropoiesis, 23 plasma non-transferrin-bound iron, iron metabolism, 36 plasmapheresis see plasma exchange plasma, preparation, 276 plasma protein antibodies, 266 blood transfusions, 287 plasma protein antigens, 266 plasma skimming, spleen, 369 plasmin, 767–768 fibrin, action on, 768 inhibitors, 770 plasminogen (PLG), 759, 767–768 activation inhibitors, 769–770 fibrinolysis, 751 haemostatic disturbance in chronic liver disease, 848, 849 plasminogen activation inhibitor type 1, 759, 769 plasminogen activation inhibitor type 2, 769–770 plasminogen activators, 768–769 see also specific types Plasmodium falciparum infection see malaria Plasmodium malariae infection see malaria Plasmodium ovale infection see malaria Plasmodium vivax Duffy glycoprotein binding, 229 infection see malaria platelet(s), 772–792 actin polymerization, 786–787 activation, 786–787, 921 adhesion, 786, 921 adhesion disorders, thrombocytopathies, 919–920 aggregation disorders, thrombocytopathies, 921–923 α-granules, 774, 774 amyloidosis, 856 animal models, 774–775 antibodies, 264–266 blood transfusions, 286–287 binding, von Willebrand disease diagnosis, 806 bleeding problems, 788 blood vessel wall interaction, 753–754

congenital disorders, 915–927 bleeding, 915 classification, 915 coagulation factor disorders vs., 915 family history, 915 gene therapy, 925–926 genetics, 925 therapy, 924–925 variable severity, 915 see also specific diseases/disorders connective tissue disorders, 946 coronary artery bypass graft, 851 count, heparin-induced thrombocytopenia, 891 cytoskeleton, 773 definition, 772 dense granules, 774 dilution of, massive transfusions, 289 disorders, viral infection, 950 fibrinogen, 772 formation, 775 functions, 772–773, 773, 915 platelet function testing, 789 function testing, 788–790 automated cell counting, 789 bleeding time, 789 flow cytometry, 789 light transmission aggregometry, 789 mean platelet volume, 789 myelodysplastic syndrome, 513 platelet function analyser, 789 genetics, 774 haemostasis, 755 HIV infection, 955 inhibition, 772, 787–788 lifespan, 774 lysosomes, 774 malignancies, 944 matching in immunological refractoriness, 266 peroxisomes, 774 post-splenectomy, 377–378 pregnancy, 953–954 preparation, 276, 277, 278 primary myelofibrosis, 698 receptors α2A-adrenoceptors, 784–785 CD36, 785 CLEC-2, 785 FcγRIIA, 785 5-HT2A receptors, 784 P2X1 ATP receptor, 785 stimulatory see below renal disease, 850, 850 second messenger factors, 785–786 secretion, 786

Index

signalling pathways, 779–783, 780 integrins, 781, 781 ITIM, 779 Src, 779, 781, 782 Syk, 779, 781, 782 thrombocytopathies, 920 stimulatory receptors, 779–783 collagen, 781–782 GPIb-IX-V, 780–781 G protein-coupled receptors, 783–784 GPVI, 779, 780, 781, 781–782 heterotrimeric proteins, 780 integrin α2β1, 781–782 integrin αIIbβ3, 782–783 P2Y1 ADP receptor, 783 P2Y12 ADP receptor, 783 PAR-1 thrombin receptor, 784 PAR-4 thrombin receptor, 784 thromboxane receptor, 783–784 tyrosine kinase-linked receptors, 780–783 storage changes, 279–280 storage granules, 774 see also specific types structure, 773–774, 919 thrombosis, 790–791 thromboxane, 772, 786 transfusions see platelet transfusions von Willebrand factor, 772 platelet-derived growth factor (PDGF) platelet α-granules, 774 primary myelofibrosis, 697 platelet suspension immunofluorescence test (PIFT), 264 platelet transfusions acquired aplastic anaemia, 214 acute promyelocytic leukaemia, 845 ‘compatible’ platelets, 466 congenital platelet disorder therapy, 924 DIC treatment, 845 HLA typing, 466 leukaemias, 466 massive blood loss, 853 myelodysplastic syndrome, 519 neonatal thrombocytopenia, 982 SCT, 733 PLCH (pulmonary Langerhans cell histiocytosis), 325–326 pLDH (parasite lactate dehydrogenase), malaria diagnosis, 960–961 PLG see plasminogen (PLG) PML–RARA fusion gene, acute promyelocytic leukaemia, 384, 386 PML (progressive multifocal leucoencephalopathy), rituximab side effects, 936

pneumococcal infections, sickle cell disease, 113–114, 114 Pneumocystis jirovecii infection ALL, 477 allogeneic SCT infections, 736, 738 CLL, 478 pneumonia, sickle cell disease, 114 PNH see paroxysmal nocturnal haemoglobinuria (PNH) POEMS syndrome (osteosclerotic myeloma), 584 poikilocytosis iron deficiency anaemia, 39 myelodysplastic syndrome, 512 point mutations haematological malignancies, 387 hereditary persistence of fetal haemoglobin, 98, 99 δβ thalassaemia, 98 β-thalassaemias, 89 α thalassaemias, 100 point-of-care testing, acquired coagulation disorders, 840–841 poly-A addition sites, haemoglobin, 85 polyacrylamide gel electrophoresis (PAGE), hereditary spherocytosis, 131 polyagglutinable red cells, 261–263 acquired, 261–262 inherited, 261–262 T activation, 262, 262 Tk activation, 262, 262 Tn activation, 262, 262–263 polyarteritis nodosa, eosinophilia, 946 polychromatic erythroblasts (Pol), 14, 17, 17 polyclonal B-cell lymphocytosis blood film, 544 CLL, 544 polycystic kidney disease, abnormal erythropoietin secretion, 693 polycythaemias, 686–693 apparent, 693 endocrine disorders, 948 hepatoma, 943 malignancies, 943 primary, 690 renal cell carcinoma, 943 renal disease, 947 secondary, 690–691 see also specific diseases/disorders polycythaemia vera, 686–690 aetiological classification, 686 bone marrow aspirates, 401 Budd–Chiari syndrome, 688 clinical features, 688–689 epidemiology, 688 essential thrombocythaemia, transformation of, 694

investigations, 689–690 pathophysiology, 686–687 thrombosis, 896–897 treatment, 690 Polycythaemia Vera Study Group (PVSG), 687 polymerase chain reaction (PCR), 410 BCR–ABL detection, 410, 410 childhood ALL minimal residual disease, 455 HLA typing, 717 molecular blood grouping techniques, 242 quantitative see quantitative polymerase chain reaction T-cell large granular lymphocyte leukaemia, 565 thalassaemia screening, 105 polymerase chain reaction sequencespecific oligonucleotides (PCRSSO), HLA typing, 718 polymerase chain reaction sequencespecific primers (PCR-SSP), HLA typing, 718, 718 polysaccharide solutions, fluid balance, 479 Pompe disease (glycogen storage disease type II), 335, 341 clinical features, 337 enzyme replacement therapy, 332 populations haemochromatosis, type 1 screening, 51 HLA polymorphism, 713 porcine factor VIII, 799 porphyria(s), 41–42, 42 congenital erythropoietic, 41–42 see also specific types porphyria cutanea tarda, 42 porphyrin synthesis, 36 portal hypertension, splenomegaly, 374 posaconazole, infection prophylaxis, 474 positive selection, T cell development, 351 positron emission tomography (PET) Hodgkin lymphoma, 643–644 response assessment, 644–645, 646 NHL, 662 Richter transformation, 549 post-thrombotic syndrome, 913 post-transfusion purpura, 287 post-translational stability mutations, β-thalassaemias, 90, 90 post-transplant lymphoproliferative disease (PTLD), 222–223, 656, 682, 739 infectious mononucleosis vs., 366 potassium blood component changes, 279 red cell metabolism, 140–141 1043

Index

potassium–chloride cotransporter, sickle cell disease, 110 PPAR-γ (peroxisome proliferator-activated receptor-γ), neutrophils, 309 pralatrexate, peripheral T-cell lymphomas, 677 pre-B cell(s), 349 pre-B-cell acute lymphoblastic leukaemia, 434 immunophenotypic classification, 451 pre-conception screening, thalassaemias, 105 prednisolone adult ALL chemotherapy, 438 ATG side effects, 216 chronic GvHD therapy, 740 clonal hypereosinophilic syndromes treatment, 706 with cyclophosphamide and vincristine see CVP (cyclophosphamide, vincristine, prednisolone) with cyclophosphamide, doxorubicin and vincristine see CHOP (cyclophosphamide, doxorubicin, vincristine and prednisolone) with melphalan see melphalan with mustine, vincristine and procarbazine see MOPP (mustine, vincristine, procarbazine and prednisolone) with rituximab, cyclophosphamide, doxorubicin and vincristine see R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone) systemic AL amyloidosis management, 609 TTP treatment, 867 prednisone adult ALL chemotherapy, 438 with bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine and procarbazine see BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine and prednisone) with melphalan, multiple myeloma management/treatment, 591 pregnancy, 953, 953–954 anaemias, 953 acquired aplastic anaemia, 207 anti-D antibody detection, 294 antiphospholipid syndrome, 895 coagulation changes, 954 essential thrombocythaemia, 696–697 factor V and factor VIII combined deficiency, 834 1044

factor V deficiency, 834 factor VII deficiency, 834 factor X deficiency, 834 factor XI deficiency, 835 factor XIII deficiency, 835 fibrinogen deficiency, 834 folate deficiency, 64, 78 folate supplements, 64–65, 78, 81 haemostasis, 767 HELLP syndrome, 953–954 Hodgkin lymphoma treatment, 651 paroxysmal nocturnal haemoglobinuria, 182 platelets, 953–954 primary immune thrombocytopenia see primary immune thrombocytopenia pyruvate kinase deficiency, 145 sickle cell disease, 117 thrombocytopenia, 953 thrombosis prevention, 881–882 venous thromboembolism, 889 therapy, 912–913 von Willebrand disease, 810 white cells, 953 preimplantation genetic diagnosis, thalassaemias, 105 prekallikrein, 758 deficiency, 748 premarital screening, thalassaemias, 105 premature ageing, dyskeratosis congenita, 195 premature infants folate deficiency, 78 folate prophylaxis, 81 iron deficiency anaemia, 39 Rhesus haemolytic disease of the fetus and newborn, 295 premature termination codons (PTCs), globin gene translation, 87 prenatal diagnosis sickle cell disease, 120 thalassaemias, 105 preoperative assessment, blood transfusion recipients, 280, 280 PRF1 gene, haemophagocytic lymphohistiocytosis, 326 priapism, sickle cell disease, 117 primaquine, malaria treatment, 962 primary CNS lymphoma, 676–677 primary cutaneous anaplastic large-cell lymphoma, 630 primary cutaneous CD30-positive T-cell lymphoproliferative disorders, 630 primary cutaneous diffuse large B-cell lymphomas, leg type, 624–625

primary cutaneous follicle centre lymphoma, 620–621, 683 primary cutaneous γδ T-cell lymphomas, 630–631 primary cutaneous large B-cell lymphoma, leg type, 683 primary cutaneous T-cell lymphomas, 682–683 primary effusion lymphoma, 625, 625 primary erythrocytosis, 691 primary familial and congenital polycythaemia (PFCP), 691 primary fibrinolysis, malignancies, 945 primary gastrointestinal lymphomas, 676 primary immune thrombocytopenia, 928–939 anti-GPIIb/IIIa antibodies, 928–929 antiplatelet antibodies, 930 CD8 T cells, 931, 931 clinical features, 928–929 CLL, 548 diagnosis, 932, 932–933 environmental trigger, 929 Helicobacter pylori infection, 930–931 heterogeneity, 928 immune response, 929 management, 933–935, 934 anti-D antibody, 933, 934 azathioprine, 934 children, 936–937 corticosteroids, 933, 934 intravenous Ig, 933, 934 long-term, 934 mycophenolate mofetil, 934 short-term, 934 splenectomy, 934–935 thrombopoietin receptor agonists, 933, 936 MHC polymorphisms, 929 multifactorial disease, 929–932 natural history, 932 new consensus terminology, 929, 929, 935 stages, 929 pathophysiology, 929 in pregnancy, 937–938 causes, 937 investigation, 937, 937 management, 937–938 refractory, 935–936 thrombocytopenia, 917, 932 thrombopoietin, 931, 931–932 viral infection, 928 primary intestinal follicular lymphoma, 620 primary lymphoid organs, 344

Index

primary mediastinal B-cell lymphoma differential diagnosis, 412 treatment, 675–676 primary mediastinal large B-cell lymphoma, 624 primary myelofibrosis, 697–701 clinical features, 697–698 diagnosis, 699 bone marrow trephine, 699 differential diagnosis, 699 epidemiology, 697 investigations, 698–699 pathophysiology, 697 prognosis, 701 treatment, 699–701 allogeneic SCTm, 699–700 anaemia therapy, 700 cytoreductive therapy, 700 splenectomy, 700 splenic irradiation, 700 thalidomide, 700–701 primary polycythaemias, 690 primary splenic hyperplasia, 374 primary β-thalassaemias, 92 primary tumours, splenomegaly, 374 primidone, folate deficiency, 79 prions, transmission, blood transfusions, 271, 274 pro-B cell(s), 349 pro-B-cell acute lymphoblastic leukaemia, 434 procainamide, warm-type AIHA, 162 procarbazine with bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine and prednisone see BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine and prednisone) with mustine, vincristine and prednisolone see MOPP (mustine, vincristine, procarbazine and prednisolone) PROC gene protein C deficiency, 873, 876 thrombosis risk, 879 procoagulant activity, thrombus formation, 779 progressive multifocal leucoencephalopathy (PML), rituximab side effects, 936 proliferation centres, WHO lymphoma classification, 615–616 promoter regions, haemoglobin, 85 pronormoblasts (Pro), 14, 17, 17

PROS gene protein S deficiency, 873, 877 thrombosis risk, 879 prostacyclin, platelet inhibition, 772, 787, 788 prostacyclin synthase, 754 prostaglandin E2 (PGE2) endothelium interactions, 753, 754 haematopoiesis, 8 protease-activated receptor 1 (PAR-1), 897 protease nexin 2, 765 proteasome inhibitors, multiple myeloma management/treatment, 596 proteasomes, antigen processing, 345 protein(s) deficiency, anaemia, 81 red cell membrane see red cell membrane protein 4.1, 129 protein 4.2 (pallidin), 129 β-protein, amyloidosis, 600 proteinase 3, 307 protein C, 759, 765–766 activated see activated protein C (APC) assays, heritable thrombophilia, 884 deficiency, 873, 876–877 assays, 884 neonates, 982–983 PROC gene, 873, 876 purpura fulminans, 882 warfarin-induced skin necrosis, 855 haemostatic disturbance in chronic liver disease, 848 inhibitors, 767 neonatal thrombocytopenia, 982 structure, 876 protein C pathway, 765–767 thrombosis in inflammation, 898 protein kinase A (PKA), platelet inhibition, 787 protein kinase C (PKC), platelet second messenger pathways, 785–786 protein kinase G (PKG), platelet inhibition, 787 protein S, 759, 766 assays, heritable thrombophilia, 884–885 deficiency, 873, 877 acquired, 858 HIV infection, 955 neonates, 982–983 PROS gene, 873, 877 warfarin-induced skin necrosis, 855 haemostatic disturbances in pregnancy, 851 neonatal thrombocytopenia, 982 proteinuria, systemic AL amyloidosis, 607 protein Z, 764 heritable thrombophilia, 878

protein Z-dependent inhibitor, 764 prothrombin, 758, 762 deficiency, 816, 818 genetics, 819 lupus anticoagulant association, 858 in pregnancy, 834 prophylaxis, 833 treatment, 833 prothrombin complex bleeding in surgery, 911 haemophilia treatment, 799–800 prothrombin complex concentrate (PCC) acquired haemophilia A therapy, 858 DIC treatment, 845 factor VII deficiency therapy, 823 factor X deficiency, 825 prothrombin deficiency, 816 vitamin K-dependent coagulation factors deficiency, 832 prothrombin time (PT), 748 acquired coagulation disorders, 840 DIC, 843 haemophilia A, 795 heritable thrombophilia, 883 in liver disease, 847–848 neonatal haemostasis, 980 rare bleeding disorders, 814 in trauma, 852 proton-coupled high affinity folate transporter (PCFT/HCP1), 76 protozoan infections haemophagocytic lymphohistiocytosis, 326 see also specific infections prourokinase, 759 pruritus polycythaemia vera, 688 Sézary syndrome/mycosis fungoides, 566 pseudo-Hurler syndrome see mucolipidosis type III (pseudo-Hurler syndrome) pseudohyperkalaemia, hereditary stomatocytosis, 135–136 Pseudomonas infections leukaemias, 472 splenectomy risks, 375 pseudo von Willebrand disease, 810, 812 genetics, 925 platelet responses, 922 psoralen with UVA irradiation see PUVA (psoralen with UVA irradiation) psychogenic purpura, 853 psychological problems leukaemia, 463, 464 sickle cell disease therapy, 124 PTCLs see peripheral T-cell lymphomas (PTCLs) 1045

Index

PTCs (premature termination codons), globin gene translation, 87 PTLD see post-transplant lymphoproliferative disease (PTLD) PTPN11 gene juvenile myelomonocytic leukaemia, 501, 527 Noonan syndrome, 391 PU.1 gene, 510 puberty, allogeneic SCT complications, 740–741 pulmonary arteriovenous fistulae, hereditary haemorrhagic telangiectasia, 854 pulmonary complications, sickle cell disease, 116 pulmonary embolism (PE) clinical scores, 902 diagnosis bilateral ultrasonography, 903 MRI, 903 spiral CT, 902–903 ventilation–perfusion (V/Q) scanning, 902 management, low-molecular weight heparin, 908 prophylaxis, 908–909 pulmonary hypertension chronic thromboembolic, 913 sickle cell disease, 116 pulmonary infections allogeneic SCT complications, 738–739 specimens, 471 see also specific infections pulmonary Langerhans cell histiocytosis (PLCH), 325–326 pulmonary oedema, allogeneic SCT complications, 738–739 pure red cell aplasia, CLL, 548 pure white cell aplasia, neutropenia, 315 purine analogues advanced follicular lymphoma treatment, 665 CLL management, 546 splenic marginal zone lymphoma, 671 Waldenström macroglobulinaemia, 672 purpura fulminans, 882 DIC, 882 neonates, 983 protein C deficiency, 882 purpura simplex, 853 PUVA (psoralen with UVA irradiation) mastocytosis treatment, 703 mycosis fungoides treatment, 682 Sézary syndrome/mycosis fungoides, 567 1046

PVSG (Polycythaemia Vera Study Group), 687 pycnodysostosis, clinical features, 338 pycnodysostosis, 335 pyknocytosis, infantile, 976 pyridoxine, sideroblastic anaemia treatment, 45 pyrimethamine, malaria treatment, 962 pyrimidine 5’-monophosphate hydrolase see pyrimidine 5’-nucleotidase pyrimidine 5’-nucleotidase, 154–155 deficiency, 154–155, 155 reactions, 154 pyropoikilocytosis, hereditary see hereditary pyropoikilocytosis (HPP) pyruvate kinase, 141–145 deficiency, 141–145, 146 clinical features, 143–144 congenital non-spherocytic haemolytic anaemia, 144 2,3-DPG accumulation, 142–143 epidemiology, 141 laboratory diagnosis, 144–145 management, 143, 145 molecular biology, 141–143 neonatal anaemia, 976 pregnancy, 145 splenectomy, 143, 145 reactions, 142, 142–143 Q quality of life, NHL treatment, 664 quantitative Buffy coat test, African trypanosomiasis, 966 quantitative haemagglutination, 241 quantitative polymerase chain reaction, 410, 411 adult ALL, 435 cytomegalovirus infection, 471 imatinib mesylate monitoring, 493 minimal residual disease, 410 Quebec platelet disorder, 923 genetics, 925 quinine, malaria treatment, 962 quinolone antibiotics allogeneic SCT infections, 736 infections in leukaemia, 472 R radiography Hodgkin lymphoma, 643 thalassaemia major, 94, 94 radioimmunoconjugates follicular lymphoma therapy, 667, 667–668 problems, 668

radionuclide investigations spleen, 378, 378 see also specific radionuclides radiotherapy aggressive NHL treatment, 681 anaplastic large-cell lymphoma, 575 classical Hodgkin lymphoma treatment, 647 cobalamin malabsorption, 74 consolidation, classical Hodgkin lymphoma, 650 Hodgkin lymphoma treatment, 645 local see local radiotherapy localized follicular lymphoma, 663 MALT lymphoma, 671 myelodysplastic syndrome development, 504 nodular lymphocyte predominant Hodgkin lymphoma treatment, 647 RAEB-1 (refractory anaemia with excess blasts 1), 506 RAEB-2 (refractory anaemia with excess blasts-2), 506 RAEBt see refractory anaemia with excess blasts in transformation (RAEBt) RAG1 gene, 349 RAG2 gene, 349 Rai staging systems, CLL, 537, 544 RANK-L (TRANCE/OPG-1), multiple myeloma pathogenesis, 582 RANTES, 363 eosinophils, 304 platelet α-granules, 774 Raph blood group, 245 antigen (CD151) absence, 229 RAPID trial, 648 Rapoport–Luebering shunt, 141, 142 RARA–PML fusion gene, 386 RARS see refractory anaemia with ring sideroblasts (RARS) RARS-T (refractory anaemia with ring sideroblasts and thrombocytosis), 527–528 rasburicase, adult ALL, 437 RAS gene/protein chronic myelomonocytic leukaemia, 526 juvenile myelomonocytic leukaemia, 389, 390, 391 leukaemogenesis, 389, 391 myelodysplastic syndrome, 509–510 RB1 gene deletion, retinoblastoma, 386 RBDD (International Rare Bleeding Disorders Database), 813 RB gene, 488 R binder, cobalamin absorption, 68

Index

RCC (refractory cytopenia of childhood), 506 R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone) aggressive NHL treatment, 681 DLBCL of stomach, 676 follicular lymphoma, 665 RCMD (refractory cytopenia with multilineage dysplasia), 505 RCMD -RS (refractory cytopenia with multilineage dysplasia and ring sideroblasts), 505 RCUD (refractory cytopenia with unilineage dysplasia), 506 receptor tyrosine kinases (RTKs), signalling, 388 recipient, blood transfusions see blood transfusion(s) recombinant activated factor VII (rFVIIa), 924 recombinant factor VIIa acquired haemophilia A therapy, 858 factor XI deficiency therapy, 827 recombinant factor VIII, 797 assays for, 797 recombinant human erythropoietin (rHuEpo) anaemia in renal disease, 947 anaemia of chronic disease treatment, 941 erythropoiesis disorders, 24 multiple myeloma, 587 recombinant human megakaryocyte growth factor (rhMGDF), primary immune thrombocytopenia management, 936 recombinant tissue plasminogen activator, venous thromboembolism therapy, 903 rectal swabs, infections in leukaemia, 471 red cell(s) alloantibodies, 280–281 antibodies, clinical significance, 236 antigen sensitization, blood transfusions, 282–286 destruction, neonatal anaemia, 974–977 development see erythropoiesis enzyme-treated, 238 enzymopathies, neonatal anaemia, 976 folate deficiency diagnosis, 79 frozen storage, 280 immunohaematology, 226–243 see also blood group antigens increased destruction, 126 increased production, 126, 127 iron utilization assay, 23–24

metabolism see red cell metabolism polyagglutinable see polyagglutinable red cells post-splenectomy, 377 preparation, 276 protoporphyrin, iron metabolism measurement, 38 quality control, spleen, 371 sickle cell disease, 110–111, 111 red cell aplasia CLL, 535, 942 large granular lymphocytic leukaemia, 942, 943 malignancies, 942 neonatal anaemia, 973–974 NHL, 942 parvovirus 9 infection, 950 thymoma, 942 red cell distribution width (RDW), megaloblastic anaemia, 66 red cell indices, 395 red cell mass, 24 red cell membrane, 128–130 acquired haemolytic anaemias, 174 cholesterol, 227 composition, 226–227 disorders, 126–139 clinical phenotype, 130–137 neonatal anaemia, 975–976 see also specific diseases/disorders lipids, 128, 128, 226–227 disorders, 136–137 see also specific diseases/disorders proteins, 128, 129, 130, 227, 227 actin, 227 ankyrin, 227 arrangement, 129 band 3, 227 Cromer glycoprotein, 227 Dombrock glycoprotein, 227 Duffy glycoprotein, 227 Gerbich glycoprotein, 227 glycophorins, 227 glycoproteins type 1, 227 glycoproteins type 3, 227 Kell glycoprotein, 227 spectrin, 227 see also specific proteins structure, 128 red cell metabolism, 128, 140–157 defects, erythrocytosis, 692 glutathione cycle, 141, 148, 153–154 oxidative damage defence, 148 glycolytic pathway, 140–147, 141, 142 ATP production, 140 disorders, 141–145, 146 see also specific diseases/disorders; specific enzymes

pentose phosphate pathway interactions, 142 potassium ions, 140–141 Rapoport–Luebering shunt, 141, 142 nucleotide metabolism, 154–155 see also specific diseases/disorders; specific enzymes oxidative stress defences, 147–148 pentose phosphate pathway, 141, 148, 148–153 glucose-6-phosphate dehydrogenase, 140 glycolytic pathway (Embden– Meyerhof pathway) interactions, 142 6-phosphogluconate dehydrogenase, 140 see also specific diseases/disorders; specific enzymes red cell transfusions erythropoiesis disorders, 24 neonatal anaemia, 978 SCT, 733 sideroblastic anaemia treatment, 45 red pulp, spleen, 368, 369 reduced intensity conditioning (RIC), SCT, 731–732 Reed–Sternberg cells, 631 referral centres, haemophilia A care, 803 refractory anaemia (RA), 504 allogeneic SCT, 521 refractory anaemia with excess blasts 1 (RAEB-1), 506 refractory anaemia with excess blasts-2 (RAEB-2), 506 refractory anaemia with excess blasts in transformation (RAEBt), 504–505 allogeneic SCT, 521 bone marrow histology, 514 WHO classification, 506 refractory anaemia with ring sideroblasts (RARS), 45, 504 allogeneic SCT, 521 bone marrow histology, 514 refractory anaemia with ring sideroblasts and thrombocytosis (RARS-T), 527–528 refractory cytopenia of childhood (RCC), 506 refractory cytopenia with multilineage dysplasia (RCMD), 505 refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS), 505 refractory cytopenia with unilineage dysplasia (RCUD), 506 1047

Index

refractory primary immune thrombocytopenia, 935–936 regulatory T-cells (Treg), 359 relapse rate (RR), ALL allogeneic SCT, 441 relapse/resistant leukaemia, adult ALL chemotherapy, 440 relapsing fever, tropical diseases, 969 remission induction adult ALL chemotherapy, 438–439, 439 AML treatment, 420–421 childhood ALL treatment, 456–457 renal amyloidosis, treatment, 610 renal cell carcinoma (RCC), polycythaemia, 943 renal disease/dysfunction, 946–978 allogeneic SCT complications, 738 anaemia, 946–977, 947 autologous SCT complications, 741 DIC, 843 dysfibrinogenaemia, 947–948 eculizumab effects, 181–182 haemostatic disturbances, 850–851, 947–948 leukaemia, 479–480 paroxysmal nocturnal haemoglobinuria, 178 platelets, 850, 850 polycythaemia, 947 sickle cell disease, 117 renal failure acquired haemolytic anaemias, 173–174 diarrhoea-related haemolytic-uraemic syndrome, 868 favism, 152 multiple myeloma, 586–587 renal transplantation abnormal erythropoietin secretion, 693 immunosuppression, 716 repeated blood transfusions, 281 replacement therapy, prothrombin deficiency, 816 reproductive complications, leukaemia, 463, 464 residual microbial risk, blood transfusions, 275–276 resistance, imatinib mesylate, 494 resistance proteins, AML relapse risk, 428 respiratory burst, phagocytes, 305 restriction endonucleases, 242 restriction fragment length polymorphisms (RFLPs), thalassaemia screening, 105 reticular dysgenesis, neutropenia, 315 reticulin assays, hairy cell leukaemia diagnosis, 551, 552 1048

reticulocyte(s), 14 counts sickle cell disease, 119 β-thalassaemias, 93 haemoglobin content, iron metabolism measurement, 38 haemolysis, 127 in spleen, 371 reticulocytopenia Diamond–Blackfan anaemia, 198 red cell aplasia, 973 reticuloendothelial function, AIHA, 161 retina neovascularization, sickle cell disease, 117 vein thrombosis, therapy, 880 retinoblastoma, RB1 gene deletion, 386 9-cis-retinoic acid receptor (RXR), erythroid cell development, 22 reverse transcriptase, dyskeratosis congenita, 194 reverse transcriptase polymerase chain reaction (RT-PCR), 410 AML minimal residual disease, 428 AML treatment monitoring, 420 Revised European–American Classification of Lymphoid Neoplasms (REAL) see World Health Organization (WHO), lymphoma classification RHAG blood group, 245 RhAG protein, 228 RhCcEe protein, 228 RHCE gene, 254 RHD–CE–D hybrid gene, 255–256 RHD gene, 254 missense mutations, 256 molecular blood grouping techniques, 242 RhD protein, 228 RHD pseudogene, 255 RhD, testing, blood donation, 275 Rhesus blood group, 245, 252–259 antibodies, 257–258 antigens, 252–253, 256 neonatal anaemia, 974 testing, 275 see also specific antigens band 3/Rh macrocomplex, 255 blood transfusion recipient testing, 280 C antigen, 253 c antigen, 253 compound antigens, 257 Cw antigen, 256 CX antigen, 256 D—, 257 D antigen, 252–253 other antigen effects, 253–254 partial D, 255

variants, 255–256 weak D, 255 E antigen, 253 e antigen, 253 fetal genotype prediction, 258–259 G antigen, 256 haplotype frequencies, 253 incompatibilities see haemolytic disease of the fetus and newborn (HDFN) molecular genetics, 254, 254–255 polymorphisms, 254–255 probable genotypes, 253–254 determination, 254 Rhmod, 257 Rhnull, 257 V antigen, 257 VS antigen, 257 rheumatoid arthritis acquired aplastic anaemia, 207 amyloidosis, 946 anaemia, 945 eosinophilia, 946 HLA-DR4 association, 713 malignancies, 946 neutropenia, 316 sideroblastic anaemia, 946 Rhmod, 257 Rhnull, 257 Rhnull syndrome, 136, 229 RHOH gene, 636 Rho/Rac GTPases, phagocyte signalling, 305 Rh proteins, 229 RIAM, 786 ribavirin, drug-induced alloimmune haemolytic anaemia, 166 riboflavin deficiency, 81 fresh frozen plasma preparation, 279 ribosomes proteins, Diamond–Blackfan anaemia, 198–199 RNA processing, inherited aplastic anaemia/bone failure syndromes, 197 Richter syndrome blood film, 549 CLL/small lymphocytic leukaemia, 616 Richter transformation, CLL management, 548–549 Rickettsia infections neutrophilia, 951 thrombocytosis, 951 RIPA (ristocetin-induced platelet aggregation), von Willebrand disease diagnosis, 806

Index

ristocetin Bernard–Soulier syndrome, 920 thrombus formation, 778 ristocetin-induced platelet aggregation (RIPA), von Willebrand disease diagnosis, 806 rituximab, 666 advanced follicular lymphoma, 665–666 aggressive NHL, 681 Burkitt lymphoma, 679 childhood ALL, 459 chronic GvHD, 740 CLL, 547, 548 cold-type AIHA, 165 with cyclophosphamide, doxorubicin, vincristine and prednisolone see R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone) follicular lymphoma, 666–667 hairy cell leukaemia, 553 mantle cell lymphoma, 673 multicentric Castleman disease, 680 nodular lymphocyte predominant Hodgkin lymphoma, 647 refractory primary immune thrombocytopenia, 935–936 side effects, 935–936 TTP, 867–868 Waldenström macroglobulinaemia, 672 warm-type AIHA, 163–164 rivaroxaban, venous thromboembolism therapy, 906 RNA interference (RNAi), myelodysplastic 5q–syndrome, 509 RNA replication, folate, 77 Romanowsky dyes B cell malignancies, 530 malaria diagnosis, 960 Rosai–Dorfman syndrome, 327 rotavirus infection, allogeneic SCT complications, 738 routine healthcare, sickle cell disease therapy, 121, 121 RPS5 gene, 974 RPS11 gene, 974 RPS14 gene, 387, 509 RPS17 gene, 974 RPS19 gene, 198, 974 RPS24 gene, 974 RPS35a gene, 974 RTKs (receptor tyrosine kinases), signalling, 388 RT-PCR see reverse transcriptase polymerase chain reaction (RT-PCR)

RUNX1 (AML-1) gene AML, 380 core-binding factor complex, 391 erythropoiesis, 15 haematopoiesis, 7–8 HSC development, 5 myelodysplastic syndrome, 509–510 transient abnormal myelopoiesis of Down syndrome, 708 RUNX1 (AML-1)–ETO gene fusion AML, 381 AML minimal residual disease, 428–429 RUNX1 (AML-1)–EVI1 gene fusion, myelodysplastic syndrome, 510 RUNX2 gene, multiple myeloma pathogenesis, 582 S SAA (serum amyloid A), amyloidosis, 599–600 Salla disease, 335 clinical features, 338 Salmonella infections, 951 neutrophilia, 951 sickle cell disease, 114 Sandhoff syndrome, 334 Sanfilippo syndrome see mucopolysaccharidosis type III (Sanfilippo syndrome) SAP scintigraphy angioimmunoblastic T-cell lymphoma, 633 systemic AL amyloidosis, 604, 605 SBDS gene, Shwachman–Diamond syndrome, 196 Sca-1, haemopoietic stem cells, 2 SCF see stem cell factor (SCF) Schindler disease, 334 clinical features, 337 schizonts, malaria life cycle, 958 Schulman–Upshaw syndrome, 917 genetics, 925 Scianna blood group, 245, 261 scintillation photography, spleen, 372, 373 SCL (TAL1) gene childhood ALL, 453 erythropoiesis, 15 HSC development, 5 T-cell acute lymphoblastic leukaemia, 385 T-cell lymphomas, 659 SCN (severe congenital neutropenia), 187, 313–314 scurvy anaemia, 81 bruising, 853–854 folate deficiency, 78

SDF-1 (stromal cell-derived factor 1), 363 multiple myeloma pathogenesis, 580–581 SDF-1α platelet α-granules, 774 platelets formation, 775 SDS, 211 SDS-PAGE, hereditary spherocytosis, 131 ‘seated immobility syndrome,’ 900–901 Sebastian syndrome, 918, 918 genetics, 925 Seckel syndrome, 191 secondary CNS lymphoma, treatment, 677 secondary erythrocytosis, 691–693 secondary lymphoid organs, 344 secondary polycythaemias, 690–691 secondary sideroblastic anaemia, 45 secondary β-thalassaemias, 92 γ-secretase, childhood ALL, 450 secretory immunoglobulin A (sIgA), effector functions, 230 selectin(s), phagocytes, 301, 302 E-selectin endothelium, 753 phagocytes, 301 P-selectin (CD62) endothelium, 753 phagocytes, 301 platelet α-granules, 774 L-selectin, phagocytes, 301 Senegal haplotype, sickle cell disease, 112 senile purpura, 853 septic shock, blood transfusions, 287 serine protease inhibitors (serpins), 763–765 see also specific types serotonin antagonists, 481 SERPINC1 gene antithrombin deficiency, 874 heritable thrombophilia, 873 thrombosis risk, 879 serpins (serine protease inhibitors), 763–765 serpocidins, phagocytes, 306 serum amyloid A (SAA), amyloidosis, 599–600 serum protein electrophoresis, myelodysplastic syndrome, 513 17p deletion, multiple myeloma, 579 17q deletion, CLL, 539 severe congenital neutropenia (SCN), 187, 313–314 Sézary syndrome, 565–567, 682–683 aetiology, 565–566 clinical course, 567 clinical features, 566 cytogenetics, 567 1049

Index

diagnosis, 566–567 differential diagnosis, 567 prognosis, 567 treatment, 567 WHO lymphoma classification, 630 see also mycosis fungoides SFLC analysis, plasma cell dyscrasia assessment, 606 Sheffield scoring system, primary myelofibrosis prognosis, 701 SHEPHERD trial, 181 Shigella infections, 951 diarrhoea-related haemolytic-uraemic syndrome, 868 SHOT, 289–292 anti D-related events, 290 error-related incidents, 290 incorrect blood component, 291 introduction, 290 mortality decline, 291 physiological reactions, 290 statutory requirements, 290 transfusion-transmitted infections, 290 Shwachman–Diamond–Oski syndrome, 315 myelodysplastic 5q–syndrome, 509 Shwachman–Diamond syndrome, 187, 196–197 genetic subtypes, 196 SBDS gene, 196 sialic acid, red cell membrane, 129 sialidosis I see mucolipidosis type I (sialidosis I) sickle cell disease, 109–125 African haplotypes, 112 Arab–Indian haplotype, 112 clinical manifestations, 112–118 acute painful episodes, 113 anaemia, 113 bone complications, 117–118 growth/development, 113 hepatobiliary complications, 116 infections, 113–114 leg ulcers, 118 neurological complications, 114, 116 ocular complications, 117 in pregnancy, 117 priapism, 117 pulmonary complications, 116 renal complications, 117 diagnosis, 119–120 haemoglobin electrophoresis, 119, 120 peripheral blood findings, 119 prenatal diagnosis, 120 geographic distribution, 109, 110 haplotypes, 109 see also specific haplotypes 1050

with HbD disease, 119 with HbE disease, 119 with Hb Lepore disease, 119 with HbO Arab disease, 119 with HPFH, 118–119 malaria, resistance to, 109 mortality, 112 newborn screening, 119–120 pathophysiology, 109–112 erythrocytes, effects on, 110–111, 111 haemolysis, 111 HbA presence, 110 molecular basis, 109–110 vaso-occlusion, 111 Senegal haplotype, 112 syndromes, 110 β thalassaemia with, 118 fetal haemoglobin, 100 therapy, 121–124 gene therapy, 123–124 haematopoietic SCT, 123, 124 hydroxycarbamide, 122–123 infection control, 121 new techniques, 123, 123 pain management, 122 psychosocial issues, 124 routine healthcare, 121, 121 transfusion therapy, 121–122 variants, 118–119 venous thromboembolism, 898–899 see also haemoglobin(s) sickle cell trait (HbAS), 118 sideroblastic anaemia, 43, 43–45 acquired, 45 autosomal mutations, 44 hypochromic anaemia vs., 37 inherited, 44–45 microscopy, 43 mitochondrial mutations, 44–45 rheumatoid arthritis, 946 secondary, 45 systemic lupus erythematosus, 946 treatment, 45 X-linked, 44 sideroblasts, 43 siderocytes, 43 signalling pathways BCR–ABL1 chimeric gene, 489 erythropoiesis see erythropoiesis inhibitors, multiple myeloma management/treatment, 596 multiple myeloma, 581, 581 phagocytes, 304–305 platelets see platelet(s) sildenafil, Waldenström macroglobulinaemia, 672

silent carriers hereditary elliptocytosis, 134 hereditary pyropoikilocytosis, 134 β-thalassaemias, 89 simple atrophic gastritis, cobalamin deficiency, 72 single base substitutions, α thalassaemias, 100 single-nucleotide polymorphisms (SNPs), 412, 413 childhood ALL, 450 cytokines, 720 myelodysplastic 5q–syndrome, 509 myelodysplastic syndrome, 510, 516 single-photon emission computed tomography (SPECT), spleen, 372 sirolimus allogeneic SCT, 744 ATG with, 218 Siwe disease, 325 Sjögren syndrome acquired aplastic anaemia, 207 malignancies, 946 skeletal effects Gaucher disease, 340, 340–341 multiple myeloma see multiple myeloma thrombocytopenia with absent radii, 202 skin GvHD, 734, 734–735 haemorrhage, acquired aplastic anaemia, 211 histology, Sézary syndrome/mycosis fungoides, 566 lesions infections in leukaemia, 471 Sézary syndrome/mycosis fungoides, 566 polycythaemia vera, 688 systemic AL amyloidosis, 602 SLC19A2 gene, sideroblastic anaemia, 44 SLC25A38 gene, sideroblastic anaemia, 44 SLE see systemic lupus erythematosus (SLE) sleeping sickness see African trypanosomiasis Sly syndrome see mucopolysaccharidosis type VII (Sly syndrome) small lymphocytic leukaemia, 614–616 small lymphocytic lymphoma (SLL), 534 small round cell tumours, childhood ALL vs., 451 smooth muscle cells, blood vessels, 753 smouldering multiple myeloma, 582–583 SMZL see splenic marginal zone lymphoma (SMZL) SNAP-23, platelet activation, 786 SNAREs, platelet activation, 786

Index

Sn-mesoporphyrin, glucose-6-phosphate dehydrogenase deficiency management, 153 SNPs see single-nucleotide polymorphisms (SNPs) social workers, haemophilia A care, 803–804 Sokal prognostic index, CML, 484 solid organ transplantation, systemic AL amyloidosis management, 610 solid-phase assays, antiphospholipid syndrome, 894–895 solid plasmacytoma of bone, 584 solvent-detergent (SD), fresh frozen plasma preparation, 279 somatic abnormalities, Fanconi anaemia, 187 somatic mutations, 385–388 aneuploidy, 386–387 chromosomal translocations, 385–386 epigenetic effects, 387–388 large deletions, 386–387 submicroscopic mutations, 387 Southeast Asian ovalocytosis, 136, 137 Southern blots, T-cell large granular lymphocyte leukaemia, 563 Southwest Oncology Group (SWOG), multiple myeloma prognosis, 590 specific factor assays, haemophilia A, 796 spectral karyotyping (24-colour FISH), 407 spectrins, red cell membrane, 129, 130, 227 SPECT (single-photon emission computed tomography), spleen, 372 spherocytic hereditary elliptocytosis, 134 spherocytosis, hereditary see hereditary spherocytosis (HS) sphingolipidoses, 330, 334 clinical manifestations, 337 see also specific diseases/disorders sphingomyelin, red cell membrane, 128 spinal cord compression, multiple myeloma, 588 spiral computed tomography, pulmonary embolism diagnosis, 902–903 spirometry, sickle cell disease therapy, 122 spleen, 368–389 AIHA, 160 blood flow, 369 blood pooling, 369–370 CD4 T-cells, 371 evolution, 368 function, 368–372 blood pooling, 369–370 erythropoiesis, 369 extramedullary haemopoiesis, 371–372 immunological function, 371 plasma skimming, 369

radionuclide investigations, 378, 378 red cell quality control, 371 granulocytes, 370 haematopoiesis, 3 hairy cell leukaemia, 551–552 irradiation, primary myelofibrosis treatment, 700 large granular lymphocyte leukaemia, 562 macrophages, 371 marginal zone B-cells, 371 removal see splenectomy sequestration hereditary spherocytosis, 132 red cells, 371 sickle cell disease, 113 size of, 372 see also splenomegaly splenic marginal zone lymphoma, 555 structure, 368–372 lymphatic organization, 370 red pulp, 368, 369 splenic cords, 369 vascular organization, 370 white pulp, 368, 369 splenectomy, 374–376 applications CLL, 547 cold-type AIHA, 165 congenital dyserythropoietic anaemia, 201 congenital platelet disorder, 924–925 hairy cell leukaemia, 553 hereditary spherocytosis, 132–133 primary immune thrombocytopenia, 934–935 primary myelofibrosis, 700 pyruvate kinase deficiency, 143, 145 splenic marginal zone lymphoma, 555, 671 T-cell large granular lymphocyte leukaemia, 565 thalassaemia major, 95 warm-type AIHA, 163 complications, 375–376, 935 delayed, 375 haematological effects, 377–378 immediate, 375 immunological effects, 378 overwhelming postoperative infection, 375–376 thrombocytosis, 375 haemoglobin structural variants, 107 immunizations, 375 indications for, 133 infection prevention, 935 response prediction, 935 risks of, 133

splenic cords, spleen, 369 splenic marginal zone lymphoma (SMZL), 554–555, 555, 671 blood film, 555 bone marrow trephine biopsy, 555 WHO lymphoma classification, 616–617 splenic marginal zone NHL, 657 splenomegaly, 372–376 causes, 372, 374, 374 see also specific causes chronic myelomonocytic leukaemia, 499, 526 essential thrombocythaemia, 694 haemoglobin disorders, 372 hairy cell leukaemia, 551 large granular lymphocyte leukaemia, 562 leishmaniasis, 968 malaria, 372, 959 malignant haematological disorders, 372 neutropenia, 370 pathology, 372 polycythaemia vera, 688 portal hypertension, 374 primary myelofibrosis, 697, 698 primary tumours, 374 warm-type AIHA, 161–162 splicing acceptor sites, haemoglobin, 85 splicing donor sites, haemoglobin, 85 spontaneous abortion anti-D antibodies development, 258 rare bleeding disorders, 814 spontaneous remission CLL management, 544 Langerhans cell histiocytosis, 326 paroxysmal nocturnal haemoglobinuria, 178–179 sporozoites, malaria life cycle, 957 sprue, tropical see tropical sprue Sprycel, CML management, 494 SPTA1 gene, 131 SPTA2 gene, 131 ‘spur-cell anaemia,’ 949 SQUID see superconducting quantum interface device (SQUID) Src tyrosine kinase phagocyte signalling, 304 platelet signalling pathways, 779, 781, 782 stabilization, acquired aplastic anaemia management, 215–216 staging see specific diseases/disorders standard surgical blood ordering schedule (SBOS), 281 Stanford trial, 649 Staphylococcus infections, sickle cell disease, 114 1051

Index

Staphylococcus aureus infections, 951 leukaemias, 472 multiple myeloma, 588 Staphylococcus epidermis infection, leukaemias, 472 Staphylococcus pneumoniae infection, sickle cell disease, 114 STAT (signal transducers and activators of transcription), BCR–ABL1 chimeric gene, 489 statutory requirements, SHOT, 290 STEAP3, 29 erythroid cell iron absorption, 35 Steel mice, haematopoiesis, 6 stem cell engraftment, SCT, 726, 726–727 stem cell factor (SCF) erythroid cell development, 22 erythropoietin receptor, 21–22 mast cell development, 321–322 stem cell mobilization, SCT see stem cell transplantation (SCT) stem cell transplantation (SCT), 8–9, 220– 224, 722–745 alternative forms, 223–224 allogeneic see allogeneic stem cell transplantation autologous see autologous stem cell transplantation see also specific types clinical management, 732–741 cryopreservative side effects, 733 mortality, 733, 733 platelet transfusions, 733 red cell transfusions, 733 conditioning regimens, 729–732 alkylating agents, 729 comparisons, 730–731 GvHD prophylaxis, 731 reduced intensity, 731–732 future developments, 744–745 graft-versus-leukaemia effect, 722 GvHD, 467–468 haemogenic endothelial cells, 8–9 haploidentical, 224 HLA-matched sibling donor, 221–223 adverse effects, 222 cell dose, 221 chimerism, 222 graft rejection, 221 graft rejection treatment, 222 GvHD, 222 immune recognition, 724 immunological basis, 722–726 acute GvHD, 724, 725 antigens, 722–723 chronic GvHD, 724, 725 effector cells, 722–723 1052

graft-versus-leukaemia effect, 724 immune recognition, 724, 726 indications for, 741–742, 742 acquired aplastic anaemia, 220–224, 221 adult ALL see acute lymphoblastic leukaemia (ALL) CLL, 547–548 CML, 497 dyskeratosis congenita, 195–196 Fanconi anaemia, 189, 191 Gaucher disease, 332 Krabbe disease, 332 lysosomal storage disorder, 336 metachromatic leucodystrophy, 332 mucopolysaccharidosis type I, 332 myelodysplastic syndrome, 519, 520–521 NHL, 668–669 sickle cell disease, 123, 124 Waldenström macroglobulinaemia, 672 long-term complications, 222–223 matched unrelated donors, CML, 497 neutropenia, 314 pretransplant assessment, 220–221 relapse management, 743–744 sources, 8–9 stem cell engraftment, 726, 726–727 stem cell manipulation/expansion, 728–729 stem cell mobilization, 727–729 autologous transplants, 727 in clinical practice, 727 cord blood transplants, 728, 728 sibling donor transplants, 728 stem cell trafficking, 727 unrelated donor transplants, 728 transfusions, irradiation, 467–468 unrelated donor, 223 steroids acute GVHD therapy, 735 antiemetic effects, 481 mycosis fungoides treatment, 682 Sézary syndrome/mycosis fungoides, 567 side effect, pernicious anaemia, 72 STI571 see imatinib mesylate stomach, DLBCL, 676 stomatocytes, 135, 135 stomatocytosis, hereditary, 135, 135–136, 136 storage iron, iron metabolism measurement, 36, 37 storage pool deficiency (SPD) syndrome, 923, 923 Streptococcus mitis infection, leukaemias, 471

Streptococcus oralis infection, leukaemias, 471 Streptococcus pneumoniae infection allogeneic SCT infections, 736 multiple myeloma, 588 polyagglutinable red cells, T activation, 262 spleen, 371 splenectomy risks, 133, 375 streptokinase, venous thromboembolism therapy, 903, 906 stroke, sickle cell disease, 114, 115, 116 stromal cell-derived factor 1 (SDF-1) haemopoietic stem cells, 3 multiple myeloma pathogenesis, 580–581 stromal cell lines, haematopoiesis, 6, 7 STX11 gene, haemophagocytic lymphohistiocytosis, 326 subcutaneous panniculitis-like T-cell lymphoma, 571 WHO lymphoma classification, 629, 629 submicroscopic mutations, somatic mutations, 387 substrate reduction therapy (SRT), lysosomal storage disorder therapy, 336–337 succinate dehydrogenase, 27 sulfasalazine, acquired haemolytic anaemias, 173 sulphatase, basophil/mast cell granules, 322 sulphonamides, malaria treatment, 962 superconducting quantum interface device (SQUID) haemochromatosis, type 1, 50 iron overload, 54 superoxide dismutase, phagocytes, 305 superoxides, phagocytes, 305 supportive therapy acquired aplastic anaemia management, 214–215 chronic myelomonocytic leukaemia, 527 DIC treatment, 844 Gaucher disease, 340–341 hairy cell leukaemia treatment, 553 myelodysplastic syndrome therapy, 519 paroxysmal nocturnal haemoglobinuria treatment, 179–180 systemic AL amyloidosis management, 610 suppurative thrombophlebitis, 951–952 suramin, African trypanosomiasis therapy, 966

Index

surgery bleeding, haemophilia A, 795 nodular lymphocyte predominant Hodgkin lymphoma treatment, 647 venous thromboembolism prophylaxis, 911 surveillance cultures, infections in leukaemia, 472 SWOG9321 study, multiple myeloma autologous SCT, 591 Syk phagocyte signalling, 304–305 platelet signalling pathways, 779, 781, 782 symptomatic multiple myeloma, 583, 583 syntaxin-4, platelet activation, 786 syntaxin 11, haemophagocytic lymphohistiocytosis, 326 syphilis testing, blood donation, 275 transmission, blood transfusions, 274 systemic AL amyloidosis, 601–610 AL fibrils, 601 clinical features, 601–602 diagnosis, 602, 603, 604–607 DNA analysis, 605–606 electron microscopy, 604 fibril identification, 604–605 histology, 602, 604 immunohistochemistry, 605 mass spectrometry, 606 organ involvement, 606, 606–607 plasma cell dyscrasia assessment, 606 SAP scintigraphy, 604, 605 differential diagnosis, 607 management, 607–610 allogeneic bone marrow transplantation, 609 autologous peripheral blood SCT, 608 bortezomib, 609 CTD, 608–609 dexamethasone, 608 lenalidomide, 609 melphalan, 608, 609 prednisolone, 609 solid organ transplantation, 610 supportive treatment, 610 VAD, 609 monoclonal light chains, 601 natural history, 607 plasma cell dyscrasia, 601 prognostic factors, 607 systemic amyloidosis, hereditary, 611–612 systemic diseases haematological aspects, 940–955 see also specific diseases/disorders

systemic hypoxia, erythrocytosis see erythrocytosis systemic lupus erythematosus (SLE) acquired aplastic anaemia, 207 antiphospholipid syndrome, 892 eosinophilia, 946 malignancies, 946 neutropenia, 316 sideroblastic anaemia, 946 thrombosis in inflammation, 898 warm-type AIHA, 945–946 systemic tuberculosis, thrombosis in inflammation, 898 T tachycardia, venous thromboembolism, 901 tachypnoea, venous thromboembolism, 901 TAFI see thrombin-activatable fibrinolysis inhibitor (TAFI) TAL1 gene see SCL (TAL1) gene T-ALL see T-cell acute lymphoblastic leukaemia (T-ALL) tamoxifen, venous thromboembolism, 890 TAP (transporter associated with antigen processing) antigen processing, 345 HLA class I antigen processing/ presentation, 711 TAR see thrombocytopenia with absent radii (TAR) tartrate-resistant acid phosphatase (TRAP), hairy cell leukaemia diagnosis, 551–552 Tasigna, 494, 495 Tay–Sachs disease, 334 TBI see total body irradiation (TBI) TCD GvHD prophylaxis, 731 unrelated donor allogeneic SCT conditioning regimens, 731 T cell(s) αβ, 353 activation, 345–346 acquired aplastic anaemia, 209 antigen recognition, 344–345 B-cell activation, 356, 357 development, 343, 351, 352 negative selection, 351 positive selection, 351 self-reactive deletion, 345 differentiation, 358 γδ, 353 hepatosplenic T-cell lymphoma, 629 homeostatic proliferation, 354

lymphoproliferative disorders, 558–576 classification, 558, 559 disease features, 559 genotypes, 561 immunophenotypes, 561 leukaemia/lymphoma syndromes, 565–570 primary leukaemias, 558, 559–565 virus infections, 558 see also specific diseases/disorders monoclonal populations, 403–404 recovery post-SCT, 726 repertoire, 353–354 in spleen, 371 see also CD4 T-cells; CD8 T-cells; T helper cells type 1 (Th1); T helper cells type 2 (Th2) T-cell acute lymphoblastic leukaemia (T-ALL) cytogenetics/molecular classification, 453–454 HOX11 genes, 385 SCL genes, 385 T-cell/histiocyte-rich large B-cell lymphoma, 624 T-cell large granular lymphocyte leukaemia (T-cell LGL), 561–565 aetiology, 561 clinical features, 561–562 diagnosis, 562–563 differential diagnosis, 563 pathogenesis, 563 prognosis, 563–564 therapy, 563–564, 565, 565 T-cell LGL see T-cell large granular lymphocyte leukaemia (T-cell LGL) T-cell non-Hodgkin lymphoma (T-NHLs), 558, 570–575 anaplastic large-cell lymphoma, 574–575 angioimmunoblastic, 572 differential diagnosis, 575 EBV-positive in childhood, 573 enteropathy-associated, 573–574 extranodal T/NK lymphoma, nasal type, 572–573, 573 hepatosplenic, 571 peripheral T-NHL not specified, 570 subcutaneous panniculitis-like T-NHL, 571 T-cell priming, 355, 356 T-cell receptor (TCR), 343 α chain, 346, 347 rearrangement, 351, 352 T-prolymphocytic leukaemia, 560 αβ, large granular lymphocyte leukaemia, 562 1053

Index

β chain, 347 enteropathy-associated T-NHL, 574 large granular lymphocyte leukaemia, 562 rearrangement, 351 chain rearrangement, molecular genetic analysis, 411 γ chain enteropathy-associated T-NHL, 574 large granular lymphocyte leukaemia, 562 γδ, 346 development, 351 large granular lymphocyte leukaemia, 562 generation, 347–349 gene rearrangements, 347, 351 leukaemogenesis, 381 genetics, 348 mutations, NHL, 658 phenotypic changes, 351 proteins, T-lineage ALL, 452 structure, 346, 347 TCF3 gene, childhood ALL, 450 TCF3–PBX1 gene fusion, childhood ALL, 450 TCF3–PBX1 (E2A–PBX1) gene rearrangements, childhood ALL, 453, 454 TCL1 gene, T-prolymphocytic leukaemia, 560 TCN1 gene, cobalamin absorption, 68, 69 TdT (terminal deoxynucleotidyltransferase), 350–351 tecarfarin, venous thromboembolism therapy, 906 TEL-AML1 gene fusion see ETV6–RUNX (TEL–AML1) gene fusion telangiectasia, conjunctival, 854–855 Tel (ETV6), erythropoiesis, 15 telomerase complex, 212 dyskeratosis congenita, 194, 194–195 temperature agglutination tests, 237 complement, lytic phase, 235 TERC gene acquired aplastic anaemia, 212–213 dyskeratosis congenita, 194 terminal deoxynucleotidyltransferase (TdT), 350–351 termination codons, globin gene translation, 87 TERT gene, acquired aplastic anaemia, 212–213 tertiary β-thalassaemias, 92–93 1054

TET2 gene myelodysplastic syndrome, 511 polycythaemia vera, 687 primary myelofibrosis, 697 tetrahydrobiopterin, nitric oxide synthase, 306 TF see tissue factor (TF) TFP1 gene, knockout animal models, 763 TFPI see tissue factor pathway inhibitor (TFPI) TFR1, iron-responsive elements, 32 TFR2, hepcidin expression, 31 TFRC gene, 28 β-TG, 363 TGF-β see transforming growth factor-β (TGF-β) thalassaemia(s), 88–106 classification, 88–89 definition, 88–89 epidemiology, 88 globin chain effects, 88 hypochromic anaemia vs., 37 inheritance, 88–89 screening, 105–106 see also specific types α-thalassaemia, 100–104 α0 thalassaemia trait, 103 distribution, 100 genetics, 101, 102 deletions, 100, 102 LUC7L, 100 point mutations, 100 single base substitutions, 100 genotype–phenotype relationship, 102–103 with mental retardation syndrome, 103–104 molecular pathology, 100–101 with myelodysplasia, 104 pathophysiology, 100–102 β-thalassaemia co-inheritance, 92 β-thalassaemia, 89–97 α-thalassaemia co-inheritance, 92 clinical findings, 93 distribution, 89 dominant inheritance, 97 fetal haemoglobin, 91 genetic basis, 89–91 β-globin gene deletions, 90, 91 post-translational stability mutations, 90, 90 processing, 89 transcriptions, 89 translation, 89–90 transposable elements, 90–91 genotype–phenotype relationships, 92–93

haemoglobin variant associations, 96–97 hyperunstable β-chain variants, 97 laboratory diagnosis, 93 management, 93–96 blood transfusions, 92, 94 bone marrow transplants, 96 carriers, 96 iron chelation therapy, 94 prognosis, 96 pathophysiology, 91–92, 92 primary, 92 secondary, 92 with sickle cell disease, 118 fetal haemoglobin, 100 tertiary, 92–93 variant forms, 97 δβ–thalassaemia, 97–100 compound heterozygosity, 97–98 deletions, 98, 98 point mutations, 98 εγδβ-thalassaemia, 100 thalassaemia intermedia, 59, 104–105 molecular pathology, 104, 104 thalassaemia major definition, 93 monitoring/review, 95–96 neonatal anaemia, 976–977, 977 radiography, 94, 94 α0 thalassaemia trait, 103 thalidomide with cyclophosphamide and dexamethasone, systemic AL amyloidosis management, 608–609 mantle cell lymphoma, 673 multiple myeloma management/ treatment, 591, 592, 593, 595, 595 primary myelofibrosis treatment, 700–701 side effects, 596 venous thromboembolism, 890 Waldenström macroglobulinaemia, 672 T helper cells type 1 (Th1) cytokine production, 358 development, 358 T helper cells type 2 balance, 358 primary immune thrombocytopenia, 929 T helper cells type 2 (Th2) cytokine production, 358 development, 358 T helper cells type 1 balance see T helper cells type 1 (Th1) T helper cells type 17 (Th17), 358

Index

thermal injury, acquired haemolytic anaemias, 174 thiamine-responsive megaloblastic anaemia, 81 thiamine transporter (THTR-1), sideroblastic anaemia, 44 thioguanine, childhood ALL treatment, 458 thiopurine S-methyltransferases, childhood ALL prognosis, 455 THPO gene, congenital amegakaryocytic thrombocytopenia, 917 thrombasthenia, Glanzmann see Glanzmann thrombasthenia thrombin blood coagulation, 748 generation, DIC, 843 protein C pathway, 749–750, 752 von Willebrand factor, 805 thrombin-activatable fibrinolysis inhibitor (TAFI), 759 in haemophilia, 793 haemostatic disturbance in chronic liver disease, 848–849 plasmin inhibition, 770 thrombin generation assays, acquired coagulation disorders, 840 thrombin time (TT), 748 acquired coagulation disorders, 840 haemophilia A, 795 neonatal haemostasis, 980 rare bleeding disorders, 814 thrombocythaemia, essential see essential thrombocythaemia (ET) thrombocytopathies, 919–924 platelet adhesion disorders, 919–920 platelet aggregation disorders, 921–923 platelet signalling transduction disorders, 920 see also specific diseases/disorders thrombocytopenias, 916–919 adult ALL, 436 adult T-cell leukaemia lymphoma, 568 African trypanosomiasis, 966 alloimmune, 916 antiphospholipid syndrome, 892 autologous SCT complications, 741 bone marrow infiltration, 916–917 CLL, 467, 534 congenital non-inherited, 916–917 definition, 916 drug-induced, 916 haemostatic disturbance in chronic liver disease, 848 HIV infection, 955 imatinib mesylate, 492 infections, 917

inherited, 917–919 increased platelet size, 918–919 normal platelet size, 917 reduced platelet size, 917 leukaemia, 464–468 malaria, 959 malignancies, 944, 944 see also specific malignancies malignancy treatment, 945 multiple myeloma, 587 myelodysplastic syndrome, 512 neonates, 981–982, 982 pregnancy, 953 primary immune thrombocytopenia see primary immune thrombocytopenia thalidomide side effects, 596 TTP, 863 viral infections, 950 thrombocytopenia with absent radii (TAR), 187, 202–203, 917 storage pool disease, 923 thrombocytosis CML association, 695–696 malignancies, 944 MDS association, 696 post-splenectomy, 377–378 reactive, 695, 695 rickettsial infections, 951 splenectomy, 375 thromboelastography, acquired coagulation disorders, 840, 840 thromboembolic pulmonary hypertension, chronic, 913 β-thromboglobulin, platelet α-granules, 774 thrombolytic therapy, venous thromboembolism therapy, 903, 906–907 thrombomodulin, 758, 766 blood coagulation feedback inhibition, 749–750 thrombosis in inflammation, 898 thrombophilias, heritable, 872–887 arterial thrombosis, 882 candidates, 878 clinical definition, 872 counselling, 885–886 environmental risk avoidance, 880 fibrinolysis, 879 gene–environment interactions, 873–874 genetic risk factors, 872 genetic testing, 885–886 homocysteine, 879 laboratory testing, 883–885 activated protein C assay, 883 antithrombin assays, 884 APTT, 883

full blood count, 883 pre-analytical variables, 883 protein C assays, 884 protein S assays, 884–885 prothrombin time, 883 prevalence, 873, 873 venous thrombosis association, 873, 873–879 therapy, 879–880 see also specific diseases/disorders thrombophilias, neonatal stroke, 882–883 thrombophlebitis blood transfusions, 288 suppurative see suppurative thrombophlebitis thromboplastin inhibition test, antiphospholipid syndrome, 894 thrombopoietin agonists, congenital platelet disorder therapy, 924 platelets formation, 775 primary immune thrombocytopenia, 931, 931–932 receptor agonists, primary immune thrombocytopenia management, 933, 936 thrombocytopenia with absent radii, 203 thrombosis eculizumab effects, 181 folate deficiency, 65 neonatal haematology, 979–983 paroxysmal nocturnal haemoglobinuria, 177–178, 180 platelets, 790–791 thrombospondin, 754 thrombotic complications essential thrombocythaemia, 694 polycythaemia vera, 688 thrombotic microangiopathy (TMA), historical aspects, 860 thrombotic therapy, contraindications, 906 thrombotic thrombocytopenic purpura (TTP), 172, 172, 862–868 acquired, 862 ADAMTS-13 deficiency, 861, 862 age of onset, 864 associated diseases, 861 autoimmune, 863 clinical findings, 863–865 haemolytic anaemia, 863 thrombocytopenia, 863 congenital, 862 differential diagnosis, 865, 865–866 historical aspects, 860 HIV infection, 955 laboratory findings, 863–865 ADAMTS-13 assays, 863–865, 864 direct Coombs test, 863 1055

Index

haematocrit, 863 lactate dehydrogenase, 863 mortality, 862 natural history, 866 pathogenesis, 862–863 anti-ADAMTS-13 antibodies, 866 pathology, 862–863 treatment, 866–868 ADAMTS-13 replacement, 868 corticosteroids, 867 fresh frozen plasma, 867 immunological therapy, 867, 867–868 plasma exchange, 866–867 prednisolone, 867 rituximab, 867–868 variants, 866 von Willebrand factor, 755 thromboxane platelets, 772, 786 thrombus formation, 777 thromboxane receptor defects, 923 genetics, 925 platelet responses, 922 platelet receptors, 783–784 platelet signalling transduction disorders, 920 thrombus formation, 775–779, 776 aggregation, 777–778 granule secretion, 777 platelet capture, 775–777, 776 collagens, 777 integrins, 776, 777 von Willebrand factor, 775 procoagulant activity, 779 spreading, 777, 778 stabilization, 778–779 thromboxane formation, 777 Thy-1, haemopoietic stem cells, 2 thymic (cortical) T-cell acute lymphoblastic leukaemia, 434 thymoma, red cell aplasia, 942 thymus, haematopoiesis, 3 thyroid gland, systemic AL amyloidosis, 602 thyroid hormone receptor, erythroid cell development, 22 TIAs see transient ischaemic attacks (TIAs) tick-borne relapsing fever, 969 ticlopidine, mechanism of action, 783 tinzaparin, venous thromboembolism in cancer, 896 tissue factor (TF), 755, 758, 760 blood coagulation, 749 DIC, 843 endothelium, 760 haemophilia A, 794 1056

tissue factor pathway inhibitor (TFPI), 759, 762–763 blood coagulation feedback inhibition, 749 endothelial cells, 763 heritable thrombophilia, 878 platelet α-granules, 774 tissue plasminogen activator (tPA), 759, 768–769 fibrinolysis, 751 inhibitor, 768 liver transplantation, 849 T-lineage adult acute lymphoblastic leukaemia, 434 adult ALL prognosis, 443 immunophenotypic classification, 452 TLX1 gene see HOX11 (TLX1) gene TMA (thrombotic microangiopathy), historical aspects, 860 TMD (transient myeloproliferative disorder), 979 TMPRSS6 see matriptase-2 (TMPRSS6) TNF see tumour necrosis factor (TNF) TNF-α see tumour necrosis factor-α (TNF-α) TNF-β see tumour necrosis factor-β (TNF-β) T-NHLs see T-cell non-Hodgkin lymphoma (T-NHLs) tobacco smoking, erythrocytosis, 693 topoisomerase, chromosome translocations, 386 tositumomab, 666 131 -I-labelled tositumomab, follicular lymphoma, 668 total body irradiation (TBI) cobalamin malabsorption, 74 cyclophosphamide see cyclophosphamide fludarabine with, 732 pain, 481 SCT conditioning, 726, 732 with fludarabine and cyclophosphamide, 731 fludarabine with, 732 toxoplasmosis (Toxoplasma infection) acquired haemolytic anaemias, 169 allogeneic SCT complications, 738 cold-type AIHA, 165 TP53 gene adult T-cell leukaemia lymphoma, 568–569 B-PLL, 550 CLL, 539, 539–540 multiple myeloma, 589 myelodysplastic syndrome, 510 tPA see tissue plasminogen activator (tPA)

TPLL see T-prolymphocytic leukaemia (TPLL) T-prolymphocytic leukaemia (TPLL), 559–561 aetiology, 559 clinical features, 559–560 cytogenetics, 560 diagnosis, 560 differential diagnosis, 560–561 disease course, 561 pathogenesis, 560 therapy, 561, 562 TRAIL (tumour necrosis factor-related apoptosis inducing ligand), myelodysplastic syndrome, 508 TRALI see transfusion-related acute lung injury (TRALI) transcobalamin(s) (TCs) cobalamin transport, 70 in disease, 70 receptors, 70 structure, 69 transcription factors erythropoiesis, 12, 15–16 globin gene clusters, 86 haematopoiesis, 2, 7–8 see also specific transcription factors transdifferentiation, HSC development, 4, 5 transferrin, 28, 30 deficiency, iron refractory iron deficiency anaemia, 41 haemochromatosis, type 1, 49–50 iron homeostasis, 33 transferrin receptor(s), 28, 29 erythroid cell iron absorption, 35, 35 iron metabolism measurement, 37, 37–38 transferrin receptor 2 (TFR2), 30 haemochromatosis type 2, 52 transforming growth factor-β (TGF-β), 362 anaemia of chronic disease, 940 hereditary haemorrhagic telangiectasia, 854 nitric oxide synthase, 306 primary myelofibrosis, 697 β-thalassaemia modifiers, 92 transfusion-related acute lung injury (TRALI), 286–287 neutrophil antibodies, 263 supportive management, 286–287 transfusions, blood see blood transfusion(s) transient ischaemic attacks (TIAs) hereditary haemorrhagic telangiectasia, 854 sickle cell disease, 114

Index

transient myeloproliferative disorder (TMD), 979 transplacental haemorrhage, anti-D antibodies, 258 transplacental passage, Igs, 232 transplant-related mortality (TRM) ALL allogeneic SCT, 441 sibling SCT, 728 unrelated SCT, 728 transporter associated with antigen processing see TAP (transporter associated with antigen processing) transthyretin amyloidosis, 611 transthyretin (TTR), amyloidosis, 600 TRAP (tartrate-resistant acid phosphatase), hairy cell leukaemia diagnosis, 551–552 trauma congenital platelet disorders, 924 haemostatic disturbances, 852–853 triazole antifungals, allogeneic SCT infections, 736 trimethoprim, malaria treatment, 962 triose phosphate isomerase, 147 deficiency, 146, 147 congenital non-spherocytic haemolytic anaemia, 147 neonatal anaemia, 976 trisomy 8, AML, 386 TRIUMPH trial, eculizumab, 181 TRM see transplant-related mortality (TRM) trophozoites, malaria life cycle, 957–958 tropical diseases, 956–970 ethnic variations in reference ranges, 956, 957 non-specific haematological abnormalities, 968–969 organisms in bone marrow, 956–968 organisms in peripheral blood, 956–968 see also specific diseases tropical sprue cobalamin malabsorption, 73 folate deficiency, 78 Trousseau syndrome, 895–896, 944–945 Trypanosoma brucei gambiense see African trypanosomiasis Trypanosoma brucei rhodesiense see African trypanosomiasis Trypanosoma cruzi infection, 966–967 trypanosomiasis African see African trypanosomiasis American, 966–967 tryptase, basophil/mast cell granules, 322 TT see thrombin time (TT) TTG-1 (LMO1) gene, 453

TTG-2 see LMO2 (TTG-2) gene TTP see thrombotic thrombocytopenic purpura (TTP) TTR (transthyretin), amyloidosis, 600 TTR gene, familial amyloidotic polyneuropathy, 611–612 tuberculosis, cobalamin malabsorption, 73 tumorigenesis, Hodgkin lymphoma, 641–642 tumour lysis syndrome, 479 Burkitt lymphoma treatment, 679 childhood ALL, 479 tumour necrosis factor (TNF) anaemia of chronic disease, 940 extranodal T/NK lymphoma, nasal type, 573 Gaucher disease, 331 thrombosis in inflammation, 897 tumour necrosis factor-α (TNF-α) anaemia of chronic disease, 940 CLL, 534 myelodysplastic syndrome, 508 receptor, CLL, 534 thrombosis in inflammation, 898 tumour necrosis factor-β (TNF-β), 362 T helper cells type 1, 358 tumour necrosis factor-related apoptosis inducing ligand (TRAIL), myelodysplastic syndrome, 508 tumour-suppressor genes, hypermethylation, 388 24-colour FISH (spectral karyotyping), 407 twins, childhood ALL, 449 twisted gastrulation protein 1 (TWSG1), 28 hepcidin regulation, 32 two-hit models, 386 tyrosine kinase inhibitors AML therapy, 432 CML, 497 second generation, chronic CML management, 494–495 tyrosine kinase, leukaemogenesis, 388–389, 389, 390 tyrosine kinase-linked receptors, platelet receptors, 780–783 T zones, lymph nodes, 355, 355 U UFH see unfractionated heparin (UFH) ultrasound abdominal, mastocytosis, 702 bilateral, pulmonary embolism, 903 deep vein thrombosis, 902 haemolytic disease of the fetus and newborn, 294, 296 mastocytosis, 702

spleen, 373 venous thromboembolism, 902 umbilical cord stem cell transplantation, 1, 8, 223, 743 conditioning regimens, 731 GvHD, 728 myelodysplastic syndrome, 521 stem cell mobilization, 728, 728 UNC13D gene, haemophagocytic lymphohistiocytosis, 326 unconjugated bilirubin, haemolysis, 126 unfractionated heparin (UFH), 764 monitoring, 904 osteoporosis, 904 thrombolytic prophylaxis, 909 venous thromboembolism therapy, 903–904 see also heparin-induced thrombocytopenia (HIT) uniparental disomy, myelodysplastic syndrome, 510 United Kingdom Haemophilia Centre Doctors’ Organization (UKHCDO), factor X deficiency, 825 ‘universal’ donors, antibodies to ABO antigens, 247–248 upper limb deep vein thrombosis, 880 uracil misincorporation, megaloblastic anaemia, 61–62 uric acid, CML, 491 urinary alkalinization, hyperuricaemia therapy, 479 urinary plasminogen activator (uPA), 769 urinary tract infections, 471 urinary urobilinogen, haemolysis, 126 urine iron excretion, iron-loading anaemias, 55 UROD (uroporphyrinogen decarboxylase), porphyria cutanea tarda, 42 urokinase, venous thromboembolism therapy, 906 uroporphyrinogen decarboxylase (UROD), porphyria cutanea tarda, 42 urticaria pigmentosa, mastocytosis, 701, 702, 702 UT-B1 protein, 228 V VAD (vincristine, doxorubicin, dexamethasone) multiple myeloma management/ treatment, 586, 591 systemic AL amyloidosis management, 609 vancomycin, with gentamicin and nystatin (GVN), infection prophylaxis, 473 1057

Index

V antigen, Rh system, 257 variable region, Igs, 232 variant Creutzfeldt–Jakob disease (vCJD) blood transfusion, 274–275 transmission, factor VIII concentrates, 796 varicella zoster virus infection, 737 vascular cell adhesion molecule-1 (VCAM-1) endothelium, 753 multiple myeloma pathogenesis, 580 phagocytes, 301 vascular endothelial growth factor (VEGF), platelet α-granules, 774 vascular organization, spleen, 370 vasculitis, haemostatic dysfunction, 854 vasoconstriction, platelets, 772–773 vaso-occlusion, sickle cell disease, 111 vasopressin, von Willebrand factor, 805 VCAM-1 see vascular cell adhesion molecule-1 (VCAM-1) VDJ gene joining, Igs, 232 VDJ rearrangements B cell development, 350 classical Hodgkin lymphoma, 641 CLL, 533 vegans, cobalamin deficiency, 70 vegetarian diet, iron deficiency anaemia, 39 VEGF (vascular endothelial growth factor), platelet α-granules, 774 veltuzumab, follicular lymphoma therapy, 667 venesection, polycythaemia vera treatment, 690 Venofer (iron–sucrose), 41 venography, venous thromboembolism diagnosis, 902 veno-occlusive disease (VOD), allogeneic SCT complications, 738, 738 venous circulation, polycythaemia vera, 688 venous thrombectomy, venous thromboembolism therapy, 907 venous thromboembolism (VTE), 888–899 cancer, 895–897 management difficulties, 896 clinical presentation, 901 clinical scores, 901 diagnosis, 901–903 chest radiography, 901 computed tomography, 902 ELISA, 901–902 fibrin D-dimers, 901 imaging, 902–903 Westermark sign, 901 drug therapy, 903–906 anticoagulant therapy, 903–906 direct thrombin inhibitors, 905–906 1058

factor Xa antagonists, 906 heparin, 903–904 see also heparin-induced thrombocytopenia (HIT) novel drugs, 906 thrombolytic therapy, 903 vitamin K antagonists, 904–905, 905 hospital-acquired, 909 iatrogenic, 890–891 immobility, 889, 900 incidence, 900 inflammation, 897, 897–898 initial therapy, 906–908 anticoagulant therapy, 907–908 inferior vena cava filters, 907 thrombolytic therapy, 906–907 venous thrombectomy, 907 management, 900–914 cancer patients, 912 drug therapy see above initial therapy see above in pregnancy, 912–913 mechanisms, 900–901 mortality, 900 non-malignant diseases, 898–899 predisposing conditions, 888, 889 pregnancy, 889 primary prophylaxis, 908–909 risk factors, 901 see also specific risk factors secondary prophylaxis, 909–912 duration, 911 low-molecular weight heparin, 911, 912 monitoring, 910 surgery, 911 vitamin K antagonists, 909–911 warfarin, 909 secondary recurrence risk, 910 splenectomy complications, 935 tachycardia, 901 tachypnoea, 901 see also deep vein thrombosis (DVT); pulmonary embolism (PE) venous thrombosis acquired see venous thromboembolism (VTE) ventilation–perfusion (V/Q) scanning, pulmonary embolism diagnosis, 902 VerifyNow test, 790 vessel repair, platelets, 772–773 Vibrio cholerae infection, polyagglutinable red cells, 262 vinblastine, with doxorubicin, bleomycin and dacarbazine see ABVD (doxorubicin, bleomycin, vinblastine and dacarbazine)

vinca alkaloids, pain, 481 vincristine with bleomycin, etoposide, doxorubicin, cyclophosphamide, procarbazine and prednisone see BEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine and prednisone) childhood ALL treatment, 457, 458 with cyclophosphamide and prednisolone see CVP (cyclophosphamide, vincristine, prednisolone) with cyclophosphamide, doxorubicin and prednisolone see CHOP (cyclophosphamide, doxorubicin, vincristine and prednisolone) with doxorubicin, dexamethasone see VAD (vincristine, doxorubicin, dexamethasone) with mustine, procarbazine and prednisolone see MOPP (mustine, vincristine, procarbazine and prednisolone) with rituximab, cyclophosphamide, doxorubicin and prednisolone see R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone) viral haemorrhagic fevers, 969 virus infections, 949–950, 950 anaemia, 949–950 haemophagocytic lymphohistiocytosis, 326 neutropenia, 316 NHL, 656–657 paroxysmal cold haemoglobinuria, 950 platelet disorders, 950 primary immune thrombocytopenia, 928 thrombocytopenia, 950 transmission, blood transfusions, 270, 271 warm-type AIHA, 163 see also specific viruses VISTA study, multiple myeloma management/treatment, 593, 594 visual disturbances, CML, 488–489 vitamin B12 see cobalamin vitamin D, mycosis fungoides treatment, 682 vitamin D receptor, β-thalassaemia modifiers, 92 vitamin E deficiency, acquired haemolytic anaemias, 174

Index

vitamin K, 845–847 as cofactor, 846 deficiency, neonates, 981 metabolism, 845–846, 846 vitamin K1, vitamin K deficiency bleeding therapy, 846–847 vitamin K antagonist-induced skin necrosis, 882 vitamin K antagonists side effects, 904–905 venous thromboembolism prophylaxis, 909–911 venous thromboembolism therapy, 904– 905, 905 vitamin K deficiency bleeding (VKDB), 846–847 classification, 846–847 vitamin K deficiency, malignancy treatment, 945 vitamin K-dependent coagulation factors deficiency (VKCFD), 814, 831–832 knockout mouse model, 815 prophylaxis, 834 treatment, 833 vitamin K epoxide reductase (VKOR), 831 V–J gene joining, Igs, 232 VKCFD see vitamin K-dependent coagulation factors deficiency (VKCFD) VKDB see vitamin K deficiency bleeding (VKDB) VKOR gene combined deficiency of vitamin K proteins, 814 vitamin K-dependent coagulation factors deficiency, 832 VMF see von Willebrand factor (VWF) VOD (veno-occlusive disease), allogeneic SCT complications, 738, 738 volunteer donor registries, 716–717 von Hippel–Lindau (vHL) protein, hypoxia sensing, 20 von Willebrand disease, 804–812 ABO effects, 809 acquired, 811, 812, 858, 945 clinical course, 810 clinical features, 806 complications, 810 laboratory diagnosis, 806, 808–809 multimer analysis, 807 molecular genetics, 810 neonates, 980–981 treatment, 809–810 type 1, 806, 808 diagnosis, 808–809 molecular genetics, 810, 811

type 2, 808 molecular genetics, 810 type 2A, 808, 808 type 2B, 808, 808, 918 type 2M, 808, 808 type 2N, 808, 808 type 3, 808 molecular genetics, 810 von Willebrand factor (VWF), 754–755, 758, 761, 804–806 acquired von Willebrand disease therapy, 858 ADAMTS-13 action, 805 agonists, 805 amyloidosis, 856 composition, 754–755 developmental haemostasis, 980 diarrhoea-related haemolytic-uraemic syndrome, 868 endothelium, 753 factor VIII complex, 805, 805–806 genetics, 804 haemolytic–uraemic syndrome, 860 haemostatic disturbances in pregnancy, 851 in homeostasis, 756 platelet α-granules, 774 platelets, 772 shear forces, 780–781 structure, 804 structure–function relationship, 805 thrombus formation, 775, 778 TTP, 860 Weibel–Palade bodies, 754, 804 VS antigen, Rh system, 257 V segment, Igs, 347–348 VTE see venous thromboembolism (VTE) W Waldenström macroglobulinaemia, 671–672 coagulation disorders, 856 WHO lymphoma classification, 617 warfarin bleeding risks, 467 paroxysmal nocturnal haemoglobinuria treatment, 180 skin necrosis, 855 venous thromboembolism prophylaxis, 909 venous thromboembolism therapy, 904 venous thrombosis in heritable thrombophilias, 879 warm-acting antibodies, 233 AIHA, 159, 160 warm-type autoimmune haemolytic anaemia, 161–164

antibody characteristics, 159, 160 autoimmune disease association, 162 systemic lupus erythematosus, 945–946 clinical features, 161–163 blood film, 161 jaundice, 161 lactate dehydrogenase, 162 splenomegaly, 161–162 drug-induced, 162–163 Evans syndrome, 162 idiopathic, 162 infancy/childhood, 162 lymphoproliferative disease, 162 malignancies, 163, 942 treatment, 163–164 prognosis, 164 viral infections, 163 WAS gene Wiskott–Aldrich syndrome, 925 X-linked agammaglobulinaemia, 925 WASP gene, neutropenias, 202 ‘watch-and-wait’ approach NHL treatment, 664 primary immune thrombocytopenia management, 936, 937 ‘waterfall’ hypothesis, blood coagulation, 748 weak D, Rh system, 255 Weibel–Palade bodies, von Willebrand factor, 754, 804 Westermark sign, venous thromboembolism diagnosis, 901 West Nile virus, blood transfusion transmission, 274 WHIM syndrome, neutropenia, 316 white cell(s) antigens, 263–264 connective tissue disorders, 946 disorders neonatal haematology, 979 tropical diseases, 969 HIV infection, 954–955 malignancies, 944, 944–945 pregnancy, 953 white cell antibodies, 263–264 blood transfusions, 286–287 white cell count adult ALL, 436 adult ALL prognosis, 442–443 AML relapse risk, 428 CLL, 466 CML, 466 ethnic variations, 956, 957 leukaemia, 465–466 malaria, 959 primary myelofibrosis, 698 1059

Index

white pulp, spleen, 368, 369 whole-brain irradiation, primary CNS lymphoma, 676 whole genome scanning, 412 Wilms tumour, acquired von Willebrand disease, 811 Wilson disease, 81 acquired haemolytic anaemias, 174 Wiskott–Aldrich syndrome (WAS) genetics, 925 storage pool disease, 923 thrombocytopenias, 917 W mice, haematopoiesis, 6 WNT signalling pathway, multiple myeloma pathogenesis, 582 Wolman disease, 335 clinical features, 338 World Federation of Haemophilia (WFH), 813 World Health Organization (WHO) blood donation, 268 classifications adult ALL, 433 ALL, 406 AML, 406, 416 B cell malignancies, 530 cytogenetic analysis, 409 glucose-6-phosphate dehydrogenase deficiency, 150 laboratory investigation, 413–414 lymphoma see World Health Organization (WHO), lymphoma classification myelodysplastic syndrome see myelodysplastic syndrome (MDS) myeloproliferative syndromes, 484 refractory anaemia with excess blasts in transformation, 506 HLA nomenclature, 714 World Health Organization (WHO), lymphoma classification, 614– 638, 615 Hodgkin lymphoma, 635–637, 640 classical Hodgkin lymphoma, 636, 636–637 nodular lymphocyte predominant, 635, 636 mature B-cell neoplasms, 614–627 ALK-positive large B-cell lymphomas, 625 B-cell prolymphocytic leukaemia, 616 borderline DLBCL and Burkitt lymphoma, 626 Burkitt lymphoma, 625–626 CLL, 614–616 1060

DLBCL not otherwise specified, 622– 624, 623 DLBCL with predominant extranodal location, 624–625 extranodal marginal zone lymphoma of MALT, 618–619 follicular lymphoma, 619–621, 620 in situ follicular lymphoma, 620 intermediate DLBCL and classical Hodgkin lymphoma, 626–627 intrafollicular neoplasia, 620 intravascular large B-cell lymphoma, 624, 625 large-cell lymphomas of terminally differentiated B cells, 625 lymphoplasmacytic lymphoma, 617 mantle cell lymphoma, 621–622 monoclonal gammopathy of undetermined significance, 618 nodal marginal zone lymphoma, 619 paediatric follicular lymphoma, 620 plasmablastic lymphoma, 625 plasma cell myeloma, 618 plasma cell neoplasms, 618 plasmacytoma, 618 primary cutaneous DLBCL, leg type, 624–625 primary cutaneous follicle centre lymphoma, 620–621 primary effusion lymphoma, 625, 625 primary intestinal follicular lymphoma, 620 primary mediastinal (thymic) large B-cell lymphoma, 624 in situ follicular lymphoma, 620 small lymphocytic leukaemia, 614–616 splenic marginal zone lymphoma, 616–617 T-cell/histiocyte-rich large B-cell lymphoma, 624 Waldenström macroglobulinaemia, 617 mature NK-cell/T-cell neoplasms, 627–635 adult T-cell leukaemia/lymphoma, 627–628, 628 aggressive NK-cell leukaemia, 627, 627 anaplastic large-cell lymphoma, ALK+, 633, 633, 634, 635 anaplastic large-cell lymphoma, ALK-, 635 angioimmunoblastic T-cell lymphoma, 631, 632, 633 EBV-positive T-cell lymphoproliferative disorders of childhood, 627, 627

enteropathy-associated T-cell lymphoma, 628, 629 extranodal NK/T-cell lymphoma, nasal type, 628, 628 hepatosplenic T-cell lymphoma, 629, 629 lymphomatoid papulosis, 630 mycosis fungoides, 630, 630 peripheral T-cell lymphoma not otherwise specified, 631, 631 primary cutaneous anaplastic largecell lymphoma, 630 primary cutaneous CD30-positive T-cell lymphoproliferative disorders, 630 primary cutaneous γδ T-cell lymphomas, 630–631 Sézary syndrome, 630 subcutaneous panniculitis-like T-cell lymphoma, 629, 629 Wra antigen, Diego blood group, 261 Wrb antigen, Diego blood group, 261 WT1 gene AML minimal residual disease, 429 mutation, AML relapse risk, 427 Wucheria bancrofti infection see filariasis X X-chromosome inactivation patterns (XCIPs) dyskeratosis congenita, 192 essential thrombocythaemia, 693 haemophilia A in females, 801 leukaemogenesis, 381 polycythaemia vera, 686 xenoantibodies, definition, 230 Xg blood group, 245, 261 antigen, 228 Xk protein, 228 absence of, 229 X-linked agammaglobulinaemia, neutropenia, 315 X-linked inhibitor of apoptosis (XIAP), neutrophil apoptosis, 312 X-linked lymphoproliferative syndrome haemophagocytic lymphohistiocytosis, 326 infectious mononucleosis vs., 366 X-linked sideroblastic anaemia, 44 X-linked thrombocytopenia (XLT), 917 genetics, 925 Y yellow fever, 969 Yersinia enterocolitica infections, 287 yolk sac, haematopoiesis, 7 Yt blood group, 245, 261 antigen, 229

Index

Z ZAP-70 CLL, 540, 541, 616 small lymphocytic leukaemia, 616 Zevalin see 90Y-ibritumomab tiuxetan zidovudine, adult T-cell leukaemia lymphoma treatment, 678

Zieve syndrome, acquired haemolytic anaemias, 174 zinc deficiency, deferiprone side effects, 58 ZNF198–FSFR1 gene fusion, chronic myelomonocytic leukaemia, 499

zoledronic acid, multiple myeloma management/treatment, 584– 585, 585–586 Zollinger–Ellison syndrome, cobalamin malabsorption, 73

1061